Examples of naïve antibody libraries applied for diagnostic applications.
\\n\\n
Released this past November, the list is based on data collected from the Web of Science and highlights some of the world’s most influential scientific minds by naming the researchers whose publications over the previous decade have included a high number of Highly Cited Papers placing them among the top 1% most-cited.
\\n\\nWe wish to congratulate all of the researchers named and especially our authors on this amazing accomplishment! We are happy and proud to share in their success!
Note: Edited in March 2021
\\n"}]',published:!0,mainMedia:{caption:"Highly Cited",originalUrl:"/media/original/117"}},components:[{type:"htmlEditorComponent",content:'IntechOpen is proud to announce that 191 of our authors have made the Clarivate™ Highly Cited Researchers List for 2020, ranking them among the top 1% most-cited.
\n\nThroughout the years, the list has named a total of 261 IntechOpen authors as Highly Cited. Of those researchers, 69 have been featured on the list multiple times.
\n\n\n\nReleased this past November, the list is based on data collected from the Web of Science and highlights some of the world’s most influential scientific minds by naming the researchers whose publications over the previous decade have included a high number of Highly Cited Papers placing them among the top 1% most-cited.
\n\nWe wish to congratulate all of the researchers named and especially our authors on this amazing accomplishment! We are happy and proud to share in their success!
Note: Edited in March 2021
\n'}],latestNews:[{slug:"webinar-introduction-to-open-science-wednesday-18-may-1-pm-cest-20220518",title:"Webinar: Introduction to Open Science | Wednesday 18 May, 1 PM CEST"},{slug:"step-in-the-right-direction-intechopen-launches-a-portfolio-of-open-science-journals-20220414",title:"Step in the Right Direction: IntechOpen Launches a Portfolio of Open Science Journals"},{slug:"let-s-meet-at-london-book-fair-5-7-april-2022-olympia-london-20220321",title:"Let’s meet at London Book Fair, 5-7 April 2022, Olympia London"},{slug:"50-books-published-as-part-of-intechopen-and-knowledge-unlatched-ku-collaboration-20220316",title:"50 Books published as part of IntechOpen and Knowledge Unlatched (KU) Collaboration"},{slug:"intechopen-joins-the-united-nations-sustainable-development-goals-publishers-compact-20221702",title:"IntechOpen joins the United Nations Sustainable Development Goals Publishers Compact"},{slug:"intechopen-signs-exclusive-representation-agreement-with-lsr-libros-servicios-y-representaciones-s-a-de-c-v-20211123",title:"IntechOpen Signs Exclusive Representation Agreement with LSR Libros Servicios y Representaciones S.A. de C.V"},{slug:"intechopen-expands-partnership-with-research4life-20211110",title:"IntechOpen Expands Partnership with Research4Life"},{slug:"introducing-intechopen-book-series-a-new-publishing-format-for-oa-books-20210915",title:"Introducing IntechOpen Book Series - A New Publishing Format for OA Books"}]},book:{item:{type:"book",id:"2610",leadTitle:null,fullTitle:"Carcinogenesis",title:"Carcinogenesis",subtitle:null,reviewType:"peer-reviewed",abstract:"Carcinogenesis covers molecular, biochemical and cellular processes that underpin this field. 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",isbn:"978-1-83768-039-9",printIsbn:"978-1-83768-038-2",pdfIsbn:"978-1-83768-040-5",doi:null,price:0,priceEur:0,priceUsd:0,slug:null,numberOfPages:0,isOpenForSubmission:!0,isSalesforceBook:!1,isNomenclature:!1,hash:"000e31f2e2f7295805e9a3864158ad63",bookSignature:"Dr. Shafizan Mohamed and Dr. Shazleen Mohamed",publishedDate:null,coverURL:"https://cdn.intechopen.com/books/images_new/11437.jpg",keywords:"Digital Parenting, Digital Education, Screen Time, Family Communication, Smart Technologies, Global Village, Crisis Communication, Emergency Management, Fake News, Media Ideologies, Crowdsourcing, Information Security",numberOfDownloads:null,numberOfWosCitations:0,numberOfCrossrefCitations:null,numberOfDimensionsCitations:null,numberOfTotalCitations:null,isAvailableForWebshopOrdering:!0,dateEndFirstStepPublish:"May 11th 2022",dateEndSecondStepPublish:"June 8th 2022",dateEndThirdStepPublish:"August 7th 2022",dateEndFourthStepPublish:"October 26th 2022",dateEndFifthStepPublish:"December 25th 2022",dateConfirmationOfParticipation:null,remainingDaysToSecondStep:"25 days",secondStepPassed:!0,areRegistrationsClosed:!1,currentStepOfPublishingProcess:3,editedByType:null,kuFlag:!1,biosketch:"Shafizan Mohamed is an assistant professor in the Communications Department, International Islamic University of Malaysia. She holds a Ph.D. in Media Studies from Monash University, Australia. She is the author, co-author, and editor of several books, journal articles, and monographs on media and communication. Her research covers new media theory, political communication, health communication, and digital media literacy. Her recent research projects examined digital media skills.",coeditorOneBiosketch:"Dr. Shazleen Mohamed is the Head of Postgraduate Studies at Universiti Teknologi MARA's Faculty of Communication and Media Studies. She is an expert in broadcasting and media studies, with a particular emphasis on children's television reception. She has more than 20 years of experience in media academia as well as industry. She has also authored numerous articles for scholarly and public journals. 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Her two recent research projects examined the digital media skills of underprivileged children and the communication of vaccination in mainstream and social media. Both studies are based on the experience of Malaysia. 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From chapter submission and review, to approval and revision, copyediting and design, until final publication, I work closely with authors and editors to ensure a simple and easy publishing process. 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Venkateswarlu",coverURL:"https://cdn.intechopen.com/books/images_new/371.jpg",editedByType:"Edited by",editors:[{id:"58592",title:"Dr.",name:"Arun",surname:"Shanker",slug:"arun-shanker",fullName:"Arun Shanker"}],productType:{id:"1",chapterContentType:"chapter",authoredCaption:"Edited by"}},{type:"book",id:"72",title:"Ionic Liquids",subtitle:"Theory, Properties, New Approaches",isOpenForSubmission:!1,hash:"d94ffa3cfa10505e3b1d676d46fcd3f5",slug:"ionic-liquids-theory-properties-new-approaches",bookSignature:"Alexander Kokorin",coverURL:"https://cdn.intechopen.com/books/images_new/72.jpg",editedByType:"Edited by",editors:[{id:"19816",title:"Prof.",name:"Alexander",surname:"Kokorin",slug:"alexander-kokorin",fullName:"Alexander Kokorin"}],productType:{id:"1",chapterContentType:"chapter",authoredCaption:"Edited by"}},{type:"book",id:"314",title:"Regenerative Medicine and Tissue Engineering",subtitle:"Cells and Biomaterials",isOpenForSubmission:!1,hash:"bb67e80e480c86bb8315458012d65686",slug:"regenerative-medicine-and-tissue-engineering-cells-and-biomaterials",bookSignature:"Daniel Eberli",coverURL:"https://cdn.intechopen.com/books/images_new/314.jpg",editedByType:"Edited by",editors:[{id:"6495",title:"Dr.",name:"Daniel",surname:"Eberli",slug:"daniel-eberli",fullName:"Daniel Eberli"}],productType:{id:"1",chapterContentType:"chapter",authoredCaption:"Edited by"}},{type:"book",id:"57",title:"Physics and Applications of Graphene",subtitle:"Experiments",isOpenForSubmission:!1,hash:"0e6622a71cf4f02f45bfdd5691e1189a",slug:"physics-and-applications-of-graphene-experiments",bookSignature:"Sergey Mikhailov",coverURL:"https://cdn.intechopen.com/books/images_new/57.jpg",editedByType:"Edited by",editors:[{id:"16042",title:"Dr.",name:"Sergey",surname:"Mikhailov",slug:"sergey-mikhailov",fullName:"Sergey Mikhailov"}],productType:{id:"1",chapterContentType:"chapter",authoredCaption:"Edited by"}},{type:"book",id:"1373",title:"Ionic Liquids",subtitle:"Applications and Perspectives",isOpenForSubmission:!1,hash:"5e9ae5ae9167cde4b344e499a792c41c",slug:"ionic-liquids-applications-and-perspectives",bookSignature:"Alexander Kokorin",coverURL:"https://cdn.intechopen.com/books/images_new/1373.jpg",editedByType:"Edited by",editors:[{id:"19816",title:"Prof.",name:"Alexander",surname:"Kokorin",slug:"alexander-kokorin",fullName:"Alexander Kokorin"}],productType:{id:"1",chapterContentType:"chapter",authoredCaption:"Edited by"}}]},chapter:{item:{type:"chapter",id:"51022",title:"Phage Display‐Derived Antibodies: Application of Recombinant Antibodies for Diagnostics",doi:"10.5772/63927",slug:"phage-display-derived-antibodies-application-of-recombinant-antibodies-for-diagnostics",body:'\nThe human body is made up of a series of complex networks or systems that involve different tissues and cells to work in harmony to regulate different functions in the human body. The immune system is one of the vital systems in the human body as its main function is to protect the body from infectious agents and pathogens. The immune system is divided into two main forms of immunity, namely the innate and acquired immunity. The innate immunity is the physical barrier that prevents foreign invasion. If the innate immunity fails to inhibit the entrance of these foreign molecules, the second line of defence being the adaptive immunity will then come into play. This response will take place with the help of both T‐ and B‐cell [1]. B‐cell activation to secrete antibodies can work through either the T‐cell dependent or the T‐cell independent pathway. The T‐cell dependent activation would require T‐helper cells to trigger the processes required for antibody production through B‐cell proliferation [2]. This will then lead to the secretion of antibodies in the body.
\nOver decades, the application and function of antibodies has expanded from being an immunologically important protein to an essential research tool. The basic application of antibodies surrounds the natural feature of antibodies being high affinity and specific binders against target molecules. This feature has allowed antibodies to be successfully applied for diagnostic and therapeutic applications. In general, diagnostic kits are likely to apply antibodies with superior affinities and specificity against a target antigen for detection [3] via different orientations. This includes either the detection of antibodies by antigens, detection of serum antibodies by the corresponding antigens or by competition [4, 5].
\nAntibodies are a form of recognition protein [6], which is ubiquitously found in serum and body fluid of vertebrates. The diverse antibody repertoire is important for the identification of antibodies against a specific target. Antibodies undergo gene rearrangement processes to generate different gene segment combinations that result in antibodies with different gene sequences. The complexity of antibody diversity is mainly attributed to the combinatorial joining of multiple V, D, J segments of the heavy chain and the V, J segment for the light chain. This process involves multiple gene‐editing enzymes to produce numerous combinations of gene segments. After gene segmentation, another process named somatic hypermutation takes place to further diversify the antibody repertoire [7, 8]. Taken together, these processes are mainly responsible for the highly diverse repertoire of antibodies found in the human body. This variation is the basis of the existence of different antigen‐binding specificities and affinities of immunoglobulins (Igs) [2, 9].
\nThe story of antibodies can be dated back to 1890, with the first report detailing the presence of antibodies and its function by Emil von Behring and Shibasaburo Kitasato. They used serum from animals immunized against diphtheria for administration to other animals infected with diphtheria and subsequently curing the infected animals [10, 11]. However, Paul Ehrlich in 1900 proposed the side‐chain theory based on his hypothesis that the binding ability of a receptor is based on the side chains available for binding [12]. The side‐chain theory was then supported by the ‘lock and key’ hypothesis by Emil Fischer that focused the hypothesis mainly on enzyme functions [12]. The constant evolution and understanding of immunology has helped open new avenues of antibody application and function.
