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Dr. Wei Wu has received awards from many national societies for the originality and quality of his projects. He has authored 70 peer-reviewed papers in international journals.",coeditorOneBiosketch:"A pioneering researcher in obstetrics and holder of three registered patents. Dr. Qiuqin Tang's research interests include genetic and epigenetic risk factors of reproductive and developmental health. She has authored over 20 papers in international journals.",coeditorTwoBiosketch:null,coeditorThreeBiosketch:null,coeditorFourBiosketch:null,coeditorFiveBiosketch:null,editors:[{id:"178661",title:"Dr.",name:"Wei",middleName:null,surname:"Wu",slug:"wei-wu",fullName:"Wei Wu",profilePictureURL:"https://mts.intechopen.com/storage/users/178661/images/system/178661.jpeg",biography:"Dr. Wei Wu is an associate professor and associate department\nchair in the Department of Toxicology, Nanjing Medical University, China, where he received his Ph.D. in Toxicology in 2012.\nHe was a guest researcher at the National Institute of Environmental Health Sciences (NIEHS) between 2017 and 2018. Dr.\nWu is a member of different national and international societies\nin the fields of human reproduction and toxicology and has\nreceived awards from many national societies for the originality and quality of his\nprojects. Dr. Wu has authored seventy-three peer-reviewed papers in international\njournals. He has edited four books and collaborated on ten others as well as seventeen patents and in the organization of three international conferences. He is a\nreviewer for ninety-eight journals.",institutionString:"Nanjing Medical University",position:null,outsideEditionCount:0,totalCites:0,totalAuthoredChapters:"4",totalChapterViews:"0",totalEditedBooks:"4",institution:{name:"Nanjing Medical University",institutionURL:null,country:{name:"China"}}}],coeditorOne:{id:"184798",title:"Ms.",name:"Qiuqin",middleName:null,surname:"Tang",slug:"qiuqin-tang",fullName:"Qiuqin Tang",profilePictureURL:"https://mts.intechopen.com/storage/users/184798/images/13334_n.jpg",biography:"Qiuqin Tang is an attending doctor of The Women’s Hospital of Nanjing Medical University (Nanjing Maternity and Child Health Care Hospital). Her research interests include genetic and epigenetic risk factors of reproductive and developmental health. 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He\nis currently a professor in the Gynecology and Obstetrics Faculty\nof Medicine, University of Kiel, Germany, and honorary doctor\nat the Democritus University of Thrace, Alexandroupoli University Hospital He previously served as chief of the Department\nof Gynecology and Obstetrics at University Hospital RWTH Aachen,\nGermany. Dr. Rath is a reviewer for numerous journals and chief editor of Geburtshilfe und Frauenheilkunde (GebFra). 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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"}}]},chapter:{item:{type:"chapter",id:"79170",title:"Attention and Learning Disabilities",doi:"10.5772/intechopen.100808",slug:"attention-and-learning-disabilities",body:'Together with hyperactivity and impulsiveness, attention deficits are the main symptoms of ADHD [1]. Most empirical studies on impaired attention are related to young people diagnosed with ADHD. Further, interventions are typically evaluated in selected groups, like ADHD. However, in an ordinary classroom, a variety of reasons may lead to attention problems. It has been claimed [2] that there is a gap between neuropsychological knowledge on attention and clinical applications in schools. Thus, this chapter aims to illustrate how Mirsky’s model of attention [3, 4] may be useful in educational institutions and further, I will add some information to inspire theoretical reflections as well as practical skills in schools.
First, I present a theoretical section on attention mainly based on Mirsky’s model of attention [5], and thereafter, a section on attention, concentration, and learning difficulties, where the two concepts attention and concentration are discussed. Next, the chapter refers to relevant interventions and highlights possible differences between laboratory experiments with selected groups and real-life interventions with mixed groups. Because of promising results, one real-life intervention comprising target shooting practice is described in more detail. Towards the end, before the conclusion, I reflect on theoretical and practical implications.
Scientists have studied attention since research on ‘reaction time’ in mid-1800 [6], and theories have been developed according to scientific evidence available at the time. Mirsky and his colleagues [4] considered impaired attention to be one of the most pervasive and least understood behavioural disturbances (p. 109). So, how can we understand attention? Parasuraman [7] refers to attention as capacities or processes of how the organism becomes receptive to stimuli and how it may begin processing internal or external stimulation. During the last two decades, the fast development of new technologies like functional magnetic resonance imaging (fMRI) has shown neural circuity in large-scale brain networks. This tells us there is communication back and forth in active networks in the cortical and subcortical brain regions [6] and leads to a neuropsychological shift from suggesting attention located in specific areas in the brain to understanding attention organised in active brain networks [8]. The new findings support Mirsky’s theory suggesting several components or elements of attention [5]. Further, and important to educational settings, Mirsky suggested that the components could be assessed by neuropsychological tests [4]. Below I will present the components, also denoted elements, together with practical examples. Actual tests are mentioned for the first two components. Others [5] have given a broader overview of actual tests related to each element.
Another Mirsky component of special importance in schools is the
Also, the next three components may be useful for teachers to recognise. First, the
Some of the elements of attention mentioned above may have blurred boundaries with other cognitive capacities. Mirsky recognised for instance that the Shift element could be seen as a feature of executive functions ([5], p. 299). ‘Executive functions’ refer to several mental processes that control and organise other mental processes [10] which are crucial for planning complex behaviours and adapting to the situation [11]. In considering attention, it may also be useful for teachers and other professionals to have features of memory in mind. ‘Short term memory’ is the simple temporary storage of information whereas ‘working memory’ implies a combination of storage and manipulation ([12], p. 4). At the same time, both short-term memory and working memory differ from the permanent storing of information, denoted ‘long-term memory’, where information can remain for the rest of our lives ([2], p. 183).
For professionals like psychologists and educators to understand possible reasons leading to learning difficulties, I consider it an advantage to know some aspects of attention. With such knowledge and insights, you have a better chance to be able to help children and adolescents with impaired attention.
Compared to the huge neuropsychological literature on attention, there has been far less interest among scientists on the term ‘concentration’. Sometimes the two terms attention and concentration are used interchangeably, both orally and in text. However, they are not synonyms although there might be some overlap between the concepts. In the last couple of decades before the millennium, neuropsychologists, especially in Germany, discussed how to differentiate the concepts of attention and concentration [13]. Translated to English, concentration was suggested to be defined as the “ability to work quickly and accurately under conditions that normally make cognitive performance difficult” ([14], p. 9). At the end of this section, I will discuss whether this suggested definition is adequate and appropriate related to observed and perceived concentration in school lessons.
Below I present and reflect on research done in a school programme intending to increase students’ concentration [15]. All 12 students aged 10–16, had harmful concentration problems, that hampered their learning. Each student, the parents, and the teacher had to agree on the student’s participation in the programme. When we talked with the students in individual tape-recorded interviews, some of them initially told us their concentration was fine, but later in the interview, they detailed out how they lost their concentration when something took place at the other side of the classroom [16].