\nAnother major breakthrough in antibody technology development is the introduction of hybridoma technology. Traditionally, antibody production for diagnostic applications involved the use of animals. The immunization of animals with an immunogenic protein with and without an adjuvant would generally produce a collection of polyclonal antibodies [13]. A polyclonal pool of antibodies is defined as a set of heterogeneous antibodies targeting a specific antigen at multiple epitopes. The ability to identify monospecific antibodies only came about after Kohler and Milstein introduced the hybridoma technology in 1975. Hybridoma technology allows for monoclonal antibodies (MAbs) to be produced by fusing myeloma cells with antibody‐producing spleen cells to create a hybrid exhibiting both characteristics [11]. This resulted in the formation of an immortal cell line with characteristics from both the spleenocytes and myeloma cells known as a hybridoma. The hybridomas are then screened until a single clone is obtained and the production of it is up‐scaled. It is well known that antibodies generated via this manner are likely to have high affinities due to the maturation process that it underwent. Although successful, the process may be cumbersome and time consuming as researchers have found it at times difficult to generate antibodies using this method against toxic antigens, self‐antigens and sensitive antigens such as membrane proteins or DNA. For all the benefits attributed to hybridoma technology, a major pitfall lies in the fact that for every new antigen a new animal host is required for immunization. Thus, it is difficult to predict or predefine the genetic information and epitope of the clone [11, 14]. This increases the difficulties, cost and time required to make antibodies, making it not the idle solution for antibody production [14].
\nThis brought about a string of alternative methods for antibody production. The turn of the century saw the booming of molecular biology due to the success of recombinant DNA technology. Researchers were hard at work to develop the next alternative method for monoclonal antibody generation. This brought about various methods including phage display and other multiple display methods such as yeast display, ribosome display and mammalian cell display methods [14]. In addition, the use of transgenic animals was also introduced mainly with the xeno‐mouse technology [11, 13, 14]. Even so, phage display is the preferred choice for recombinant antibody (rAb) production in most laboratories. Phage display allows for a faster and cost‐effective solution towards antibody generation using Ff filamentous phage. In general, rAb production involves several steps including the generation of an antibody library, selection and enrichment of phage‐displaying antibodies against specific antigens through the panning process, screening of monospecific antibodies and recombinant production of the antibodies via expression systems [15]. As phage‐derived rAb may suffer from lower affinities, an additional stage of affinity maturation may be introduced to improve the antibodies produced. A major advantage to the use of phage display for rAb generation in contrast to conventional animal‐derived methods is clearly the omission of animal use in the process. Another advantage of phage display is the lower downtime required for antibody production in between antigens. Conventional methods require immunization that may take up to weeks if not months to yield sufficient immune response for antibody production. This makes phage display rather efficient in the long term for antibody production process. However, one must acknowledge that phage‐derived antibodies suffer from lower affinity when compared to conventional antibodies. This is due to the absence of affinity maturation in phage‐derived antibodies as animal‐derived antibodies are produced post maturation.
\nThis chapter highlights monoclonal antibody development for diagnostic applications via phage display technology. This includes the different types of antibody libraries associated with antibody phage display. The chapter also highlights the different methods used to isolate antibodies against target antigens. The application of recombinant antibodies in different diagnostic platforms is also discussed briefly.
\nPhage display makes use of the natural replication cycle of bacteriophages to fuse a specific peptide or protein with the coat protein on the surface of the filamentous phage particles for selection. This design allows the presentation of a predefined foreign phenotype on the phage surface with the genotype being retrievable in the phage. This allowed a physical linkage between the phenotypic characteristics with the genotypic information to be established [11, 16–22].
\nThere are two main methods for the display mechanism on phage. The first is with the use of a phage vector system, which allows the expression of coat protein III (pIII) to the foreign protein, in this case the antibodies are driven by the natural phage promoter [23, 24]. Additional helper phages are not required for phage packaging with phage vector systems. Unlike the phage vectors, the expression of the antibody‐pIII protein by phagemid systems requires an artificial promoter such as the lac promoter. In addition, phagemid systems also require helper phage for phage packaging. As phagemid vectors do not carry the phage genome, complete phage packaging can only be done with the presence of the helper phage that carries the genetic information for the other proteins required for phage packaging. Therefore, a competition between wild‐type pIII with the mutant pIII will occur. This difference in design of both phage and phagemid vectors also contributes to the display efficiency as phage systems are able to provide a multivalent display of the antibodies on pIII, whereas phagemid systems only allow monovalent display.
\nThe isolation of antibody‐presenting phages post binding with a target antigen allows simple identification of the clones by standard sequencing. Therefore, this approach has been utilized to introduce a collection of different antibody sequences into the phagemid vector to produce a collection of varying clones known as an antibody library [25]. The generation of antibody libraries will bring together a different set of considerations that is outlined in the following section.
\nFor antibody isolation with phage display technology, a collection of antibodies has to be first made available. This involves the initial investment to generate a library of antibody clones to be presented on the surface of bacteriophages. The choice of library to be generated is rather dependent on its application, which would influence the subsequent decision‐making process. This is because the type of library required would determine the source required and the minimum library size required ranging from 106 to 1010 [26]. In general, there are four main types of antibody libraries, namely naïve, immunized, synthetic and semi‐synthetic library. Naïve and synthetic antibodies are known as ‘single‐pot’ libraries, which can be screened against any antigen [22, 26]. Figure 1 shows the overall summary of all the libraries and their differences. However, each different library has its own particular characteristic that makes it preferred for certain applications. The application of phage display for antibody generation is not confined only to human antibodies but can also be applied to animal‐derived antibodies.
\nTypes of antibody library design.
Naïve antibody libraries are by definition a collection of antibody genes that are naïve in nature. In other words, the V‐gene repertoire originates from IgM isotype of unimmunized or healthy donors [21]. The main characteristic of a naïve library is the unbiased nature of the repertoire. The antibody repertoire of healthy donors would mean no prior exposure or infection of the donors to any form of infection that could skew the immune response. As antibody production by the immune system is a direct response to the exposure of the individual to any pathogen, it is necessary for naïve libraries to obtain its repertoire from truly healthy donors.
\nThe main advantage of a naïve or single‐pot library is its large repertoire [22] for monoclonal antibody identification against numerous targets such as self, non‐immunogenic or toxic substances. A technical bottleneck associated with naïve libraries is the sheer number of donors required as well as the number of theoretical diversity required. This would involve a large number of ligation and transformation experiments to achieve such numbers. A common problem of naïve libraries is the number of unknown and uncontrollable contents, such as the presence of memory cells of past infections that might influence the true nature of the naïve repertoire due to the huge naïve repertoire [27]. Another issue common to naïve libraries is the isolation of antibodies with varying degrees of affinities. It is common to obtain antibodies of modest affinities using naïve libraries, as the repertoire would have not undergone affinity maturation processes as opposed to hybridoma‐derived or immunized library‐derived antibodies.
\nThe adaptation of naïve antibody libraries for diagnostic applications is not a new concept with several antibodies successfully being isolated for various diagnostic targets. Naïve libraries are useful for diagnostic as it can be used for selection against haptens, foreign and self‐antigens. The ErBb2 protein, which is a tumour marker expressed by breast tumour, was selected against a naïve antibody library to obtain antibodies for immunoassay applications [28, 29]. Table 1 summarizes the application of naïve antibody libraries to generate antibodies against a variety of antigens for diagnostic purpose.
\nLibrary | \nLibrary size | \nSource | \nFormat | \nTarget | \nRef | \n
---|---|---|---|---|---|
2 × 107 | \nAlpaca | \nsdAb (VHH) | \n[27] | \n||
6.86 × 1011 | \nCamel | \nsdAbs | \nHuman procalcitonin (PCT) | \n[28] | \n|
1 × 1012 | \nHuman | \nFabs | \nReceptor‐binding domain of the MERS‐CoV spike glycoprotein | \n[29] | \n|
n/a | \nHuman | \nFabs | \nTES1 domain of the oncogenic LMP1 | \n[30] | \n|
n/a | \nIlama | \ndsAbs | \nGlycoprotein of the Rabies virus (RABV) | \n[31] | \n|
5 × 109 | \nHuman | \nscFv | \nAlphavirus species (virus) | \n[32] | \n|
n/a | \nHuman | \nscFv | \nSin Nombre Virus nucleocapsid protein (SNV‐N) | \n[33] | \n|
2 × 109 | \nHuman | \nFabs | \nReceptor tyrosine kinase Met | \n[34] | \n|
1.47 × 108 | \nHuman | \nscFv | \nMicrocystins | \n[35] | \n|
6.7 x 109 | \nHuman | \nscFv | \nErbB2 protein, Malaria (rPfHRP2) | \n[25, 36] | \n
Examples of naïve antibody libraries applied for diagnostic applications.
The antibody‐binding region is located at the three complementarity‐determining regions (CDRs) of the variable region in both the light chain and heavy chain. The gene sequences along the CDR are highly heterogeneous as a consequences of gene diversification such as V(D)J gene recombination, class switching and somatic hypermutation. The randomization in the CDR is responsible for the varying affinities as well as specificity to all target antigens. DNA technology advancement encourages synthetic antibody design and synthesis at a reasonable cost in laboratory with the help of structural bioinformatics. This is because the prediction of antibody‐antigen interaction can be done after considering the antibody structural constraints and preference.
\nIt is with this information that antibody engineers are now able to design, improve and generate customized frameworks for antibody production. The ability to synthesize specific gene sequences and codons has made it possible to introduce randomization at the CDR. In general, CDR3 are found to be the region with the highest diversification other than CDR1 and CDR2 [30]. In addition to gene randomization, antibody‐binding site was altered by inserting preferential amino acids that can also be used to introduce specific criteria to the antibody‐binding sites. Another key factor is the CDR length that can also be predefined by the user.
\nIn the context of synthetic antibody libraries, the natural immune maturation and somatic hypermutation process can be elevated to generate similar quality antibodies. To construct a synthetic library, the unarranged and randomized V‐gene segments are synthetically assembled ex vivo normally by polymerase chain reaction (PCR). However, customization can be done on the genetic sequence, local variability and overall diversity for synthetic libraries unlike naïve libraries. Even specific codon usage can be applied to suit the needs of the user. Modifications to the framework can be carried out to improve features such as solubility and heat stability [31]. The main criterion for synthetic libraries to be beneficial is the theoretical diversity of the library. As the repertoire is largely naïve, the potential combinations generated by synthetic methods are able to rival the process carried out naturally with the large library size. However, the sheer size of the library diversity makes it difficult to re‐culture without eventual loss of diversity. Even so, the antibodies enriched from synthetic libraries showed comparable affinities to those derived naturally [32]. Another major bottleneck associated with the use of synthetic libraries is the cost of generating a synthetic library. Even so, the turn of events in genetic engineering over the past few years has seen the cost for oligonucleotide synthesis reduced tremendously. Therefore, now it is a plausible solution for most laboratories to generate their own version of a synthetic antibody library.
\nA key subset of synthetic libraries is the production of semi‐synthetic libraries. The first semi‐synthetic library was reported in 1992, in which rearrangement of 49 human VH gene segments with five to eight residues of synthetic CDR was carried out to yield a semi‐synthetic single‐chain fragment variable (scFv) library [33]. The key difference between semi‐ and fully synthetic libraries is the source of the diversity. In semi‐synthetic libraries, the diversity is largely obtained from natural sources whereby the genes encoding the CDR are isolated. These CDR genes are then inserted to a fixed framework sequence, which encodes the antibody backbone [27, 34]. The diversity is still natural, taking advantage of the maturation processes of antibodies
This is evident with approximately three billion clones in the ETH‐2‐Gold library that were used against a wide range of recombinant antigens, such as extracellular glycoprotein of tenascin‐C (TNC) [35]. Other versions have been reported such as the Tomlinson I library with 18 amino acid side‐chain diversity on the CDR, and the Griffin library [36] was constructed by using six diverse synthetic CDR3 regions [37]. The main difference between Tomlinson I and J libraries is the choice of randomization used before clone into pIT2 phagemid system. The Tomlinson I library uses the DVT degeneracy to introduce diversity at the CDR. The Tomlinson J library makes use of the NNK degeneracy in the CDR design. Nucleotide degeneracy is commonly used to introduce mutations in the codon at the CDR regions that are responsible for antigen‐binding specificity and affinity. DVT and NNK degeneracy are generic codons abiding a specific formula for base usage. The base usage of each degeneracy is as follows: D is 33% G/33% A/33% T; V is 33% G/33% A/33% C; N is any nucleic acid and K is 50% G/50% T. When the degeneracy is translated as a codon, it will yield multiple combinations of amino acids to increase the diversity of the gene. The application of different degenerate codons also allows the application of different groups of amino acids in the CDR design. The antibodies isolated from these libraries could then be used as a diagnostic tool [37, 38].