The in-depth work with the qualitative material gathered before the intervention programme started, enwidened my insight into students with so-called concentration problems. Half the students had an ADHD diagnosis, some had dyslexia or other diagnoses, and some had no medical diagnoses. None was diagnosed with dyscalculia, although most of the students had challenges in Mathematics and clearly expressed that they disliked the subject [9]. We can look at one citation where the student finds it difficult to cope with arithmetic: “Mathematics, I don’t like maths because it is very difficult … for instance some multiplication and division tasks. But I know how to do it.” This student had the self-confidence that the knowledge was there, that he/she
Regarding foreign language (English), only one student liked this subject, half the students disliked it, and two of those claimed it was because of dyslexia. Among the subjects, there was one favourite: Physical Education (P. E.). Eight students spontaneously said they loved P. E. and they argued it was because of activity: “It is activity, and we get to be outside.”; “Because then I can move around and such things.”
However, for me as a professional (psychologist, researcher, and lecturer in teacher education) the diversity in the students’ likes and dislikes of subjects was interesting. For instance, a subject that one student might appreciate, another disliked and vice versa. The authors could not find any pattern related to subjects except the ratios mentioned above on Mathematics, foreign language, and P. E. On the other hand, we found an interesting pattern when we related perceived concentration with the theory of play [17, 18] and the theory of motivation; the situated expectancy-value theory [19]. There was a clear relationship between perceived concentration and enjoyment (like we find enjoyment in the theory of play as well as in intrinsic motivation). With higher enjoyment, the concentration was perceived as better, and when the student felt concentration was bad, the enjoyment was correspondingly low. The qualitative data indicated a high correlation, illustrated below in the previously published Figure 1 [9].
The importance of content and context for concentration and enjoyment © Løhre, Vedul-Kjelsås, Østerlie ([
What is special with this qualitative material? On one hand, the group is mixed, meaning that some students do not have medical diagnoses whereas others have one or more diagnoses. Both genders were included, seven boys and five girls, representing a relatively large age span from 10 to 16 years. Despite all those differences, we find the clear relationships presented in Figure 1. Another aspect I want to highlight from the empirical data is the importance of context versus content. Illustrated in Figure 1, we see that content is related to the whole range of concentration/enjoyment whereas the context was more important when the student had challenges with the tasks. To cope with those challenges, the empirical data showed both institutional strategies and personal strategies [9]. And worth noticing, all strategies were about shielding.
The institutional strategies are well known to educators. They included going to a separate room to work in small groups or individually with a teacher. Those students who told they had the opportunity to be alone with the teacher in some lessons or together with a few others, appreciated this situation. We found two reasons for their appreciation of the small room. First, they felt calmer because often there were many disturbances in the classroom, like one student said: “It is no problem if I sit by myself or together with only a few others. But if I sit in the classroom and there are many students talking I kind of become more interested in what they are doing.” ([9], p. 91). This corresponds to the Focus/executive element in Mirsky’s model of attention [5]. It was difficult for the students to screen out peripheral stimuli and simultaneously allocate attentional resources to their work. Additionally, the next reason was adapted help when needed, for instance in Mathematics. Municipalities and counties in Norway are obliged to offer special education to students considered to have this right [20]. However, it has for years been debated whether it is necessary to anchor special education as a right in the school laws. Some argue it is important to secure the rights of students with learning disabilities whereas others claim each school must support every student without binding resources to individuals [21, 22].
As we see, the institutional strategies included both mental and physical shielding. The personal strategies, on the other hand, were mostly some sort of mental shielding. Some students consciously paused from studying when they felt exhausted and unable to concentrate. Several of them started to draw or scribble and some raised to move around a little. The following citations ([9], p. 92) illustrate the students’ autonomy in a shift of activities: “I cannot sit too long, then my concentration disappears, it does. Then I need to take pauses and such”; “[…] Oh, yeah, I need to have a little pause now and then.”; “Then I take a few minutes off.” Regarding this last citation, the interviewer asked what the student did in those “few minutes” and the student answered: “Draw a little. When I draw, I become quiet. My thoughts sort of disappear.” The interviewer followed up the conversation and wondered if someone had suggested for the student to take pauses, upon which the student answered: “No, it’s something I have found out for myself.” The empirical data does not tell us if any of the other students’ shifts in activities were agreed upon with the teacher, but obviously, teachers accepted.
From a professional point of view, I find the conscious shift in activities interesting. Like teachers who typically think students are daydreaming when they sit scrabbling on a paper and do not listen, I had thought the same when I was observing in classrooms. The finding of autonomous shifts was thought-provoking to me. I had often in my years as a school psychologist advised teachers to find ways to give a student pause when needed, for instance, leave the classroom to do something else, but it was new to me that impaired attention trigged some students to initiate pauses, almost to survive. Going back to Mirsky and the Encode Element [5], the exhausted student was not even able to register new information. In accordance with Danish findings [23], this shows us that classroom behaviour is not always what we think it is. The researcher who studied students with ADHD and their peers from a sociocultural perspective suggested that the behaviour and actions of students with ADHD often were intentional and led to interaction with peers in the classroom. Thinking of Martha, the little girl who introduces this chapter, I might have drawn the wrong conclusions the first time. I considered it obedient to eat grapes and spit stones on the floor. Perhaps she needed a mental pause. Talking with Martha thirty years or so later, she did not remember the grape episode, but reflecting around it she proposed the lesson might have been boring to her and therefore she started to eat the grapes.
Martha was a quick, smart girl and creative with a fabulous fantasy. She did the best to make her world funny. This is in line with what we found in the material with the 12 students [16]. Those with hyperactive behaviour often initiated something to have fun, and their high levels of activity were perceived as nurturing friendships. When the interviewer asked how friends would characterise him/her, one student answered: “hyper, funny and frisky” (p. 12). Concerning Martha, she did not have specific learning difficulties, no dyslexia or dyscalculia, but the impaired attention gave her challenges in learning. Hence, she did not go straight forward and finish an education. She needed more years than her peers.
At this point, I would like to go back to the earlier presented definition of concentration ([14], p. 9): “ability to work quickly and accurately under conditions that normally make cognitive performance difficult”. We must keep in mind that the suggested definition was derived from experimental tests. For our purpose, we need to analyse the content, and therefore I divide it into parts:
ability
to work quickly and accurately
under conditions
that normally make cognitive performance difficult
I fully agree that ability (point 1) is necessary to be able to concentrate. This corresponds to Mirsky’s model of attention [4]; you need the ability to succeed in the different components. In terms of the second point, I think you should not ask a student with impaired attention to work quickly and accurately. In my opinion, the student must be allowed to work in an individual tempo to avoid getting stressed. The empirical material cited above [9], showed that some students consciously paused from studying when they felt exhausted and unable to concentrate: “I cannot sit too long, then my concentration disappears […].”
The third point indicates
Concerning students with perceived concentration problems, our data, as well as field observations, indicate that both ability and effort are necessary to be able to concentrate. The term effort is what the person does and may comprise the person’s energy and motivation to engage. Based on the knowledge and experiences presented above, I suggest the following definition of concentration: the ability and effort to apply cognitive resources to an object or topic of interest. As proposed by others [13], I think there are overlaps between attention and concentration. Furthermore, the definition indicates that concentration is a broader concept that may include other cognitive resources in addition to attention.
Although associations of attention with academic achievements have been explored in population studies, including mixed groups, in municipalities [24, 25], most intervention studies measure effects on attention in selected groups. Three recent reviews report intervention effects among children and adolescents diagnosed with ADHD. Two of them are quantitative and one is a literature review, and all applied rigorous inclusion criteria.
The literature review [26] explored 29 studies published before April 2016. The authors looked at the effects of physical activity on cognitive capacities. They divided the material into cardio activities, such as cycling or treadmill running, versus non-cardio activities, for instance yoga. The cardio activities showed some improvements on various outcomes whereas the results of non-cardio activities were questionable. In terms of attention in children, the results were inconclusive. Some studies reported no effects whereas others found significant effects.
One of the quantitative reviews [27] also studied the effects of physical activity. This review included nine of the same studies as the literature review [26] above. The meta-analysis of 20 studies demonstrated no significant effects on children’s attention, and there were neither any effects on academic achievements nor disruptive behaviour. However, the analyses showed significant improvements for internalising problems. In general, the cardio activities with running and cycling (here denoted aerobic activities) benefitted the children more than relaxation training and yoga (denoted non- aerobic activities).
Exploring studies with cognitive outcomes in several types of non-pharmacological interventions, the second meta-analysis [28] is of special interest to discussions in Section 5. With rigorous inclusion criteria including objective neuropsychological outcomes, the analysis comprised 18 out of 854 records published in the period 1980–2017. The authors had two research questions. First, they asked which non-pharmaceutical intervention was most effective for ADHD’s cognitive symptomology and secondly, they asked which cognitive symptoms were most amenable to change (p. 42). The analysis showed physical exercise (Morris
In addition to the main analyses, the review [28] presents an overview of studies that specifically assessed attention outcomes (p. 52). These studies altogether produced 14 effect sizes, showing an average Morris
In accordance with the recommendation from Lambez and colleagues [28], target shooting practice is conducted in real-life situations. As far as I know, we find target shooting practice integrated with public-school education only in two Scandinavian countries. Denmark was the first country to introduce this type of intervention for students with ADHD or ADHD-like symptoms. In the years 2012–2015, altogether 462 students were included in the FOKUS project [29] where instructors participated voluntarily. As both students and leaders reported positive experiences, further research was planned [30].
The Danish initiative soon spread to Norway, and in 2014, a public school in Mid-Norway started an educational programme for students with harmful concentration problems [15]. The programme is still running, and the selection of students is based on the agreement between the student, parents, and the school, assisted by professionals in the counselling services. Students are recruited from classes 5 to 10, corresponding to the age-group from 10 to 16 years. All training takes place outside the school area, in localities about five minutes’ walk from the school, and consists of theoretical lessons as well as shooting exercises. Step by step throughout the school year, the theoretical lessons prepare the students to improve in the target shooting practice. The theory includes how to behave, how to treat the weapon, and how to breath to be able to focus on the target. And above all, security is highlighted. All instructors in the programme are certificated by the Norwegian Civilian Marksmanship Association (Det frivillige Skyttervesen, DFS), and both certificated teachers and certificated volunteers from the local club of the NGO organisation DFS participate.
At the shooting range, each student gets adapted help by one of the instructors, see Figure 2. Typically, the students appreciate the care and involvement they receive from the instructors. The young adolescents thrive and perceive mastery [15, 31].
Adapted help by the certificated instructor. Photo Trond Jære.
Researchers at the NTNU Department of Teacher Education were hired to evaluate the implemented intervention. Data were gathered in the school year 2016/2017 and included questionnaires, individual interviews, observation at the shooting range, and objective neuropsychological tests [15]. Two qualitative publications on pre-data [9, 16], gathered before the seven months’ intervention started, have already informed this chapter. Additionally, quantitative results have shown statistically significant improvements on neuropsychological tests [32]. To the best of our knowledge, those results are the first worldwide to show significantly improved attention after one school year of target shooting practice. Further, the improved attention corresponds to basic components in the Mirsky model of attention [5], indicating that the intervention made everyday life easier for the students. It must be added that controls showed no statistically significant improvements on the neuropsychological tests. Concerning the intervention students, qualitative post-data from individual interviews support the quantitative results [32]. Moreover, Danish results [33] have recently demonstrated increased differences between intervention students and controls on a couple of other cognitive tests, not included in our study.
Getting promising results after target shooting practice, urge us to analyse what can be possible active ingredients in the intervention. We have suggested [32] that immediate neurofeedback is one of the important ingredients. When the student shoots and the bullet reach the target, an electronic visualisation of the target, placed beside the student, immediately shows the result, see Figure 3. Supporting our suggestions, the importance of neurofeedback is shown in the review by Lambez and colleagues [28]. Otherwise, we think the whole package included in the intervention; theoretical lessons, and adapted help by caring instructors, contribute to the results.
Also of great interests, are qualitative Danish findings [34] that report inhibition of impulsiveness after two or more years with target shooting practice. Our preliminary findings [15] indicate the same. However, a one-year intervention is possibly too short to impact impulsiveness outside the shooting range. In accordance with the Danish findings [34], the Norwegian instructors underline that they see this sort of change in students who have participated in the programme for at least two years. Maybe the length of intervention time is one of the reasons why a review [27] found no effects of physical activity on disruptive behaviour. Except the length of intervention, another reason behind the promising Danish results [34] as well as the Norwegian instructors’ experiences, maybe the theoretical lessons advising how to breathe and behave.
Immediate response on electronic target. Photo Mona Isene.
I agree with Mirsky [4], who thirty years ago argued that impaired attention is a pervasive behavioural disturbance. Attention seems to be one of the ground pillars for learning. Having impaired attention the student will meet a lot of challenges, and so will parents and teachers. To help the student, there are two main roads to follow. One is to facilitate improved attention through interventions, and the other is to facilitate learning in the here and now situations.
The great variety among students with perceived concentration problems [9], points to the benefits of knowing each student. The teacher needs to know individual preferences and learn to understand signs of poor attention as well as individual signs of a student losing attention during work. This is of course a high-hanging star and aim to reach but anchored in the Norwegian school laws [20], school leaders and teachers must do their best to reach the aim.
Previous research [9] has shown the importance of enjoyment in subjects and tasks for students with perceived concentration problems. The better they enjoyed the subject, the better was their concentration, cf. Figure 1. Further, the students highlighted adapted help and shielding, in accordance with their preferences. It was obvious that students who were to be included in the target shooting programme, administered their pauses in class when perceived as necessary to calm down, like one of them said: “When I draw, I become quiet. My thoughts sort of disappear.” This reminds me of the title: “ADHD attention deficit hyperactive disorder: an autobiography of survival” [35] and corresponds to autonomy as the suggested main force in motivation [36]. More autonomy could be given to students who struggle with impaired attention, − making agreements on self-regulated pauses and other individual learning strategies might create trust and strengthen the relationship between teacher and student.
At the first glance, ideas of student autonomy might seem to contradict previous recommendations [37] of structure and predictability for students with ADHD. Nevertheless, the two strategies can be combined, as exemplified in an ordinary public classroom [38]. Teachers make the frame and rules, and students fill the reserved time, for instance three hours, with self-chosen activities in the tempo and order they decide for themselves. Comprising the whole class, every child in class, is a great advantage of this educational programme, and as such, it is a health-promoting strategy aiming for thriving and wellbeing among all students. At the same time, there are some indications of better concentration for students with ADHD attending the programme [39].