\nThe HuCAL library from Morphosys is a famous fully synthetic antibody library with predefined randomized frameworks [39]. The different versions of the HuCAL libraries, namely HuCAL Gold and HuCAL Platinum, were generated with six trinucleotide‐randomized CDRs [40, 41], whereas HuCAL and HuCAL Gold libraries were made with seven VH, four VK and three Vλ germline families. This allows the generation of 49 framework combination of the VH‐VL [42]. Structural diversity of the CDR was introduced by randomization of CDR1, CDR2 and CDR3 [39]. Another version of the HuCAL library is the HuCAL Platinum that consists of seven VH, three VK and three Vλ framework sequences. HuCAL Platinum was designed without the use of VH4 and VK4 as they are found to be rare [43]. The HuCAL libraries have been used also for antibody generation for the diagnosis of bovine insulin [39, 40, 42]. Table 2 shows a list of some known fully-synthetic and semi-synthetic libraries used for diagnostic applications.
\nLibrary | \nType | \nDiversity generation | \nFormat | \nTarget | \nRef | \n
---|---|---|---|---|---|
Synthetic | \nDegenerate codon NNK | \nscAbs (VHH) | \nHuman prealbumin (PA) and neutrophil gelatinase‐associated lipocalin (NGAL) | \n[50] | \n|
Synthetic | \n– | \nscFv | \nCoronary artery disease (CAD) | \n[51] | \n|
Synthetic | \n– | \nscFv | \nExtracellular glycoprotein C‐domain of tenascin‐C (TNC) | \n[42] | \n|
Synthetic | \nFixed VH/VL framework pairs selected on biophysical characteristics | \nFab | \nRecombinant human (rh) ErbB4, rhFZD4/Fc, rhTNFalpha, M‐CSF, eGFP | \n[49, 52] | \n|
Synthetic | \n7 VH, 4 VK and 3 Vλ germline family | \nscFv, Fab | \nIL18R‐Fc, ᵝ‐Gal, Est‐BSA | \n[46, 48] | \n|
Synthetic | \n7 VH, 3 VK and 3 Vλ framework | \nFab | \nHuman TRAIL R2/Fc and rhIL‐2Rαalpha;/Fc fusion protein | \n[47] | \n|
Synthetic | \nDVT side chain | \nscFv | \nMicrocystins, ferritin, SARS‐associated corona virus | \n[35, 51, 53] | \n|
Semi‐synthetic | \n– | \nscFv | \nMicrocystins | \n[43, 44] | \n|
Semi‐synthetic | \nNNK side chain | \nscFv | \nSARS‐associated corona virus | \n[53] | \n
A list of fully‐synthetic and semi‐synthetic libraries and application in diagnostics.
The immunized library repertoire originates from V‐gene of immunized donors [27] or disease‐infected donors. In immunized libraries, the immunized repertoire would be specific for one antigen or a collection of antigens specific for a particular disease. This limits the use of the libraries when compared to naïve and synthetic libraries. Even so, the V‐genes are collected from donors that have been exposed to the target antigen allowing isolation of higher‐affinity antibodies using such libraries. These antibody libraries mainly produce antibodies of good affinities with high clonal diversity due to
Several different libraries have been developed for various diseases such as hepatitis B [44] and those listed in Table 3. These libraries contain a plethora of useful antibodies that are specific to the disease making it a valuable asset for infectious diseases. The generation of immunized libraries is not restricted to humans but can also be carried out in animals such as mice. Immunization of mice with the target antigen would likely yield a library of clones against the specific target protein. Although this may not differ much from the conventional hybridoma technology, however, conversion to a recombinant version would allow easy up‐scaling for production and also for modification.
\nLibrary | \nSource | \nFormat | \nTarget for diagnosis | \nRef | \n
---|---|---|---|---|
Camel | \nsdAbs (VHH) | \nH7N2 virus | \n[61] | \n|
Human | \nsdAbs | \nMesothelin cancer biomarker | \n[62] | \n|
Chicken | \nscFv | \nBursal disease virus (VP2 protein) | \n[63] | \n|
Buffalo | \nscFv | \nSchistosome infection | \n[64] | \n|
Rainbow trout | \nscFv | \nViral haemorrhagic septicaemia, VHS (viral haemorrhagic septicaemia virus, VHSV) | \n[65] | \n|
Ilamas/camelid | \nscAbs (VHH) | \n[66] | \n||
Camel | \nscAbs (VHH) | \nCoronary artery disease, CAD (Apolipoprotein B‐100) | \n[67] | \n|
Camel | \nsdAbs (VHH) | \nTuberculosis (TB) | \n[68] | \n|
Camel | \nscAbs (VHH) | \nBotulinum neurotoxin E (BoNT/E) | \n[69] | \n|
Mice | \nscFv | \nHIV‐1 capsid protein p24 | \n[70] | \n|
Chicken | \nscFv | \nNewcastle disease virus (NDV) | \n[71] | \n|
Camel | \nscAbs (VHH) | \nPorcine circovirus type 2 (PCV2) | \n[72] | \n|
Camel | \nscAbs (VHH) | \nInfluenza H3N2 | \n[73] | \n|
Chicken | \nscFv | \nMalaria | \n[74] | \n|
Mice | \nscFv | \n[75] | \n
A list of immunized antibody libraries and application in diagnostics.
Immunized libraries have been used successfully in developing antibodies against diagnostic biomarkers. This can be seen with the application of this concept for epitopes of immunogenic antigen, such as carcinoembryonic antigen (CEA) [32, 45]. Anti‐CEA antibodies can be applied for targeting and to image colorectal tumours. This transcends the conventional diagnostic platforms allowing
Panning is an
Antibody selection bio‐panning protocol.
In this respect, (Figure 2) shows that the target antigen must first be immobilized on solid surfaces such as nitrocellulose, magnetic beads, column matrices or plastic surfaces in the form of polystyrene tube or 96‐well microtitre plates [22]. In fact, the major difference among all the panning selection strategies is the immobilization surface where antigens are coated on. Other than this, parameters such as washing, elution and enrichment steps can be optimized for a successful selection process. As for evaluation, polyclonal and monoclonal antibody phage enzyme‐linked immunosorbent assay (ELISA) is usually used to determine the presence of a positive clone after panning [53].
\nTo date, there have been a number of different panning strategies reported for the isolation of MAbs against various antigens. Traditionally, the favoured method for most researchers is to immobilize or coat antigens on solid supports such as polystyrene immunotube and polystyrene immunoplate [53].
\nThe attachment of antigen to the polystyrene surface can either be direct using surface‐treated plates or by using intermediate capture mechanisms. This includes streptavidin‐coated plates to capture biotinylated ligands (protein or peptide). The biotin‐streptavidin mechanism is useful to avoid epitope disconformation during antigen immobilization on a plastic surface [54]. After the immobilization of antigen, phage‐displayed libraries are incubated with the bound antigens for affinity capture. This was then continued with the washing of unbound phages before phage elution for rescue. Then, the eluted phage was subjected to amplification and precipitation steps for the following panning round until a positive clone is obtained [55]. Here, the wash step can be modified to introduce different degrees of stringency to specify certain characteristics required for the antibodies. This way, customization of the strategy helps to determine the final output characteristics.
\nTo increase the screening effectiveness, streptavidin magnetic beads can be used to coat the biotinylated antigens. A major advantage in using nanoparticles is the higher surface area to volume ratio for the capture of higher amount of targets. Magnetic‐based panning allows multiple antigen screening at the same time. A semi‐automated panning process includes manipulation of magnetic beads by multi‐pin method, robotic arm or a robotic system during the panning process to increase the panning efficiency [56]. The main concept of panning using the semi‐automated process applies a similar concept of affinity‐based selection as the conventional method. The incubation, wash and elution steps are carried out with automation to improve reproducibility and accuracy. However, the phage rescue process is still carried out offline by manual infection. Even so, the process still utilizes significant automation to lower labour involvement and allows for high‐throughput screening to be carried out.
\nThe concept of full automation refers to a pipeline process without the involvement of any human, whereas semi‐automation requires human involvement at some point of the process [53]. The main benefit that high‐throughput panning brings to the process is a higher efficiency in selection with minimum labour as all the parameters can be easily programmed for repetitive steps. This also increases reproducibility of the incubation time, temperature, washing and elution condition. This allows for easy handling of multiple targets at the same time. Such protocols can help to reduce from 2 days to a single day for one selection cycle, which means only a week is required for the entire panning process. This method is easily automated with the use of magnetic particle processors such as the Kingfisher Flex system [56].
\nThe next‐generation phage display allows differentiation of unselected and selected phage after enrichment rounds [57] against a target antigen for both large combinatorial peptide and antibody libraries through DNA sequence analysis of the phenotype‐bearing phage [51]. There are some similarities of this platform with conventional phage panning, where both includes laborious colony picking and functional ligand screening. The sheer number of clones to be analysed is easily overcome by the use of next‐generation sequencers (NGSs). This strategy is cost‐effective, fast and less labour intensive as compared to conventional phage display selection. Moreover, this technology improves the overall accuracy for large quantification. In terms of coverage, DNA deep sequencing through NGS offers a high coverage for full repertoire of ligand particles. In short, high‐throughput DNA sequencing through NGS method is cost‐effective, provides higher accuracy and high coverage for large quantification especially for library screening [57].
\nAnother method utilizing the mass spectrometry immunoassay (MSIA™) system was introduced where the separation of antibodies and antigen for mass spectrometry (MS) analysis is done via affinity. Previously, the MSIA™ method is an immune affinity method used in protein analyte purification for MS detection purposes. The MSIA™ tip was successfully used as a solid phase to carry out semi‐automated panning for antibody enrichment. The MSIA™ tips that contain streptavidin that are covalently linked to a porous monolithic solid support will function as the capture molecule. The streptavidin capture molecules are best known for the easy capture of biotinylated target through biotin‐streptavidin interaction. The MSIA™ tip method only requires the use of a standard electronic multichannel pipettor and an adjustable pipette stand. This method is also cost‐effective for phage display panning as it does not require investments on instrumentation. However, the method can also be incorporated to larger pipetting instruments for antibody panning also. The panning protocol uses the similar concepts as conventional panning that includes incubation, washing and final elution. Then, bound phages are then amplified and used in the following panning rounds to obtain clonal enrichment. This method was reported to successfully identify antibodies against the hemolysin E antigen of
Cell panning is an innovative screening method using whole fixed or live cells, tissue section or live animals expressing the antigen of interest for panning [22]. In other words, whole live cells serve as an antigen carrier to screen phage antibodies [59, 60]. All the selection methods mentioned were panned against purified antigens, but whole cell antigen is used in whole cell panning. This panning strategy is usually an alternative method for complex and difficult antigens which cannot be purified with similar properties, for example, cell surface receptor or antigen [60] is only functional when retained in lipid bilayers [59]. There are several parameters to consider when carrying out cell panning such as (1) quality of antibody library, (2) display manner, (3) antigen concentration and (4) cell‐surface antigen density. This is to ensure the success of the panning process.
\nAntibodies are normally identified in a Y‐shape configuration. The variability is mainly due to the V‐region (two arms of Y‐end) as this is where the antigens bind [61]. Thus, smaller versions of antibody formats have been developed to take advantage of the binding specificities of the V‐region. Due to advancement in recombinant DNA technology, a number of new antibody formats such as domain antibodies, single‐chain fragment variable, tandem scFv, diabody, tetrabody, minibody and single‐chain fragment antigen binding (scFab) have been introduced (Figure 3).
Antibody fragment design. (A) Full antibody; (B) fragment antibody binding (Fab); (C) single‐chain fragment variable (scFv); and (D) single‐domain antibody (sdAbs).