For cognitive capacities in general, hard exercise has proved to be better than relaxing practices [26, 27]. Turning back to impaired attention, the theoretical and empirical knowledge we have to day points to specifically designed programmes to improve attention. Being recognised as one of the cognitive capacities most difficult to change [28], it is important to search for effective programmes. Interventions offering neurofeedback are among those with the most positive results [28]. In target shooting practice referred to above, immediate neurofeedback plays an essential role together with adapted help and caring. For countries that have target shooting sport in their communities, it might be an idea to initiate a partnership between local NGO shooting clubs, schools, and the municipality. The human resources found in the local shooting clubs in Denmark and Norway have provided valuable individual and adapted help at the shooting range, meaning a lot to young adolescents. Offering human resources, materials, and localities for free, the local NGO clubs additionally contribute to the school economy.
We have many challenges related to attention and impaired attention among students in educational institutions. First, the knowledge is scare on the impact of attention in different age groups. Next, we need to do more real-life interventions to investigate possible effects on attention. Further, more controlled longitudinal studies are needed to measure the effects of target shooting practice.
Although our research on target shooting practice is small-scaled, it has opened some windows into the understudied world of attention in students, and opening windows, gives room for new questions: Are our results on improved attention reliable? Will studies in other communities and other countries show corresponding results? What about the time span; is more than one school year of target shooting practice necessary to observe changes in the classroom? With higher numbers of students included, will possible differences between age groups and gender be uncovered?
Aiming to improve attention with neurofeedback seems promising. Thus, alternative interventions comprising neurofeedback should be developed, for instance with computer-based programmes in schools. Moreover, we need to expand the knowledge on autonomy and self-regulation in students with impaired attention.
In a paper reporting on results after target shooting practice [32], we hypothesised that the sequential order in shooting is beneficial to students with impaired attention. Hence, the role of sequential order must be further investigated, and if this point is crucial, it could be adopted to other educational situations. Furthermore, it is interesting in a theoretical perspective to study the impact of sequentially ordered activities versus activities based on simultaneous handling. Do persons with impaired attention prefer and succeed better in sequentially ordered activities?
The last theoretical question for me to present is about relationships between attention and concentration. Will the suggested definition of concentration be reliable in other settings and is it appropriate to see concentration as a broader concept than attention?
In this chapter, I have highlighted that more students than those diagnosed with ADHD have impaired attention. This may apply to students with other diagnoses like dyslexia, dyscalculia, or for example, undiagnosed or not well-medicated hypothyroidism that can occur in childhood or adolescence. Thus, we understand that many students in school and an ordinary classroom may have challenges due to impaired attention.
To help students with learning disabilities, it is beneficial to have knowledge on attention. One main goal of this chapter was to inform professionals in educational institutions by illustrating attention through Mirsky’s model of attention. Insight into the different components of the model can support teachers to better understand academic achievements, behaviour, and emotional reactions in students with impaired attention, and thus, give foundations for individually adapted teaching. For instance, realising that an otherwise smart 15-year-old student register and recall information (c.f. the Encode element in Mirsky’s model) at the mean level of students 8-year-old, can be thought-provoking for the teacher.
The term concentration is widely used by students and teachers in Norwegian schools. In our research, concentration was closely linked to enjoyment in schoolwork, despite great variations among the students in a heterogenous community group [9]. On the other hand, concentration did not seem to influence wellbeing or friendship [16]. The terms concentration and attention are sometimes used interchangeably, but they are not synonyms. I have suggested a definition of concentration for natural settings.
Further, the chapter presents interventions designed to improve cognitive capacities. Attention is found to be among the capacities least amendable to change [28]. Nevertheless, a seven month’s intervention comprising target shooting practice demonstrated increased attention on objective tests, and the results were supported by qualitative data [32]. Reasons of the success may be related to immediate neurofeedback in a caring and educational context at the shooting range. The suggested effect of neurofeedback finds support in other studies [28]. Proposals for practical implications and further research are put forward.
In this text, my purpose was to write in a language understandable for professionals outside neuropsychological circles, and thus, reduce what is pointed to as a gap of knowledge [2] in the field of practice. Additionally, one goal has been to remind me and the reader about possibilities of turning impairment into success.
The educational programme comprising target shooting practice has provided me with new insights and knowledge. Therefore, I am grateful to the students, parents, and teachers who participated in the evaluation of the programme. I will also thank the volunteers in the local shooting club for their valuable contributions.
Enzyme linked immunosorbent assay (ELISA) has existed for 50 years and ELISAs with different technical solutions are still being developed, which improves and expands the range of application.
The test was first described by Engvall and Perlmann in 1971 [1, 2, 3] and was based on the work of Avrameas, who used enzyme linked antibodies in histochemistry [4, 5]. The method was quickly developed for sero-diagnosis of trichinosis [6] and antibodies to
Since the discovery there have been numerous applications of ELISA, used to detect both antigens and antibodies. Besides the detection of protein antigens ELISAs that permit the determination of antibodies to native and denatured DNA [8, 9], polysaccharide antigens [10, 11, 12] and phospholipids [13] have been optimized. In fact, sometimes the name ELISA is applied to tests in which there are no antibodies, but instead specific protein–protein interactions are used. From the perspective of optimization, validation and standardization such tests can be treated in the same way. Regarding protein antigens the sensitivity of ELISA is usually in the pg/ml range [14].
When developing a diagnostic test, precise and optimal performance conditions must be found for all the steps within the test protocol. This ensures that the entire procedure is optimal. Before routine usage in diagnostics, for example, the newly developed, or a newly modified procedure must be proven to be accurate, precise and reproducible. Also, in order to measure the values obtained with the test, it is necessary to standardize the test. Therefore, optimization, validation and standardization (OVS) of ELISA are extremely important and necessary, especially if it is to be used in clinical or veterinary medicine. This chapter will present the procedures by which ELISA is characterized in an understandable and precise way.
Reviewing the literature, we noticed that the described boundaries between optimization, standardization and validation are not clear enough. The reason for this is that in certain situations performing a single ELISA can lead to a completion of both validation and optimization characteristics, which is completely valid. Before going into more details and in order to avoid confusion it is suitable to clearly define these three terms.
According to Merriam-Webster dictionary,
ELISA most often serves to measure the presence or quantity of antibodies or antigens, or biomolecules in general which can be recognized by antibodies. In biological matrices (such as serum, plasma, blood, urine and saliva) ELISA is an important diagnostic tool used to detect various antigens and antibodies. Indirect or direct ELISAs are used in medical product development, particularly for testing vaccines and new drugs. ELISA with specific antibodies can be designed to measure impurities within the medical products resulting from the production process. Antibody assays against these impurities should also be developed and validated for testing the levels of the impurities, which should be kept at a minimum in order to avoid adverse immune responses. For immunogenic substances with expected low concentrations, such as cytokines, hormones, toxins etc., sandwich ELISA is used.
Irrespective of the ELISA design (indirect, direct or sandwich), OVS principles are the same. Of paramount importance for any bioanalytical method is that it is well characterized, fully validated and documented to a satisfactory standard in order to yield reliable results.
The first step in ELISA development is optimisation, which is followed by standardization and finaly validation.
Optimization of an ELISA is essential to its success. Since ELISA is a multistep procedure, each component can be individually tested prior to the start of an experiment.
ELISA procedure consists of antigen or antibody coating, saturation, analyte application, detection with appropriate antibodies, primary or secondary and signal detection. Between each step the plate is washed. A variety of samples can be tested with ELISA, and the choice of assay conditions will depend upon the complexity of the sample and the expected amount of analyte present. Optimization is the establishment of ideal concentrations of each assay reagent and ideal conditions for each step and that must be done empirically. The cornersotne of any ELISA is the selection of the protocol type: direct, indirect or sandwich; which is dependent on the type of sample, avaliable reagents and the concentration of the analyte, keeping in mind that the procedure should be as straight forward as possible.
Numerous factors should be tested, such as the concentration of antigen, or antibody used for coating, temperature, the duration of individual steps the type of coating buffer, such as phosphate-buffered saline (PBS) or carbonate buffer, sample preparation methods (with or without EDTA, decomplementation, serum or plasma or whole samples). Plate saturation is also a step which requires optimization such as different concentration of bovine serum albumine (BSA), nonfat-dried milk, or whole serum from different animals. Here we will discuss the most important steps of the optimization procedure.
The first step in ELISA is coating wells with antigen or capturing antibodies. Most often this consists of applying a protein solution in PBS or carbonate buffer to microttiter plate wells. The microtiter plates for coating with proteins are special plates with modified surface, i.e. highly charged polystyrene surface with high affinity to molecules with polar or hydrophilic groups. This kind of surface has a high binding capacity for proteins, including globular antibodies and ensures proper antibody orientation. On the other hand ELISA for lipid antigens is performed on a hydrophobic surface, suited for non-protein antigens, which are not soluble in PBS or carbonate buffer, but are dissolved in an apropriate alcohol. Irrespective of the type of antigen the whole surface of the well bottom must be covered. If the whole surface is not covered the absorbance read will be lower, and if excess antibody/antigen is present, layers of antibody/antigen may form and wash away in subsequent steps, which again leads to lower signal. Figure 1 shows the dependance of absorbance on the ammount of antibody/antigen used for coating. For the optimized protocol it is important to select that antigen/antibody concentraion that gives the highest absorbance, marked with a red circle in Figure 1, which ensures that the complete well surface availiable for binding is covered in a monolayer. This principle should be followed regardless of the type of antigen/antibody or the ELISA type. For axample, in sandwith ELISA the wells are covered with capture antibodies, either whole IgG or Fab fragments and in direct and indirect ELISA with the antigens.
Dependance of absorbance on the ammount of antibody/antigen used for well coating in ELISA.
The process of coating an ELISA plate with antigen relies on the binding activity of the solid phase of the well, which immobilizes biomolecules on the well surface. Step after that must be blocking. During blocking free binding sites at the bottom of the wells become saturated with a blocking buffer in order to prevent the possibility of nonspecific binding and the residual binding capacity of the wells, thus greatly improving the signal-to-noise ratio and specificity. Without appropriate blocking the detection antibody could bind nonspecifically alongside the antigen, resulting in high background signal and low sensitivity.
There is a variety of blocking buffers, to choose from, not one of which is ideal for every situation. Although these buffers are called blocking buffers they usually contain a blocking component such as BSA, nonfat-dried milk, casein or whole serum. Every blocking buffer represents a compromise between reducing the background and maintaining specificity. Whole sera and serum protein albumin can cause non-specific ELISA signals in certain circumstances [15].
Even different BSA preparations show variations in the blocking activity of non-specific binding in ELISA. To prevent false positive results from cross reactive antibodies or non-specific binding of ELISA reagents to BSA, alternative blocking agents can be used and even no protein can be included in the blocking buffer [1]. These different blocking agents, (as well as their different concentration, incubation time, etc) should be tested in parallel, to discover the best way of saturation for each individual ELISA system.
It is almost always necessary to dilute samples for ELISA test, so the choice of the diluent is important. Generally, standard diluent should be as similar as possible to the matrix of the sample. For example, PBS with BSA is a good serum replacement in ELISA and is most often used for biologycal samples. The next important diluent component is non-ionic detergent (Tween 20, Triton X-100, CHAPS) that, in low concentrations, prevents non-specific (hydrophobic) protein–protein interactions. The specific binding is usually more resistant to the detergent. Detergents in one step do not provide a permanent barrier to biomolecule non-specific attachment in the following steps because it washes away with water or aqueous buffer, so in certain situations, detergents should be present in all the diluents/buffers.
It may be necessary to choose a different diluent than PBS/Tween/BSA, if the analyte is not serum. In that case, it is necessary to check the standard curve and linearity of dilution for the experimantal sample. The reason for this is the influence of the components of a standard diluent or matix on antigen/antibody interactions. In such cases spike-and-recovery or linearity-of-dilution experiments should be performed.
The goal in assay development is to achieve high signal-to-noise ratio while maintaining optimal responses. The sample matrix may contain interfeering components that affect assay response to the analyte by introducing a difference in comparison to the standard diluent. In order to asses this phenomenon, spike-and-recovery experiment is designed.
The idea of spike-and-recovery is that you add (spike) a certain amount of standard into the sample buffer or the samples, and measure them in parallel with samples with no standard added. Sometimes one can compare the same amount of analyte added into the natural test sample matrix and identical spike added to the standard diluent. So it can be seen whether you can measure (recover) the exact amount again, and how much you can recover from it in percentages. If, for any reason, you can not recover the same amount in comparison to a control, this means that something in the test solution is not in favor of the assay, so one should proceed with finding the right standard diluent.
Linearity-of-dilution experiments provide information about the precision of the assay results for different diluted samples in the chosen sample diluent. These experiments are performed to demonstrate that highly concentrated samples can be accurately measured by diluting into the assay’s quantitative range and the concentration can be calculated by multiplying the measured concentration by the dilution factor. Linearity-of-dilution experiment in practise means the measurement of at least three dilutions in the appropriate range in the selected diluent. There are two different ways to perform a linearity-of-dilution experiment, both with the same outcome. The usual method implies using a highly concentrated sample and then testing several different dilutions of that sample in the chosen sample diluent. Alternatively one can first prepare several different dilutions of a low concentration sample and then spike it with the same amount of the analyte before testing. If a sample does not exhibit linear dilution (i.e. linear dependence of absorbance on dilution), the situation can be that one has missed the range of linearity, as generally speaking linearity rarely or never exists over the entire range of concentrations; or that the matrix component is interfering with the measurement at the given dilution. Sometimes, matrix interference occurs if an interfering factor is present at concentrations above a certain threshold, and when the sample is diluted, interference is no longer observed. This kind of testing of a novel bioanalytical method is required by the EMA [16, 17].
When testing an experimental sample it is important to test several dilutions, all in duplicate or triplicate in conjunction with a known standard to ensure that the final results fall within the linear portion of the standard curve. This ensures the accuracy of the result. In highly concentrated samples underestimation of the concentration can occur, while in highly diluted samples overestimation can occur. Prepare different concentrations of the sample, keeping in mind the detection limit of the substrate. At this point, it is very suitable to detect maximal quantity of sample that can be detected, that is the last concentration after which there is no further absorbance increases (the same principle as for antigen coating optimization), Figure 1. This way the upper limit of the method is determined which enables the optimization of the next step.