A main consideration of antibody formats is the size; smaller fragments have an advantage that they are able to retain the antigen‐binding specificity and can be produced economically [26, 62]. This is important for application in diagnostics, as this will in turn contribute to lowering the production cost during manufacturing. The main consideration for any antibody to be used on a diagnostic platform is mainly the specificity and affinity of the antibody against the target antigen. These two characteristics are not lost with the use of smaller fragments albeit there will be no avidity effect with the monomeric smaller fragments.
\nBoth scFv and Fab formats are commonly used in research and industrial applications, and are the preferred formats for presentation on phage [16, 20, 34, 62, 63]. This is because the expression of complete IgG is not suitable for
Domain antibodies are small (11–15 kDa) and consist only of either the VH or the VL domain [34]. Thus, they will only have three out of a possible six CDR from a full variable region consisting of both VH and VL. It was found that single‐domain antibodies (sdAbs) are able to provide better stability as well as solubility when specific families such as the VH3 framework are used. The stability of the human VH domain antibody can be further enhanced by extending the length of CDRH3 loop [65], much like the hypervariable region of camelid VHH that are longer than the human VH. According to Ponsel and Neugebauer [34], camelid and cartilage fish\'s single‐domain antibodies are more stable, with a high resistance towards aggregation and temperature due to the framework sequence [34, 66].
\nDomain antibodies can also penetrate tissue efficiently as compared to full‐length IgG due to their smaller size. VHHs are commonly used as detection units on biosensor or immune‐adsorbent to identify the presence of lysozyme, carbonic anhydrase, alpha‐amylase [67], beta‐lactamase or even act as a cancer‐imaging agent. It was also used to detect the surface antigen of different hepatitis serotypes [66]. In addition, single‐domain antibody is used due to an easier production system when compared to conventional antibodies because of the size and folding [56, 68]. As domain antibodies are devoid of any quaternary structure, production and stability of domain antibodies allows it to be used at extreme conditions. This provides great benefit for diagnostics, as the improved heat stability would allow easy transportation of antibodies without cold chain. This is especially beneficial for diagnostic kit development for in‐field diagnosis of infectious diseases in areas with limited resources.
\nSingle‐chain antibody variable fragment is made up of VL and VH domain with a glycine‐serine flexible linker in between to hold the domain in proximity to form the binding cavity upon folding [3]. In addition, GS linkers are known to improve the folding, flexibility and stability of the single‐chain fragment variable as compared to proline‐rich linker. This is because pro‐rich sequence exhibits rigid and stiff conformation due to the absence of hydrogen at the amine, which forms hydrogen bonds with other amino acids [69]. Therefore, the scFv fragments are designed with six CDRs, thus increasing the diversity and repertoire of the antibodies. The scFv has been shown to bind to a variety of antigens, such as hapten, protein, carbohydrate, receptor, tumour antigen and viruses [63]. Moreover, small scFvs are easily folded and producible in
As a linker joins the VH and VL domain physically, the single polypeptide composition helps to facilitate the production and folding in
As a comparison to scFv, Fab fragments are composed of the VH, CH1 and entire VL fragment held together by interchain disulphide bonds. The Fab fragments have a tendency to form dimers which could cause problems with binding and affinity as it exerts the avidity effect [13].
There are several designs used for Fab presentation that includes either the monocistronic or the bicistronic arrangement of antibody. The gene arrangement of a Fab fragment for phage display requires fusion of either VL or VH to the phage pIII coat protein. The production of the accompanying chain will be carried out simultaneously as an independent protein allowing it to locate each other at the periplasmic cavity to form disulphide bonds between them for proper presentation. This challenge is overcome by alternative approaches using molecular chaperones to improve the presentation of full Fab fragments during phage display and protein expression [74].
\nIn diagnostic applications, Fab‐peptide epitope and Fab‐Fab bifunctional reagent were used to detect the HIV‐1, HIV‐2 and hepatitis B surface antigen, which were known as antibody‐based reagent [71] in agglutination assays. Table 4 shows the broad application of different antibody formats for diagnostic applications. There is no actual best format for diagnostics but is mainly subjected to the preference of the users and the diagnostic platform set‐up.
\nAntibody fragment format | \nSource | \nAntigen | \nDisease | \nRef | \n
---|---|---|---|---|
Camel | \nProstate‐specific antigen (PSA) | \nProstate cancer | \n[98] | \n|
Semi‐synthetic | \n[99] | \n|||
Ilama | \nMarburg virus variants | \nHaemorrhagic fever | \n[100] | \n|
Human | \nProtein C3a, cancer antigen, carcinoembryonic antigen, MUC family glycoproteins, autoantibodies, sialyl‐LewisX and cytokines | \nBreast cancer | \n[101] | \n|
Alpaca (VHH) | \nFood‐borne pathogens | \n[27] | \n||
Camel (VHH) | \nH7N2 virus | \nAvian influenza virus subtype H7N2 | \n[61] | \n|
Mice | \nAnthrax | \n[60] | \n||
– | \nDomoic acid | \nAmnesic shellfish poisoning (ASP) | \n[102] | \n|
Mouse | \n[103] | \n|||
Mice | \nProstate‐specific membrane antigen (PSMA) | \nProstate cancer | \n[104] | \n|
Human | \nAnti‐carcinoembryonic antigen (CEA) | \nColorectal carcinoma | \n[105] | \n|
Mouse | \nprostate‐specific antigen (PSA) | \nProstate cancer | \n[106] | \n|
Hepatitis B surface antigen (HBsAg) | \nHepatitis B | \n[54] | \n
Application of antibody fragment as diagnostic probe.
Recombinant antibodies are used in diagnostics due to its binding specificity and affinity. There are many platforms, such as lateral‐flow assay (LFA), ELISA and cell imaging available in the market today, which are rapid and accurate in identifying the target antigens found in sample. Most of the platforms make use of either the antigen‐capture assay or the antibody‐capture assay to diagnose the presence of certain diseases [75].
\nLateral‐flow assay or immunochromatography assays are normally found as a test strips with the most common being the pregnancy test strip. The theory behind lateral‐flow assay is based on the capillary action that occurs in the nitrocellulose membrane to migrate molecules along the membrane to cause a reaction and detect target antigen [75, 76]. This is because the presence of antigens in the sample matrix against a specific antibody reflects the onset of certain disease and treatment should be carried out immediately. (Figure 4) shows the actual design, polyclona antibodies against the target antigen were conjugated with gold nanoparticles and are deposited on the membrane. The migration of the sample when mixed with the antibody‐coated particles will allow the particles to flow along the membrane until it is captured by a secondary antibody that is permanently fixed along the membrane as a line. Therefore, the presence of the antigen will be reported by the appearance of a band on the dipstick that represents the concentration of the gold nanoparticles on the target line.
\nLateral‐flow dipstick assay design.
The conventional design of the lateral‐flow strips will show a single control line and another test line. The control line indicates the control assay to show that the lateral‐flow system is in order. The working assay requires buffer to improve the performance as well as the compatibility with other components used in assay. There are two formats used in LFAs, sandwich and competitive format [77]. In short, dipstick assay is sensitive enough to detect the antigen constituent within 10 min besides the simple usage that does not require professional personnel to carry out the test. The conjugation of recombinant antibodies to gold nanoparticles is essential for the generation of lateral‐flow assays. The ability to produce recombinant antibodies easily using bacterial expression systems would facilitate rapid kit production at a lower cost.
\nMicrofluidics makes use of the movement of small amounts of fluid along a small diameter channel. Microfluidics was vastly applied due to the small sample volume needed for fast and precise result. The microfluidic platform is highly sensitive, efficient and portable [78].
\nRecent developments in microfluidic technology including on‐chip detection and imaging, on‐chip flow cytometry, on‐chip immunoassay and nanosensor for point of care (POC) diagnostic application [79] help to overcome conventional ELISA\'s limitation for immunoassay‐based diagnosis; micro‐ELISA systems have been proposed for sensitive and rapid diagnostics using a fluidic chamber. This modified ELISA platform uses a microfluidic platform that reduces the amount of sample required by 10 times (<10 µL), 20 times faster analysis (<20 min) with a higher sensitivity range (Um–pM) as compared to conventional ELISA [79, 80]. Rapid diagnosis is the most important selling point for biomarkers such as protein, carbohydrate, lipid, metabolites, genomic DNA and RNA by using immunoassay due to the obvious advantage in disease management.
\nTo improve the quantitative result, fluid‐handling components and data acquisition software were used. In addition, microfluidic kits are integrated with electrical, optical and mechanical transducer to improve the platform [79, 80]. Other than that, the development of on‐chip diffusion assay allows the measurement of small molecules within the microfluidic channels. This is done by detection of the labelled probe by antibodies against the probe itself. This study also proves that microfluidic diffusion is suitable for blood sample analysis [81]. There have been other versions of assays utilizing recombinant antibodies in fluidic platforms such as malaria detection with on‐card dry reagent storage of microfluidic immunoassay from blood samples [79].
\nThe ELISA platform takes advantage of the specific interaction between antibody and antigens to allow a capture base for detection. The method requires a series of incubation and wash steps, which can be time consuming and tedious. Thus, antigens are first coated on a microtitre plate and then blocked overnight before incubation with antibodies. To detect binding between antigen and antibody, bio‐conjugate and chemical‐conjugate proteins coupled with reporter enzymes such as horseradish peroxidase (HRP) or alkaline phosphatase (AP) are used. Lastly, 2,2’‐azino‐bis(3‐ethylbenzothiazoline‐6‐sulphonic acid) (ABTS) is used as a colour indicator for successful binding between antigen and antibody. However, the conventional ELISA approach has setbacks such as long assay time, large amount of expensive antibodies, chemicals, plastic ware and liquid‐handling platforms. This process although straightforward may still require professional training in order to minimize any false‐positive result and ensure reproducibility [82].
\nThe advent of nanotechnology has brought about the introduction of nanosized particles and with it a series of new diagnostic platforms. There are many nanoparticles that have been used for diagnostic platforms. This includes gold nanoparticles (AuNPs) [83–86], carbon nanotubes and carbon nanoparticles (CNPs) [87]. The chemical property of these nanoparticles has allowed the expansion of the diagnostic platforms from simple colorimetric assays to the introduction of electrical signal, fluorescence and even phase transition readouts [83–85].
\nAlthough the use of AuNPs is associated with its application in lateral‐flow dipstick assay as the immobilization surface for DNA, antibodies and proteins for the line development, it has also been used independent of the lateral flow. An assay was developed using AuNPs with influenza‐specific antibodies acting as labels to detect influenza virus. This will result in the AuNPs probe aggregating and causing a readout in dynamic light‐scattering (DLS) spectroscopy. Thus, the detection of AuNP aggregation was analysed using the DLS to determine the concentration of virus present. This method monitors the size change of aggregated nanoparticles and not change colour. However, the DLS approach is more suitable in detecting larger viruses with multiple epitopes [88].
\nThe latex antigen detection or latex immunoagglutination test established in 1959 makes use of protein‐conjugated latex microspheres to magnify the antigen‐antibody interaction [89]. This LAT assay is fast and simple to use for the diagnosis of circulating antigens in patients with systemic infection because latex is sensitized with the serum of an immune donor [90]. The latex complexes will agglutinate if the target antigen is present [91]. The LAT platform has been used for the diagnosis of systemic candidiasis [90, 91], visceral leishmaniasis [89], invasive pulmonary aspergillosis [92],
Enzyme‐linked immunosorbent assay is an assay that uses the enzymatic reaction as a basis of reporting. However, such assays sometimes suffer from a lack of sensitivity to detect low‐dose drugs or biomarkers [95]. To overcome this issue, the fusion of DNA technology with protein engineering has brought about newer reporter systems that can increase the sensitivity of assays. This includes modified hybrid methods such as antigen‐DNAzyme, immuno‐PCR, immuno‐QPA and immuno‐RCA.