At this point of optimisation, if sample is sera, high unspecific absorbance can occur, which is not related to the concentration of the sample/analyte. This can occur if the sera is not decomplemented, because active complement binds to antibody Fc. Heat-inactivation of serum for 30 minutes at 56°C eliminates complement activity, but one must keep in mind that different immunoglobulin isotypes and immunologbulins from different species show different sensitivity to heat treatment [18]. So, it is important to carefully consider or test the inactivation step.
ELISA is largely dependent on the choice of antibodies used, so antibodies should be carefully chosen. Based on the type of sample and the expected analyte concentration, the choice of monoclonal or polyclonal antibodies, or even the combination of both, should provide optimal signal-to-noise ratio [19]. Each antibody type offers distinct advantages.
The interaction between antibodies and their antigens is described by specificity, affinity, and avidity.
Specificity is an indication of whether an antibody binds solely to a unique epitope from a single antigen in a single species, or whether it binds to similar epitopes present on several molecules from the same or a few different species, i.e. whether it is cross-reactive. Specificity is the most important quality of an antibody, and this is the principle that ELISA is based upon, so a carefull selection should be made.
Affinity describes the strength of binding of an antibody with an antigen. This binding is a reversible interaction and affinity determines how much antigen is bound by an antibody at any particular moment, which is dependent upon how quickly this binding occurs, and for how long the interaction lasts. High affinity antibodies should be used in all types of immunoassay because they rapidly produce a large number of stable interactions and provide the most sensitive detection.
Avidity is a less intuitive term than affinity as it is based on affinity, but is highly influenced by the the total number of antigen binding sites or valency, which determines the overall stability of the antibody–antigen interaction. Therefore, avidity varies with antibody isotype and whether it is intact or fragmented. Additional factors which determines avidity are the structure of the antibody, the length and motility in the hinge region and the space between the Fab fragments.
When available, one should always choose monoclonal antibodies over polyclonal antibodies, in fact, commercial ELISA kits almost always utilize monoclonal antibodies. Monoclonal antibodies have specificity for a single epitope, usually a small part of the antigens’ surface. Monoclonal antibodies are therefore less likely to interact with closely-related proteins and are not generally expected to trigger non-specific signals in an immunoassay. Polyclonal antibodies are a mixture of antibodies with increased specificity to the antigen, therefore they bind different epitopes. Commercial polyclonal antibodies are often affinity purified or cross-adsorbed, but still the posibility of crossreactivity is higher. In addition, polyclonal antibody preparations can show batch to batch variations which should not be the case with monoclonal antibodies.
The advantage of using polyclonal antibodies is that they rarely fail to bind to the antigen due to a single blocked antibody binding site, antigen configuration change, or misfolding, although the latter are more important in tests other than ELISA. When combining monoclonal antibodies as in sandwich ELISA it is important to check literature or to test experimentaly the compatibility of the antibodies in terms that they do not share an epitope or for steric hinderance. Matched pairs are the basis of many sandwich ELISAs, either in kits or for in house assay set up. Matched antibody pairs means they are capable of detecting different epitopes on the same protein antigen, so they can be used together in a sandwich ELISA.
Sometimes the ELISA sensitivity can be increased by using indirect detection with polyclonal antibodies instead of direct detection with a monoclonal antibody, due to higher levels of polyclonal antibody binding to the target antigen. For cost reduction it can also be the combination of monoclonal capture with polyclonal detection.
After careful antibody selection, serial dilutions of capture antibodies should be carefully prepared for proper titration of antibody concentration. This is performed according to the previously mentioned principle of detecting maximum ammount of the component (in this case detection antibody) after which there is no further absorbance increase, Figure 1. Again, the ideal concentration should provide the highest signal and lowest noise.
As ELISA is a method which basicaly consists of overlaying different components which specificaly interact in each step (except washing) an optimization is required which follows the principle of titration until the complete coverage of the previous layer. Often the enzyme conjugate, i.e. enzyme responsible for color development, is already chemically bound to the detecting antibody, thereby enabling its direct use as a detection antibody in immunoassays. If this is not the case then enzyme concentration should be optimized too.
In this step, the first point is choosing the apropriate enzyme conjugate, depending on the needs of the researcher. The enzymes should be stable at typical assay temperatures: 4°C, 25°C, and 37°C; have a shelf life greater than six months when stored at 4°C; be inexpensive and commercially available. The enzymes should also survive the necessary conjugation conditions and yield productive conjugation. The enzymes should have an easily measurable activity; with high substrate turnover number. Horse radish peroxidase (HRP) and calf intestine alkalne phosphatase (ALP) are two most widely used enzymes for detection in ELISA assays [20]. HRP is usually conjugated to an antibody in a 4:1 ratio. For ALP the ratio is a little more unfavorable, 2:1, but the conjugate is more stable [21]. These enzymes are typically used because they each meet most, if not all, of the criteria necessary to produce a sensitive, inexpensive, and easily performed assay.
All enzyme-linked immunoassays, imply the usage of the enzyme substrate. Colorimetric ELISAs usually require soluble colored reaction products. The decision which substrate to choose depends on the desired sensitivity, reaction time, and the detection device. For colorimetric detection the most desirable substrates quickly produce intensely colored reaction products. When the analyte amounts span a wide range of concentrations (large dynamic range), then it is more suitable to use substrates that produce color over a longer time period (15 to 30 minutes) because then, one is able to detect the wider range of analyte-dependent color intensities. For assays with a timed endpoint, the reaction is stopped with an inhibitor suitable for the specific enzyme substrate combination after a defined time period that stops further color development. This allows detection to be performed within a reasonable time; for this, a substrate that has a “slow” reaction rate (15 to 30 minutes to completion) is optimal.
Both HRP and ALP have substrates that yield soluble colored reaction products.
The most common substrates that produce soluble reaction products with HRP are: TMB (3,3′,5,5′-Tetramethylbenzidine), ABTS (2,2′-azino-di[3-ethylbenzthiazoline] sulfonate), and OPD (o-phenylenediamine). TMB is a highly sensitive substrate, safe for laboratory workers. Due to its rapid reaction rate, it is ideally suited for on-line kinetic analysis. TMB can also be used in endpoint assays by stopping the reaction with 1 M phosphoric acid. ABTS is considered an all-purpose substrate. Although it is less sensitive than either TMB or OPD, it has the widest working range of any substrate currently available for peroxidase or alkaline phosphatase. Its reaction rate is suitable for endpoint assays and is easily stopped with 1% SDS (sodium dodecyl sulfate), which does not change the color or the absorbance of the reaction product. OPD was once the most popular substrate for peroxidase. It is slightly less sensitive than TMB, but it is cancerogenic.
The most commonly used substrate that produces a soluble reaction product with ALP is p-NPP (p-nitrophenylphosphate). pNPP is a substrate with a low reaction rate, so it usually takes 30 to 60 minutes for the dye to develop optimally. This property makes it possible to increase the sensitivity by increasing the reaction time period. At the same time, this property makes the pNPP substrate unsuitable for kinetic analysis [22].
Factors that affect the measurement of enzymatic activity are temperature, buffer composition (pH, ionic strength), build-up of product inhibitors, the increase in back-reaction as the product concentration increases, stability of the enzyme and sometimes exposure to light. As most of these facors such as pH and substrate depletion, are known, commercially available reagents are optimized for composition and concentration in order to control these parameters. For novel ELISA optimization of the most concern are reaction time and temperature.
If the antigen can clearly be detected then the substrate is appropriate. If the antigen is below the threshold for detection then one should select a more sensitive substrate.
It should be noted that the detection methodologies for ELISA are few, but the most prevalent in the laboratories is colorimetric. In addition, fluorescent and luminescent are also used.
In colorimetric detection the amount of color in each well is read by a spectrophotometer and samples are compared relative to one another or with the use of a standard curve derived from known analyte concentrations.
Fluorescent substrates [23] for ALP and HRP can potentially yield a higher signal, leading to increased sensitivity and broader dynamic range. This kind of detection requires black plates, which are also availiable with various degrees of hydrophobicity and a fluorescent plate reader is required. Fluorescence yielding substrates have a shorter half-life than colorimetric substrates, so the signal is declining over time. This kind of ELISA is useful for measuring immune responses because of broader dynamic range [19].
The same detection antibodies conjugated with ALP or HRP, can also be used for chemiluminescent assays [24]. In this type of experiment, ALP, for example, will modify a substrate, forming a chemiluminiscent product which creates light emission. ALP chemiluminiscent substrates can have pg/ml sensitivity. The signal can be read in black or white opaque ELISA plates and a luminometer is required. The advantages of this detection type are typically a higher dynamic range and lower background signal. The signal is not as stable as the colorimetric or fluorescent detection and must be read within a short time of generating the signal.
The type of substrate used depends on several factors, most notably the desired assay sensitivity and signal to bakground ratio.
Many laboratories have independently developed ELISA techniques for their own purposes. For results to be valid they must be comparable with results of the same ELISA test performed in different laboratories. Consistency in the assessment of ELISA results in different areas of application (diagnostics, production control, scientific research, immunogenicity assessment etc.) requires standardized and acknowledged methodological protocols. Protocol harmonization progress with respect to the international standardization and validation of this technique has been made.
Today, leading regulatory agencies for specific guidance on immunogenicity assessment of biotherapeutic products are part of EMA and WHO, [25] and there are other agencies. The National Institute for Biological Standards and Control (NIBSC), for example, part of UK Medicines and Healthcare products Regulatory Agency (MHRA), is of great importance to the field of biological standardization. It produces over 90% of the biological international standards in use around the world. The WHOs’ Biological Reference Materials are established through a standard procedure, [26] in which representative materials are tested by participating laboratories using their own methodologies and coordinated by a responsible WHO Collaborating Center [27]. Upon establishment of the reference preparation by the Expert Committee on Biological Standardization (ECBS), the material is assigned a unitage and serves as the comparator against which results from laboratories can be standardized and compared, irrespective of the location or the methods employed. This enables the results of bioanalytical methods, including ELISA, to be comparable. Based on international standards „ working standard” (i.e. in-house or secondary standards) are evaluated and compared, and subsequently adequately used.
At first glance, it is very simple to explain the process, i.e. the term of standardization in ELISA: comparing the absorbance of a sample with the absorbance of the known concentration of the standard (in-house or commercial) and based on that, determining the unknown concentration.
If the ELISA is intended for the measurement of the final detectable dilution, as in titration experiments, and not for the measurement of biomolecule quantity a reference standard may not exist.
Then the need exists for establishing a reference standard. For any ELISA, consideration must be given to the selection of standards which represent, on average, what would be expected of an immune response of the organism in question. Immunogenicity assessment relies on the measurement of antigen induced antibodies in serum or plasma. Such antibodies are heterogeneous in terms of classes, subclasses and alotypes, concentration as well as antigenic specificity. Some will neutralize the biological activity of the antigen, others will not, despite the high affinity/avidity. Irrespective of the type of ELISA system used, endpoint titration is a function of both antibody concentration and avidity. And finally, as every sample is unique with vast individual differences among humans, for example, it is not possible to make a straightforward comparison with standard antibodies. Nevertheless, although the ideal is unreachable, if wanting to produce valid and reproducible data a reference standard must be established.
The physical quantity to be measured in ELISA is absorbance. Absorbance is influenced by test parameters and photometric instrumentation, so raw, corrected or normalized OD values [28] cannot be used for inter-laboratory standardization. This is why end-point titration or determination of highest serial dilution which demonstrates a minimum of antibody activity is often used for measuring the immune response in diagnostics and vaccinology. Under some circumstances, quantitative data are not required for diagnostic purposes and sometimes end-point titration is sufficient, with an adequate semi-quantitative standard. End-point titrations are labor-intensive, costly and impractical for most routine diagnostic purposes.
In order to overcome the relativity of the measured absorbance a notion of “percent positivity” (PP) is accepted, this way the absorbance of each sample tested is expressed as a percentage of a highly positive reference standard. Although semi-quantitative, PP is expressed on a continuous scale of 0–100 and has two major advantages, first, it requires only a single dilution and second, it does not assume parallelism or uniform background activity. Therefore, it may be used for inter-laboratory standardization.
Even with measurements with qualitative standard curve, it is not correct to determine the result from a single sample dilution measurement. This can only be acceptable if there is a parallelism in dilution curves between the sample and the standard. If more quantitative data are needed, PP values can be converted to units which are directly proportional to antibody activity.
Sometimes an elegant and appropriate way to quantify samples is competitive or inhibitory ELISA. When performing competitive ELISA, one applies the sample preincubated with the same antigen used for plate coating and measures the amount of non inhibited antibodies. There is a negative relationship between color intensity and the amount of test sample antibody inhibited by antigens. Percent inhibition (PI) of the color produced by the standard competing antibody is more widely used. The development of consistent standard curves for this kind of assay is extremely difficult, but still possible.
The specific guidance on immunogenicity assessment of biotherapeutic products has been elaborated by leading regulatory agencies such as the EMA and U.S. Food and Drug Administration (US FDA) [29, 30, 31, 32].
Validated analytical methods such as ELISA for quantification of biomarkers, drugs, biological products, and their metabolites in a given biological matrix (e.g. blood, plasma, serum, or urine) are critical for the successful conduct of nonclinical and clinical studies. Validating the analytical method ensures that the data are reliable [33]. Validated methods provide critical data to support the safety and effectiveness of drugs and biological products.
Although there is abundant literature relating to immunochemical methods, [34] EMEA [35, 36] and US FDA [8] have clearly defined the characteristics of the validation procedure for bioanalytical methods, which also applies to the validation of ELISAs, which are intended for use in diagnostics, toxicology, basic or applied research [37] or production control [38]. Metodology for the validation of bioanalytical methods must follow clear recomendations from reference institutions such as the EMEA [35, 39] or the WHO because that provides important measurements to be of satisfactory quality all over the world.