\nThe antigen‐DNAzyme‐based probe reporter system is a simple and rapid immuno‐based assay that depends on the peroxidase activity as a reporter signal and the affinity of antigen‐antibody for binding. DNAzymes also known as deoxyribozyme, founded by Ronald Breaker and Gerald Joyce in the year 1994, are catalytically active DNA molecules, which are able to function mimicking enzymatic reactions [96]. G‐quadruplex (G‐quad) structures are formed by guanine‐rich nucleic acids. The guanine‐rich sequence will form a guanine tetrad structure with intra‐Hoogsteen hydrogen bonding. This will then allow two or more guanine tetrads bind to form a G‐quad. The G‐quad structure is further stabilized by the presence of cations. The complexation of G‐quad structures with a hemin will form a peroxidase mimicking DNAzyme that catalyses the peroxidase‐mediated oxidation of ABTS [97, 98]. The main difference of this antibody‐antigen detection assay is the use of G‐quad DNA structures in association with hemin as a reporter system. The addition of hemin to a G‐quad structure will allow the transfer of electrons from the guanine to hemin in the presence of peroxide to oxidize the ABTS to form a green complex that is visible to the eye [98].
\nThis assay design allows the fusion of the G‐quad to function as a DNAzyme to generate a colorimetric readout as a reporter system for the rapid detection of small haptens such as hormones or drug molecules [99]. Thus, DNAzyme can be conjugated with antibodies for signal enhancement with the help of hemin and is suitable to be used as an immunoassay in biodiagnostic platforms [98]. As the G‐quad sequence is made up of oligonucleotides, the sensitivity of the assay can be greatly improved via external DNA amplification processes to generate more G‐quad sequences for reaction with hemin.
\nImmuno‐quadruplex priming amplification is another hybrid method that couples both the calorimetric DNAzyme detection with an effective DNA amplification for improved sensitivity. IQPA is an immunoassay platform that generates G‐quad reporter molecules via an isothermal quadruplex‐priming amplification process. The reporter G‐quad forming sequences, which were amplified from isothermal QPA, allow QPA to fuse with an immunoassay platform. In other words, we can avoid reporter conjugate enzyme in the immunoassay platform. QPA employs a set of primer and enzyme to maintain the stringent condition for target amplification to generate multiple copies of the G‐quad sequence at an isothermal condition. Streptavidin can be sandwiched between the antigen and antibody DNA to form the binding for IQPA to work. The hemin molecules are then used complex with the amplified G‐quad structures to catalyse the colour change of ABTS in the presence of hydrogen peroxide [100].
\nImmuno‐PCR is another hybrid immuno‐based assay that combines ELISA‐type ligand‐binding assay (LBA) technologies with PCR amplification signal without the use of antibody‐enzyme conjugates. As a replacement, antibody‐DNA conjugates were used whereby the DNA marker is physically linked to the capture antibody and a polymerase chain reaction step is introduced to generate copies of the DNA sequence. This allows improvements of 100–10,000‐fold in limit of detection (LOD) as compared to conventional ELISA [95, 101]. Although the LOD of IPCR is almost in line with the ligand‐binding assay, IPCR assay has been considered as challenging. Thus, various modifications are required to increase its sensitivity. This includes technical issues such as the availability of thermostable enzymes, high protein‐binding capacity microplates and minimizing cross‐contamination by avoiding plate transfer steps. IPCR has been reported in detecting human interleukin 6 (IL‐6) for neurological disease [95].
\nA further enhancement includes the immuno‐RCA method that is another diagnostic platform, which utilizes DNA amplification steps to enhance the signal of immunoassay. This method employs similar concepts to IPCR but the method of DNA amplification varies. As IPCR utilizes the standard PCR amplification cycles, IRCA uses an isothermal amplification method, which does not require a thermal cycler. This makes it more attractive as there is a reduction of dependency on high‐end instrumentation. IRCA was used to develop assays in detecting ovalbumin (OVA) allergens [102] and foot‐and‐mouth disease virus (FMDV) [103]. In IRCA, oligonucleotide primers are attached covalently to the antibody. However, DNA circularization is required to allow binding of the DNA primer to work with DNA polymerase and nucleotides for amplification to start [104]. The detection sensitivity of IRCA exceeds the conventional ELISA and microparticle formats. Thus, IRCA is a system that can allow detection of specific antigens using antibodies at high sensitivity with a wide dynamic range to detect a single molecule [104]. In short, DNA‐fusion technologies with recombinant antibodies could potentially aid in the development of newer assays with improved sensitivities.
\nPhage display technology is commonly used for recombinant antibody production. The ability to produce antibodies via recombinant methods can help to improve the speed at which newer antibodies are produced at a fraction of the conventional cost. The freedom associated with recombinant antibodies would also allow the customization of antibodies for various downstream applications in diagnostics. This is particularly important as the development of newer technologies in sensing and reporting mechanisms, the flexibility of recombinant antibodies towards modifications would allow the antibodies to evolve with the advancement of sensing technologies. Therefore, phage display‐derived recombinant antibodies provide an important platform for antibody generation for current and future diagnostic applications.
\nThe authors would like to acknowledge support from the Malaysian Ministry of Higher Education under the HICoE program and Universiti Sains Malaysia under the Research University Individual Grant scheme (1001/CIPPM/812173).
\nAn estimated 1–3% of people in Canada are transgender, and just over 7% of Canadians identify as lesbian, gay or bisexual [1]. Approximately 32% of them report having unmet healthcare needs [2]. In the United States, there are over 1,000,000 transgender people, a third of which report avoiding healthcare for fear of discrimination [3]. Transgender people and sex and gender minorities (SGM) experience unnecessary and preventable health inequities including higher rates of mortality, depression, anxiety, substance use, chronic disease and suicidality which are, in part, the summary result of atrocious experiences of discrimination and stigmatization on the part of healthcare staff and institutions [1, 4, 5, 6]. Institutionalized cisheteronormativity, a term which refers to the culturally-driven normalization of cisgender, heterosexual status and the invisibilization of non-cis, non-heterosexual people [7, 8], is a primary social bias encoded into digital health structures and care cultures that prevents meaningful knowledge development about the health status and needs of this population [1, 6, 7]. Poorly defined data elements, conflated sex and gender concepts, constrained representation of gender variation, and lack of cultural understanding on the part of health information professionals and clinicians are all conditions contributing to healthcare systems, environments and interactions that stigmatize transgender people, and that drive health inequities [1, 6, 7, 8, 9, 10]. Since 2019, technical, data and messaging standards development organizations (SDOs) have been working to modernize gender, sex and sexual orientation (GSSO) information practices in electronic health records (EHRs) to enable inclusive and affirming care for transgender people and SGM [1, 8, 11]. In this chapter, I will provide an overview of these recent innovations, including work of Canada Health Infoway’s Sex and Gender Working Group and the HL7 Gender Harmony Model.
The global population of transgender people and SGM live with higher burdens of disease and lower health than many other populations, especially cisgender people [12, 13]. Despite of the fact that the human rights of transgender people are protected by national and internal law [14], transgender people continue to face severe discrimination. The experiences of transgender people in healthcare, at the hands of healthcare staff, are atrocious [1], and ought to be a central concern to healthcare decision makers, policy makers and legal supports, and to society at large [15, 16]. Healthcare, as an organizational, regional, national and global social system with a specific ethical orientation that ought to adhere to, uphold, and advocate for the Universal Human Rights of transgender people, and
Modernization of outdated GSSO information practices is an equity-oriented endeavor [19] intended to address the technical conditions that enable structural and institutional bias and that contribute to health inequities for transgender people and SGM [16]. Information practices include the definition, collection, organization, sharing and use of health information [17]. The term “Gender, Sex and Sexual Orientation” or “GSSO” was coined by C. Kronk, PhD of Yale University, who has created and published a Queer Ontology to facilitate increased awareness and improved understanding and consistency of use of GSSO terms [14, 20]. Outdated GSSO information practices include:
Poor definition of GSSO information
Conflation of the meaning of distinct gender and sex concepts.
Problems related to collection of GSSO information
Confusion as to who should be collecting what GSSO information, when, and for what purpose(s) in healthcare workflows.
Problems related to the organization of GSSO information
Use of sex and gender codes as though they are interchangeable and synonymous in administrative and clinical domains.
Problems related to information sharing and exchange
Problematic mapping of sex and gender codes between code systems in exchange standards.
Inadequate representation of sex and gender diversity in exchange standards.
Problems of use
Unconscious bias and assumptions related to sex and gender concepts.
Inadequate emphasis on quality clinical care and the therapeutic relationship for SGM.
Overemphasis on business requirements and efficiency at the expense of clinical care quality.
Building on the momentum of the work of such expert agencies as the Fenway Institute, Trans Care BC and Rainbow Health Ontario, there has been a recent increase in activity related to raising the critical awareness about the importance of diversity, equity, and the role of healthcare in creating the conditions for the well health of transgender people and SGM in North America and beyond [11, 12]. A diverse international community of people and organizations, including expert agencies, SDOs, researchers, EHR and solutions vendors, healthcare professionals and health information professionals, have dedicated their time and expertise to making healthcare more inclusive. In this section, I will discuss key developments within this community and key activities in relation to the healthcare of transgender people and SGM beginning with my own country, Canada.
Heading up the co-development, maintenance and support of technical and interoperability standards that support healthcare in the country, Canada Health Infoway (Infoway) has been a key organization enabling digital health in Canada since 2001. As Canada’s National Release Center for such technical standards as the HL7 International information exchange standards, the Systematized Nomenclature of Medicine—Clinical Terms (SNOMED CT) and Logical Observation Identifiers, Names and Codes (LOINC), Infoway’s mandate includes bringing a Canadian perspective to international standards development with SDOs such as HL7 International, SNOMED International, and the Regenstrief Institute. At the national level, Infoway collaborates with a network of pan-Canadian Healthcare Organizations, provinces, jurisdictions, vendors and governance bodies to support the implementation of technical standards in the Canadian digital health ecosystem and develops, maintains and publishes pan-Canadian and jurisdictional subsets via the Terminology Gateway service. Infoway convenes stakeholders by establishing communities for collaboration and working groups on a central information sharing and engagement platform, InfoCentral. One such working group is Infoway’s Sex and Gender Working Group (SGWG).
Infoway’s SGWG was established in 2019 with the primary aim of modernizing outdated GSSO information practices in Canadian electronic health records [17] to support the health of all Canadians. Since then, this international community of researchers, pan-Canadian Health Organizations, healthcare agency representatives, subject matter experts, and people with lived experience have been convening monthly to share recent and relevant research, discuss the process of modernization [17] and lead modernization by taking evidence-informed action.
After 2 years of convening, Infoway’s Sex-Gender Working Group produced
Envisage an equity- and SGM-oriented health system that embraces diversity and aligns with other SGM-related initiatives.
Engage organizations and communities across Canada to modernize GSSO information practices in EHRs that support equity-oriented healthcare and meet SGM needs.
Establish a precise, inclusive, appropriate, evolving and multi-level GSSO terminology with standardized data definitions, coding schemes and value sets to support affirming patient care, provide complete and accurate health system use of data and inform health research.
Adopt a common set of EHR functions that support the collection and use of standardized GSSO data, SGM-oriented clinical care guidelines (e.g. radiation shielding in imaging exams, cancer screening, lab reference ranges, reminders, and reports), clinical quality improvement, data-driven analytics, health system performance tracking and health evidence generation.
Integrate and tailor GSSO data collection and use including secondary uses within all organizational structures, policies, practices, governance, use cases and workflow processes in order to be responsive to specific care needs of SGM.
Educate and train healthcare staff to enhance their capacity to provide culturally competent and safe care, and implementers, policy makers and researchers to ensure required safeguards in place to protect these data. Inform patients on the need for GSSO data collection and protections for safe access and use.
Establish a central hub to liaise, guide, assist and monitor the progress of this action plan over time [21].
Although there remains much work to do, members of Infoway’s SGWG are using the Action Plan and its aims as a guide to specify and cultivate adoption of modern, consensus-based data, technical and exchange standards that address structural cisheternormativity in Canadian healthcare. The community continues to meet on a monthly basis and drive new research, insights, and the development of modern approaches and practices to realize the aims outlined in the Action Plan.
The continued support of standards organizations such as Canada Health Infoway will play a foundational role in the ongoing process of modernization of healthcare. More information about Canada Health Infoway and Infoway Communities can be found on InfoCentral.
The beliefs, attitudes and organizational practices required to create culturally and psychologically safe, quality healthcare for transgender people will be adopted by individual health organizations. However, meaningful change will not happen unless the standards that we use to communicate health information from one service to another—the standards we use to connect a continuum of care services—are also modernized such that they are inclusive of transgender people and SGM and are designed to address the problems related to outdated information practices. Health information exchange standards (sometimes referred to as messaging standards) are powerful drivers of systemic change. As a world-leading exchange standard development organization, HL7 International plays an important role in the specification and implementation of health information exchange standards that support
In 2019, HL7 International created the Gender Harmony Project as an open, international collaboration of volunteers and experts from international agencies and SDOs dedicated to creating gender-inclusive standards in healthcare, and to addressing harms associated with biased exchange standards [11]. Members of this group have been meeting regularly to define, specify, model and explicate the Gender Harmony Logical Model since its establishment.
In early 2021, the Gender Harmony Project produced the informative-ballot-for-comment that outlines the Gender Harmony Logical Model. Input on the document was collected from international stakeholders, and the first release of the informative document was published by the HL7 International Vocabulary Working Group in August of 2021:
Unambiguous, evidence and consensus-based definitions of key sex and gender concepts that support model elements.
Avoidance of stigmatization in value sets by enabling nonbinary options without the use of such labels as “other” or “complex” in model elements.
Explicit description of model concept and element attributes means that organization of administrative and clinical GSSO information is clearly defined, streamlining GSSO data collection use cases.
Clear definition of administrative and clinical sex and gender data.
Clear division between administrative and clinical sex and gender data in model elements.
Clear articulation of essential elements that support affirmative care interactions.
Unambiguously defined concepts and clear organization of model element concepts and attributes reduces risk for transformation of sex and gender concepts during sharing and exchange.
Representation of gender diversity in messaging standards.
Reduces unconscious bias and unchecked assumptions related to sex and gender.
Emphasizes care quality by supporting gender-affirming patient-provider interactions.
Supports business requirements by separating administrative data from clinical data.
The consistent application of the Gender Harmony Model in the design of joined-up health information systems is a shift in paradigms—from outdated GSSO information practices to modern ones, and a shift that enables quality healthcare for transgender people and SGM.
The Gender Harmony Paradigm in healthcare is implementing systems design that addresses conditions resulting in health inequities for SGM. It involves meaningful integration of the HL7 Gender Harmony Logical Model, modern GSSO information practices and associated standards enabling culturally safer institutional policies and practices that support inclusive healthcare for transgender and SGM people. The Gender Harmony Model reduces real and latent harms associated with current gender constructs in health by: (1) decoupling administrative and clinical concepts in digital health systems; (2) addressing the conditions that lead to negative clinical interactions that deter transgender people from seeking care (e.g. being misgendered, misnamed or outed); and finally (3) by addressing conditions that make transgender people and people with nonbinary gender identities invisible to clinicians, policy makers, analysts and researchers who use health information for system improvement. At the patient level, the Gender Harmony Model enables clinicians’ use of person-centred language in clinical interactions and provides them with the information necessary for objective and appropriate preventive screening, treatment and referral options based on unambiguous sex and gender health information. At the organizational level, implementation of the Gender Harmony Model involves the adoption and use of logical, technical and data standards necessary to support gender affirming care cultures and practices. At the health system level, implementation of the model orients clinical and institutional healthcare toward gender equity by addressing problems associated with the omission of data elements required to generate knowledge for health system improvement.
The Gender Harmony Logical Model, which I will call ‘the Model’ throughout the remainder of this chapter for ease of reading, does not exclusively benefit transgender and nonbinary people. All patients benefit from higher quality care and analytics enabled by the Model. Application of the Model can broadly be considered a step toward personalized medicine and more person-centred care since it enables more granular use and understanding of complex social and medical phenomena. The data elements of the Model [22] are designed to address the conditions that are currently leading to health inequities for transgender people such as problems associated with omission of data elements required for affirming care (i.e. Name to Use, Pronouns), conflation of administrative sex and gender concepts (i.e. Gender Identity, Recorded Sex or Gender) and by creating opportunities for clinicians to provide care by specifying care needs, rather than making assumptions related to outdated binary constructs (i.e. Sex for Clinical Use) [11]. In this section, I will provide an overview of each data element and its specific attributes that support design and implementation decisions in digital health systems such as EHRs. Model data elements will be capitalized to improve readability.
In healthcare, knowing a patient’s gender identity allows clinicians to provide informed and affirming, quality care, without misgendering them based on assumptions or pre-existing and out of date information in the patient record. All people have a gender identity. Gender Identity is a person or patient’s personal sense of being a man, woman, boy, girl or something else [22]. Gender identity must be declared by the person or patient, rather than them being labeled with it. Gender identity is fluid and can change, and so must be collected routinely [23]. People can identify with none, one, or many genders, and can set time constraints on specific gender identities. Gender Identity should be used in conjunction with the other Model data elements to support gender-affirming, quality healthcare and should be validated privately with the patient to avoid outing them. Outing is when information about a person’s gender or sexual identity is made known to specific people without permission. If this is done by a clerk or a clinician in the context of an emergency room or a waiting room, it may be directly harmful to the person being outed or lead to harm. As with all EHR data, informed consent about collection, sharing and use of Gender Identity information should be acquired before it is entered in the patient chart.
In order to ensure international applicability of the Model, the terms
Gender Identity is included in the Canadian Institute for Health Information CIHI Reference Data Model [24] (CRDM) and the United States Core Data for Interoperability Version 2 [25] (USCDIv2).
Name to Use is the name that the person accessing healthcare services uses and wishes the care team to use. Importantly, Name to Use may not simply be a person’s preferred name. Name to Use may be a name associated with the person’s gender identity, and so should be used in healthcare interactions to avoid misnaming or outing people. Name to Use may or may not be consistent with a person’s legal name. Misnaming people based on legal records can be harmful, so it is best to ensure that the name the patient uses is validated privately, and that this validated information is updated in the patient record routinely and promptly upon patient arrival to prevent misnaming or outing in subsequent care interactions. Use of the Name to Use may be time-limited [22].
Pronouns (also called third person pronouns, personal pronouns and incorrectly often called preferred pronouns) and possessives are language tools used to communicate with and about people. They include such options as he/him/his, she/her/hers, and they/them/theirs, among many options [7]. Pronouns come in sets. The most common pronouns used are gendered, and so by validating pronouns used by a patient and updating the care record before clinical interactions occur, intake staff can help ensure that patients are not misgendered by clinicians in subsequent interactions by the use of incorrect pronouns, and maintain the integrity of the therapeutic relationship. The Pronouns data element of the Model complements Gender Identity and Name to Use to ensure that communications with and about a patient are person-centred and affirming of their gender [21]. A useful rule of thumb is that until pronouns are confirmed by the patient, the gender-neutral options they/them/theirs can be used in interpersonal interactions to avoid misgendering. Like Name to Use and Gender Identity, patients may use more than one pronoun or set of pronouns, and their use of specific pronouns may be time delimited.
One of the primary problems associated with outdated GSSO information practices is that sex and gender concepts, the code values that represent them, the labels on the data fields they occupy and their distinct meanings, are conflated and used as though they are synonymous [6]. Administrative values cannot be used for clinical decision making with 100% reliability, particularly when used in the care of people whose sex at birth does not align with their gender identity and people who identify as transgender. Separating the sex or gender value that has been recorded on identification and other administrative or legal artifacts from clinical sex and gender identity helps to reduce unchecked assumptions about anatomy based on inaccurate recorded sex or gender information. The Model data element called Recorded Sex or Gender does just this: it separates administrative sex and gender information from the clinical sex and gender information, and is necessary to provide safe, quality, and gender-affirming clinical interactions.
Because there are so many different possibilities in the documentation that patients can present to support identification, it may be important to know the provenance of the Recorded Sex or Gender information. Provenance includes details about the jurisdiction that issued the record, its acquisition date, its validity period, and information about the source record including the name and definition of the field name and definition. Sex or gender information that is displayed on drivers’ licenses, government IDs, birth certificates and other documents are Recorded Sex or Gender information. In many cases, this information is acquired through active interfaces between health information systems and government administrative databases via exchange standards. Inconsistencies between Recorded Sex or Gender data values and Gender Identity should not automatically be regarded as erroneous and should be validated with the patient.
Birth sex, or sex assigned at birth, as an example of recorded sex or gender that is present in most care records, is included in the CRDM [24] and the USCDIv2 [25].
Sex for Clinical Use is a novel clinical data element that supports person-centred care by enabling clinicians to specify clinical sex beyond binary options based on different observations. Much like the explicit inclusion of a patient-declared Gender Identity, the key value of Sex for Clinical Use is that it enables the removal of unchecked assumptions about a person’s sex that may be present in administrative binary male/female constructs. To achieve this, a provider may refer to an anatomic inventory, a surgical history, a hormonal inventory or other artifacts that inform the context for the observation. Four such examples include:
ordering a screening mammography for a transman who has had top surgery;
specifying testicular protection for a transgender woman receiving an imaging procedure;
referencing a nonbinary hormonal reference range for a nonbinary person in transition; or
ordering a Pap smear for a man whose sex assigned at birth does not match his gender identity.
Full transition is not always required to address gender dysphoria [10], but the ability for providers to reduce unchecked assumptions based on recorded sex or gender, and provide agile, appropriate and quality care to all people is an important feature that the model supports. Value options that are proposed for the model include ‘male,’ ‘female’ and ‘specified’ sex for clinical use. The term ‘specified’ was chosen for the Model to eliminate stigma associated with a value options of ‘complex’ (not all patients who may benefit from the use of this element are particularly complex), to align with the use case where an option is specified by the provider, and to enable backwards compatibility with (and avoid use) of the stigmatizing term ‘other,’ as is so commonly used in health information systems [8].
When used together, the five data elements of the Gender Harmony Logical Model facilitate the tight alignment between social concepts, code systems, health terminology standards, exchange standards, EHR design and healthcare practices required to address stigma and drive health equity through the provision of gender-affirming care. However, code systems and health terminology standards are dynamic in nature and remain prone to the inappropriate codification of stigmatizing or pathologizing terms, codes or concepts [4]. As society becomes more complex and technology become more ubiquitous, the importance of remaining vigilant for standards that exclude marginalized populations and intersectional patient identities will become increasingly important. Primary care providers, in particular, are instrumental to the implementation of changes that will close the equity gap for transgender people because they provide the bulk of preventive screening and treatment related care and serve as a primary coordination point for patients. Modernized GSSO information practices help primary care providers to ensure that patients are appropriately matched to their care needs, and that assumptions related to conflated sex and gender concepts are avoided [13]. However, regulated health information professionals that have healthcare-specific training and awareness of healthcare models, ethics and practice will play a critical role in supporting modernization efforts. Certified Terminology Standards Specialists (CTSS), Certified Classification and Coding Specialist (CCCS), Certified Healthcare Information Management (CHIM) professional credentials, signal professional adherence to a base set of demonstrated competencies and ethics, and professionals with health information credentials such as these are more likely to have knowledge about the importance of the therapeutic relationship and downstream impacts of technical design on patient care [26]. A major responsibility of certified health information professionals is to align technical design with clinical requirements such that the integrity of this most sacred and fundamental component of healthcare—the therapeutic relationship—is protected. Like credentialed and regulated clinicians, certified health information professionals are accountable for upholding the core evidence-based values and ethics that support healthcare (i.e., person-centred care, trauma-informed care, the Gender Harmony Logical Model). The Digital Health Revolution has arrived [27], and the ongoing datafication of healthcare and society means that informatics professionals must regularly visit their ethics to ensure they are aligned with healthcare values, develop critical awareness of the impact of structural bias in data and technical design and standards on social justice, cultivate ethical communities around the design and analysis of information systems and standards, and evaluate the impact of their work on the lives of the patients they serve [28].
Transgender people experience unnecessary and preventable health inequities that are, in part, the result of social bias encoded into the technical and data structures that connect systems in the digital health ecosystem. Poorly defined data elements, conflated sex and gender concepts, constrained representation of gender variation in code systems, outdated terminology standards and health information exchange standards contribute to this harm. Canada Health Infoway’s Sex and Gender Working Group is an international, transdisciplinary community of people and agencies with an Action Plan to modernize gender, sex and sexual orientation information practices in healthcare. The HL7 International Gender Harmony Project has recently published the first release of the informative document for the Gender Harmony Logical Model, a consensus-based, evidence-informed logical model developed to address outdated information practices and support the modernization of healthcare for sex and gender minorities. Though there remains much work to do, this dedicated international community of professionals, researchers and people with lived experience are undertaking actions that enable inclusive and affirming care for transgender people and SGM.
I gratefully acknowledge the ongoing support of these efforts by Health Canada, Canada Health Infoway, all the members, agencies and presenters participating in Infoway’s Sex-Gender Working Group, the Canadian Institute of Health Information, the Canadian Health Information Management Association, the Office of the National Coordinator, HL7 Canada, HL7 International and the University of Victoria GSSO Research Team, the Canadian Institutes for Health Research, Michael Smith Health Research BC, the Community-Based Research Centre, and the large community of people, providers and agencies dedicated to equitable health for transgender people and sex and gender minorities for their continued work toward a better humanity, and a better future.
The author(s) declare no conflict of interest.
My thanks to Dr. Francis Lau for his review of this chapter as I was drafting it. The information provided in this chapter has been derived from my participation as Co-Chair in Infoway’s Sex and Gender Working Group and participation in the HL7 Gender Harmony Project. Please report any mistakes or inconsistencies to me at
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Singh",profilePictureURL:"https://mts.intechopen.com/storage/users/329385/images/system/329385.png",institutionString:"Punjab Technical University",institution:{name:"Punjab Technical University",institutionURL:null,country:{name:"India"}}}],equalEditorOne:null,equalEditorTwo:null,equalEditorThree:null},{type:"book",id:"8018",title:"Extracellular Matrix",subtitle:"Developments and Therapeutics",coverURL:"https://cdn.intechopen.com/books/images_new/8018.jpg",slug:"extracellular-matrix-developments-and-therapeutics",publishedDate:"October 27th 2021",editedByType:"Edited by",bookSignature:"Rama Sashank Madhurapantula, Joseph Orgel P.R.O. and Zvi Loewy",hash:"c85e82851e80b40282ff9be99ddf2046",volumeInSeries:23,fullTitle:"Extracellular Matrix - Developments and Therapeutics",editors:[{id:"212416",title:"Dr.",name:"Rama Sashank",middleName:null,surname:"Madhurapantula",slug:"rama-sashank-madhurapantula",fullName:"Rama Sashank Madhurapantula",profilePictureURL:"https://mts.intechopen.com/storage/users/212416/images/system/212416.jpg",institutionString:"Illinois Institute of Technology",institution:{name:"Illinois Institute of Technology",institutionURL:null,country:{name:"United States of America"}}}],equalEditorOne:null,equalEditorTwo:null,equalEditorThree:null},{type:"book",id:"9759",title:"Vitamin E in Health and Disease",subtitle:"Interactions, Diseases and Health Aspects",coverURL:"https://cdn.intechopen.com/books/images_new/9759.jpg",slug:"vitamin-e-in-health-and-disease-interactions-diseases-and-health-aspects",publishedDate:"October 6th 2021",editedByType:"Edited by",bookSignature:"Pınar Erkekoglu and Júlia Scherer Santos",hash:"6c3ddcc13626110de289b57f2516ac8f",volumeInSeries:22,fullTitle:"Vitamin E in Health and Disease - Interactions, Diseases and Health Aspects",editors:[{id:"109978",title:"Prof.",name:"Pınar",middleName:null,surname:"Erkekoğlu",slug:"pinar-erkekoglu",fullName:"Pınar Erkekoğlu",profilePictureURL:"https://mts.intechopen.com/storage/users/109978/images/system/109978.jpg",institutionString:"Hacettepe University",institution:{name:"Hacettepe University",institutionURL:null,country:{name:"Turkey"}}}],equalEditorOne:null,equalEditorTwo:null,equalEditorThree:null}]},subseriesFiltersForPublishedBooks:[{group:"subseries",caption:"Proteomics",value:18,count:4},{group:"subseries",caption:"Metabolism",value:17,count:6},{group:"subseries",caption:"Cell and Molecular Biology",value:14,count:9},{group:"subseries",caption:"Chemical Biology",value:15,count:13}],publicationYearFilters:[{group:"publicationYear",caption:"2022",value:2022,count:8},{group:"publicationYear",caption:"2021",value:2021,count:7},{group:"publicationYear",caption:"2020",value:2020,count:12},{group:"publicationYear",caption:"2019",value:2019,count:3},{group:"publicationYear",caption:"2018",value:2018,count:2}],authors:{paginationCount:301,paginationItems:[{id:"116250",title:"Dr.",name:"Nima",middleName:null,surname:"Rezaei",slug:"nima-rezaei",fullName:"Nima Rezaei",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/116250/images/system/116250.jpg",biography:"Professor Nima Rezaei obtained an MD from Tehran University of Medical Sciences, Iran. He also obtained an MSc in Molecular and Genetic Medicine, and a Ph.D. in Clinical Immunology and Human Genetics from the University of Sheffield, UK. He also completed a short-term fellowship in Pediatric Clinical Immunology and Bone Marrow Transplantation at Newcastle General Hospital, England. Dr. Rezaei is a Full Professor of Immunology and Vice Dean of International Affairs and Research, at the School of Medicine, Tehran University of Medical Sciences, and the co-founder and head of the Research Center for Immunodeficiencies. He is also the founding president of the Universal Scientific Education and Research Network (USERN). Dr. Rezaei has directed more than 100 research projects and has designed and participated in several international collaborative projects. He is an editor, editorial assistant, or editorial board member of more than forty international journals. He has edited more than 50 international books, presented more than 500 lectures/posters in congresses/meetings, and published more than 1,100 scientific papers in international journals.",institutionString:"Tehran University of Medical Sciences",institution:{name:"Tehran University of Medical Sciences",country:{name:"Iran"}}},{id:"180733",title:"Dr.",name:"Jean",middleName:null,surname:"Engohang-Ndong",slug:"jean-engohang-ndong",fullName:"Jean Engohang-Ndong",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/180733/images/system/180733.png",biography:"Dr. Jean Engohang-Ndong was born and raised in Gabon. After obtaining his Associate Degree of Science at the University of Science and Technology of Masuku, Gabon, he continued his education in France where he obtained his BS, MS, and Ph.D. in Medical Microbiology. He worked as a post-doctoral fellow at the Public Health Research Institute (PHRI), Newark, NJ for four years before accepting a three-year faculty position at Brigham Young University-Hawaii. Dr. Engohang-Ndong is a tenured faculty member with the academic rank of Full Professor at Kent State University, Ohio, where he teaches a wide range of biological science courses and pursues his research in medical and environmental microbiology. Recently, he expanded his research interest to epidemiology and biostatistics of chronic diseases in Gabon.",institutionString:"Kent State University",institution:{name:"Kent State University",country:{name:"United States of America"}}},{id:"188773",title:"Prof.",name:"Emmanuel",middleName:null,surname:"Drouet",slug:"emmanuel-drouet",fullName:"Emmanuel Drouet",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/188773/images/system/188773.png",biography:"Emmanuel Drouet, PharmD, is a Professor of Virology at the Faculty of Pharmacy, the University Grenoble-Alpes, France. As a head scientist at the Institute of Structural Biology in Grenoble, Dr. Drouet’s research investigates persisting viruses in humans (RNA and DNA viruses) and the balance with our host immune system. He focuses on these viruses’ effects on humans (both their impact on pathology and their symbiotic relationships in humans). He has an excellent track record in the herpesvirus field, and his group is engaged in clinical research in the field of Epstein-Barr virus diseases. He is the editor of the online Encyclopedia of Environment and he coordinates the Universal Health Coverage education program for the BioHealth Computing Schools of the European Institute of Science.",institutionString:null,institution:{name:"Grenoble Alpes University",country:{name:"France"}}},{id:"131400",title:"Prof.",name:"Alfonso J.",middleName:null,surname:"Rodriguez-Morales",slug:"alfonso-j.-rodriguez-morales",fullName:"Alfonso J. Rodriguez-Morales",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/131400/images/system/131400.png",biography:"Dr. Rodriguez-Morales is an expert in tropical and emerging diseases, particularly zoonotic and vector-borne diseases (especially arboviral diseases). He is the president of the Travel Medicine Committee of the Pan-American Infectious Diseases Association (API), as well as the president of the Colombian Association of Infectious Diseases (ACIN). He is a member of the Committee on Tropical Medicine, Zoonoses, and Travel Medicine of ACIN. He is a vice-president of the Latin American Society for Travel Medicine (SLAMVI) and a Member of the Council of the International Society for Infectious Diseases (ISID). Since 2014, he has been recognized as a Senior Researcher, at the Ministry of Science of Colombia. He is a professor at the Faculty of Medicine of the Fundacion Universitaria Autonoma de las Americas, in Pereira, Risaralda, Colombia. He is an External Professor, Master in Research on Tropical Medicine and International Health, Universitat de Barcelona, Spain. He is also a professor at the Master in Clinical Epidemiology and Biostatistics, Universidad Científica del Sur, Lima, Peru. In 2021 he has been awarded the “Raul Isturiz Award” Medal of the API. Also, in 2021, he was awarded with the “Jose Felix Patiño” Asclepius Staff Medal of the Colombian Medical College, due to his scientific contributions to COVID-19 during the pandemic. He is currently the Editor in Chief of the journal Travel Medicine and Infectious Diseases. His Scopus H index is 47 (Google Scholar H index, 68).",institutionString:"Institución Universitaria Visión de las Américas, Colombia",institution:null},{id:"332819",title:"Dr.",name:"Chukwudi Michael",middleName:"Michael",surname:"Egbuche",slug:"chukwudi-michael-egbuche",fullName:"Chukwudi Michael Egbuche",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/332819/images/14624_n.jpg",biography:"I an Dr. Chukwudi Michael Egbuche. I am a Senior Lecturer in the Department of Parasitology and Entomology, Nnamdi Azikiwe University, Awka.",institutionString:null,institution:{name:"Nnamdi Azikiwe University",country:{name:"Nigeria"}}},{id:"284232",title:"Mr.",name:"Nikunj",middleName:"U",surname:"Tandel",slug:"nikunj-tandel",fullName:"Nikunj Tandel",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/284232/images/8275_n.jpg",biography:'Mr. Nikunj Tandel has completed his Master\'s degree in Biotechnology from VIT University, India in the year of 2012. He is having 8 years of research experience especially in the field of malaria epidemiology, immunology, and nanoparticle-based drug delivery system against the infectious diseases, autoimmune disorders and cancer. He has worked for the NIH funded-International Center of Excellence in Malaria Research project "Center for the study of complex malaria in India (CSCMi)" in collaboration with New York University. The preliminary objectives of the study are to understand and develop the evidence-based tools and interventions for the control and prevention of malaria in different sites of the INDIA. Alongside, with the help of next-generation genomics study, the team has studied the antimalarial drug resistance in India. Further, he has extended his research in the development of Humanized mice for the study of liver-stage malaria and identification of molecular marker(s) for the Artemisinin resistance. At present, his research focuses on understanding the role of B cells in the activation of CD8+ T cells in malaria. Received the CSIR-SRF (Senior Research Fellow) award-2018, FIMSA (Federation of Immunological Societies of Asia-Oceania) Travel Bursary award to attend the IUIS-IIS-FIMSA Immunology course-2019',institutionString:"Nirma University",institution:{name:"Nirma University",country:{name:"India"}}},{id:"334383",title:"Ph.D.",name:"Simone",middleName:"Ulrich",surname:"Ulrich Picoli",slug:"simone-ulrich-picoli",fullName:"Simone Ulrich Picoli",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/334383/images/15919_n.jpg",biography:"Graduated in Pharmacy from Universidade Luterana do Brasil (1999), Master in Agricultural and Environmental Microbiology from Federal University of Rio Grande do Sul (2002), Specialization in Clinical Microbiology from Universidade de São Paulo, USP (2007) and PhD in Sciences in Gastroenterology and Hepatology (2012). She is currently an Adjunct Professor at Feevale University in Medicine and Biomedicine courses and a permanent professor of the Academic Master\\'s Degree in Virology. She has experience in the field of Microbiology, with an emphasis on Bacteriology, working mainly on the following topics: bacteriophages, bacterial resistance, clinical microbiology and food microbiology.",institutionString:null,institution:{name:"Universidade Feevale",country:{name:"Brazil"}}},{id:"229220",title:"Dr.",name:"Amjad",middleName:"Islam",surname:"Aqib",slug:"amjad-aqib",fullName:"Amjad Aqib",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/229220/images/system/229220.png",biography:"Dr. Amjad Islam Aqib obtained a DVM and MSc (Hons) from University of Agriculture Faisalabad (UAF), Pakistan, and a PhD from the University of Veterinary and Animal Sciences Lahore, Pakistan. Dr. Aqib joined the Department of Clinical Medicine and Surgery at UAF for one year as an assistant professor where he developed a research laboratory designated for pathogenic bacteria. Since 2018, he has been Assistant Professor/Officer in-charge, Department of Medicine, Manager Research Operations and Development-ORIC, and President One Health Club at Cholistan University of Veterinary and Animal Sciences, Bahawalpur, Pakistan. He has nearly 100 publications to his credit. His research interests include epidemiological patterns and molecular analysis of antimicrobial resistance and modulation and vaccine development against animal pathogens of public health concern.",institutionString:"Cholistan University of Veterinary and Animal Sciences",institution:null},{id:"62900",title:"Prof.",name:"Fethi",middleName:null,surname:"Derbel",slug:"fethi-derbel",fullName:"Fethi Derbel",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/62900/images/system/62900.jpeg",biography:"Professor Fethi Derbel was born in 1960 in Tunisia. He received his medical degree from the Sousse Faculty of Medicine at Sousse, University of Sousse, Tunisia. He completed his surgical residency in General Surgery at the University Hospital Farhat Hached of Sousse and was a member of the Unit of Liver Transplantation in the University of Rennes, France. He then worked in the Department of Surgery at the Sahloul University Hospital in Sousse. Professor Derbel is presently working at the Clinique les Oliviers, Sousse, Tunisia. His hospital activities are mostly concerned with laparoscopic, colorectal, pancreatic, hepatobiliary, and gastric surgery. He is also very interested in hernia surgery and performs ventral hernia repairs and inguinal hernia repairs. He has been a member of the GREPA and Tunisian Hernia Society (THS). During his residency, he managed patients suffering from diabetic foot, and he was very interested in this pathology. For this reason, he decided to coordinate a book project dealing with the diabetic foot. Professor Derbel has published many articles in journals and collaborates intensively with IntechOpen Access Publisher as an editor.",institutionString:"Clinique les Oliviers",institution:null},{id:"300144",title:"Dr.",name:"Meriem",middleName:null,surname:"Braiki",slug:"meriem-braiki",fullName:"Meriem Braiki",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/300144/images/system/300144.jpg",biography:"Dr. Meriem Braiki is a specialist in pediatric surgeon from Tunisia. She was born in 1985. She received her medical degree from the University of Medicine at Sousse, Tunisia. She achieved her surgical residency training periods in Pediatric Surgery departments at University Hospitals in Monastir, Tunis and France.\r\nShe is currently working at the Pediatric surgery department, Sidi Bouzid Hospital, Tunisia. Her hospital activities are mostly concerned with laparoscopic, parietal, urological and digestive surgery. She has published several articles in diffrent journals.",institutionString:"Sidi Bouzid Regional Hospital",institution:null},{id:"229481",title:"Dr.",name:"Erika M.",middleName:"Martins",surname:"de Carvalho",slug:"erika-m.-de-carvalho",fullName:"Erika M. de Carvalho",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/229481/images/6397_n.jpg",biography:null,institutionString:null,institution:{name:"Oswaldo Cruz Foundation",country:{name:"Brazil"}}},{id:"186537",title:"Prof.",name:"Tonay",middleName:null,surname:"Inceboz",slug:"tonay-inceboz",fullName:"Tonay Inceboz",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/186537/images/system/186537.jfif",biography:"I was graduated from Ege University of Medical Faculty (Turkey) in 1988 and completed his Med. PhD degree in Medical Parasitology at the same university. I became an Associate Professor in 2008 and Professor in 2014. I am currently working as a Professor at the Department of Medical Parasitology at Dokuz Eylul University, Izmir, Turkey.\n\nI have given many lectures, presentations in different academic meetings. I have more than 60 articles in peer-reviewed journals, 18 book chapters, 1 book editorship.\n\nMy research interests are Echinococcus granulosus, Echinococcus multilocularis (diagnosis, life cycle, in vitro and in vivo cultivation), and Trichomonas vaginalis (diagnosis, PCR, and in vitro cultivation).",institutionString:"Dokuz Eylül University",institution:{name:"Dokuz Eylül University",country:{name:"Turkey"}}},{id:"71812",title:"Prof.",name:"Hanem Fathy",middleName:"Fathy",surname:"Khater",slug:"hanem-fathy-khater",fullName:"Hanem Fathy Khater",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/71812/images/1167_n.jpg",biography:"Prof. Khater is a Professor of Parasitology at Benha University, Egypt. She studied for her doctoral degree, at the Department of Entomology, College of Agriculture, Food and Natural Resources, University of Missouri, Columbia, USA. She has completed her Ph.D. degrees in Parasitology in Egypt, from where she got the award for “the best scientific Ph.D. dissertation”. She worked at the School of Biological Sciences, Bristol, England, the UK in controlling insects of medical and veterinary importance as a grant from Newton Mosharafa, the British Council. Her research is focused on searching of pesticides against mosquitoes, house flies, lice, green bottle fly, camel nasal botfly, soft and hard ticks, mites, and the diamondback moth as well as control of several parasites using safe and natural materials to avoid drug resistances and environmental contamination.",institutionString:null,institution:{name:"Banha University",country:{name:"Egypt"}}},{id:"99780",title:"Prof.",name:"Omolade",middleName:"Olayinka",surname:"Okwa",slug:"omolade-okwa",fullName:"Omolade Okwa",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/99780/images/system/99780.jpg",biography:"Omolade Olayinka Okwa is presently a Professor of Parasitology at Lagos State University, Nigeria. She has a PhD in Parasitology (1997), an MSc in Cellular Parasitology (1992), and a BSc (Hons) Zoology (1990) all from the University of Ibadan, Nigeria. She teaches parasitology at the undergraduate and postgraduate levels. She was a recipient of a Commonwealth fellowship supported by British Council tenable at the Centre for Entomology and Parasitology (CAEP), Keele University, United Kingdom between 2004 and 2005. She was awarded an Honorary Visiting Research Fellow at the same university from 2005 to 2007. \nShe has been an external examiner to the Department of Veterinary Microbiology and Parasitology, University of Ibadan, MSc programme between 2010 and 2012. She is a member of the Nigerian Society of Experimental Biology (NISEB), Parasitology and Public Health Society of Nigeria (PPSN), Science Association of Nigeria (SAN), Zoological Society of Nigeria (ZSN), and is Vice Chairperson of the Organisation of Women in Science (OWSG), LASU chapter. She served as Head of Department of Zoology and Environmental Biology, Lagos State University from 2007 to 2010 and 2014 to 2016. She is a reviewer for several local and international journals such as Unilag Journal of Science, Libyan Journal of Medicine, Journal of Medicine and Medical Sciences, and Annual Research and Review in Science. \nShe has authored 45 scientific research publications in local and international journals, 8 scientific reviews, 4 books, and 3 book chapters, which includes the books “Malaria Parasites” and “Malaria” which are IntechOpen access publications.",institutionString:"Lagos State University",institution:{name:"Lagos State University",country:{name:"Nigeria"}}},{id:"273100",title:"Dr.",name:"Vijay",middleName:null,surname:"Gayam",slug:"vijay-gayam",fullName:"Vijay Gayam",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/273100/images/system/273100.jpeg",biography:"Dr. Vijay Bhaskar Reddy Gayam is currently practicing as an internist at Interfaith Medical Center in Brooklyn, New York, USA. He is also a Clinical Assistant Professor at the SUNY Downstate University Hospital and Adjunct Professor of Medicine at the American University of Antigua. He is a holder of an M.B.B.S. degree bestowed to him by Osmania Medical College and received his M.D. at Interfaith Medical Center. His career goals thus far have heavily focused on direct patient care, medical education, and clinical research. He currently serves in two leadership capacities; Assistant Program Director of Medicine at Interfaith Medical Center and as a Councilor for the American\r\nFederation for Medical Research. As a true academician and researcher, he has more than 50 papers indexed in international peer-reviewed journals. He has also presented numerous papers in multiple national and international scientific conferences. His areas of research interest include general internal medicine, gastroenterology and hepatology. He serves as an editor, editorial board member and reviewer for multiple international journals. His research on Hepatitis C has been very successful and has led to multiple research awards, including the 'Equity in Prevention and Treatment Award” from the New York Department of Health Viral Hepatitis Symposium (2018) and the 'Presidential Poster Award” awarded to him by the American College of Gastroenterology (2018). He was also awarded 'Outstanding Clinician in General Medicine” by Venus International Foundation for his extensive research expertise and services, perform over and above the standard expected in the advancement of healthcare, patient safety and quality of care.",institutionString:"Interfaith Medical Center",institution:{name:"Interfaith Medical Center",country:{name:"United States of America"}}},{id:"93517",title:"Dr.",name:"Clement",middleName:"Adebajo",surname:"Meseko",slug:"clement-meseko",fullName:"Clement Meseko",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/93517/images/system/93517.jpg",biography:"Dr. Clement Meseko obtained DVM and PhD degree in Veterinary Medicine and Virology respectively. He has worked for over 20 years in both private and public sectors including the academia, contributing to knowledge and control of infectious disease. Through the application of epidemiological skill, classical and molecular virological skills, he investigates viruses of economic and public health importance for the mitigation of the negative impact on people, animal and the environment in the context of Onehealth. \r\nDr. Meseko’s field experience on animal and zoonotic diseases and pathogen dynamics at the human-animal interface over the years shaped his carrier in research and scientific inquiries. He has been part of the investigation of Highly Pathogenic Avian Influenza incursions in sub Saharan Africa and monitors swine Influenza (Pandemic influenza Virus) agro-ecology and potential for interspecies transmission. He has authored and reviewed a number of journal articles and book chapters.",institutionString:"National Veterinary Research Institute",institution:{name:"National Veterinary Research Institute",country:{name:"Nigeria"}}},{id:"158026",title:"Prof.",name:"Shailendra K.",middleName:null,surname:"Saxena",slug:"shailendra-k.-saxena",fullName:"Shailendra K. Saxena",position:null,profilePictureURL:"https://s3.us-east-1.amazonaws.com/intech-files/0030O00002bRET3QAO/Profile_Picture_2022-05-10T10:10:26.jpeg",biography:"Professor Dr. Shailendra K. Saxena is a vice dean and professor at King George's Medical University, Lucknow, India. His research interests involve understanding the molecular mechanisms of host defense during human viral infections and developing new predictive, preventive, and therapeutic strategies for them using Japanese encephalitis virus (JEV), HIV, and emerging viruses as a model via stem cell and cell culture technologies. His research work has been published in various high-impact factor journals (Science, PNAS, Nature Medicine) with a high number of citations. He has received many awards and honors in India and abroad including various Young Scientist Awards, BBSRC India Partnering Award, and Dr. JC Bose National Award of Department of Biotechnology, Min. of Science and Technology, Govt. of India. Dr. Saxena is a fellow of various international societies/academies including the Royal College of Pathologists, United Kingdom; Royal Society of Medicine, London; Royal Society of Biology, United Kingdom; Royal Society of Chemistry, London; and Academy of Translational Medicine Professionals, Austria. He was named a Global Leader in Science by The Scientist. 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