ELISA validation according to these recommendations means determining the following method caracteristics:
Specificity
Linearity –
Sensitivity
Accuracy
Precision (repetability = intra assay, inter assay, reproducibility = inter laboratory assay)
Robustnes
Acceptance criteria should be prospectively defined based on the intended use of the method.
Specificity means that the method must differentiate the targeted analyte from all other matrix components. Which is why it is important to test wether
Evaluation of specificity may be conducted during optimization and validation, when more data on the behavior of the analyte become available. Specificity should be tested with quality control (QC) samples [40]. QC samples are the samples with known amounts of the analyte, in identical matrix like the sample. These are usually in-house produced samples, with a lower amount of the analyte. When the method is performed with these QC samples and satisfactory results are obtained, then the method is also good, i.e. valid. If the method does not give good enough results with the QC samples, it means that the method is not of sufficient quality, so it must be investigated why the method worked poorly. The shortcomings must be corrected, and then again checked with QC samples. Still it needs to be defined what is satisfactory. The criterion for accepting the results obtained with QC samples is that the measured value does not deviate by more than 25% from the nominal value [40].
Linearity is the ability of the analytical method to produce results by calculating a direct proportion, within the working range. Linearity is described by range and detection limits.
Linearity is a function of values that can be graphically represented by a straight line. The linearity of an analytical method can be explained as its capability to show “results that are directly proportional to the concentration of the analyte in the sample” [39].
Unfortunately, the analytical response of a method is not always linear. Sometimes when the data are not linear they can be mathematically transformed, e.g. by applying logarithms but in some cases or some range of immunoassays transformation is not appropriate.
Linearity is important as it confirms the sensitivity of the method for the analysis of concentration within a defined range. According to the EMEA International Council for Harmonization ICH Q2(R1) guideline, linearity of a given response must be evaluated using a minimum of 5 concentrations of the analyte (multi-point calibration). Then, the collected data must be statistically analyzed, by performing regression analysis using the method of the least squares, in order to mathematically determine the line that best fits a set of data. For linearity, the results are required to be represented as linear equation (Eq. (1)).
In a linear regression line, the regression coefficient is the constant “k” that represents the rate of change of one variable “y” as a function of change in the other “x” (thus the slope), while “n” is the Y-intercept. The correlation coefficient r, a value without units, expresses the precision of the linearity fit of the experimental data. In case of a value being less than 0.95, it may either be a result of a broad spreading during measurement or due to a non-linear correlation. Often, the coefficient of determination (R2) is used, which is the square r. For most methods applied at R2 ≥ 0.98 can be achieved. If there is a perfect linear relationship, it has a value of 1 (100%). Linearity studies are important because they define the range of the method within which the results are obtained accurately and precisely.
To summarize, linearity is one major aspect in the quantitative method validation procedures. It describes the range of concentrations for which the method can function reliably. If the data are non-linear, transformation into a linear form may be performed, or the data can be accepted as is while demonstrating a clear relation between the analyte concentration and the measured absorbance [41].
Senzitivity or limit of detection, (LD) for ELISA is defined in the same way as for other bionalaytical methods. At this point, it is appropriate to underline the difference between the limit of detection (LD) and lower limit of quantification nominal (LLOQ). LD is the lowest analyte concentration that can be distinguished from the assay background, while the LLOQ is the lowest concentration at which the analyte can be quantitated at defined levels for precision and accuracy. LD is determined from standard deviation of the sample blank and the slope of the linear curve (Eq. (2)).
LD—LD (detection limit) nominal
k—slope of the linear curve Eq. (1)
SD(b)—standard deviation of the blank [39]
There are bioanalytical methods which have the same values for LD and LLOQ, but with ELISA, especially when biologycal samples are measure this is not the case, and LD is lower than LLOQ. For liretature reference of these terms one should read Armbruster and Pry [42].
The accuracy of an analytical method describes the closeness of the value determined by the method to the nominal concentration. In practice, as the reference material is precious and universally needed, the first step is to make a sufficient amount of the quality control (QC) samples, previously standardized against the reference material. Then the QC sample can be used for determining validation characteristics. Accuracy should be assessed on samples spiked with known amounts of the analyte, the QC samples. The accuracy can be expressed as the difference between the obtained experimnental value and the nominal value (which is acurate), using the absolute or even better the relative error.
Δxi—absolute error of individual measurement
μ— nominal value
xi—measured value
It is important to perform multiple measurements for a single sample, in order to present the absolute error as the mean value of absolute errors of individual measurements (Eq. (4)).
n—number of measurements
Δx—mean value of absolute or standard error
Because of the numerical nature, the absolute value of the difference does not give insight into its significance for the accuracy of measurement, so it is always important to calculate the relative error as well.
The level of accuracy must be determined for the whole range of the analytical procedure. Minimal requirements for this are three concentrations one close to ULOQ, one close to LLOQ and one in the middle of the range, each in triplicate.
Today it is common practise to develop an ELISA as an internal laboratory assay without the standards or the QC samples or for titration experiments for the determination of the last measurable dilution. In this situation there is no measurable quantitifier for accuracy testing. For accuracy to be calculated as % that shows how much the obtained results corresponds with the actual value, it is necessary to use concrete, absolute and measurable quantity such as analyte concentration. In practise this can be achieved [43] with inhibitory ELISA, which is based on the dependance of the absorbance on inhibitor concentration. The difference between the described calculations is in the reverse proportion, as described in the ELISA standardization section [37].
Precision is a validation characteristic which describes the reproducibility of the measurement, in other words the closeness of two measurements of the same sample. Precision is higher if the results are closer to one another. At first glance it is easy to confuse accuracy with precision, because in both cases it is about the absolute and the relative error of the obtained results. Figure 2 shows the difference between accuracy and precicion, where accuracy describes the deviation from the actual (nominal) value, while precision describes the deviation from the mean value. Precision is determined by simply repeating the measurement.
Accuracy and precision defined.
Standard deviation, relative standard deviation (coefficient of variation) and confidence interval should be reported for each type of precision (intra, intermediate or inter) investigated [35]. The three parameters are dependent on the closeness of individual results to the mean value, and give the complete picture of the precision of the test.
DEVIATION is the difference between the measured value from the mean value, and has the same units as the measured value (Eq. (6)).
x̅—mean value of repeated measurements of the same sample
xi—one measured value
di—deviation, the difference between the mean value and one measurement
Standard deviation is the mean value of all measurement deviations Eq. (7).
COEFFICIENT OF VARIATION CV (relative standard deviation) is standard deviation expressed in percentages and is calculated based on the measured mean value x̅ (Eq. (8)).
CONFIDENCE INTERVAL (CI) is the range of values within which the “actual” result is found. A CI of 95% means that if the measurement was to be repeated an infinite number of times, 95% of the results would fall within this range of values. For validation purpose, higher CI, 95% or 99% is needed, with optimal performance within the middle part of the range. A wide CI can be caused by small number of samples or by a large variance between sample measurements. Range of values for the given CI shows precision. This parameter is easily calculated by statistical programmes, or by a profesional statistician.
Intra-assay validation shows the reproducibility between wells within an assay plate. Data resulting from intra-assay validation helps ensure that repeated measurement of the same sample on a single plate gives comparable results. Repeatability should be assessed using a minimum of 6 determinations covering the specified range for the procedure (e.g. 3 concentrations, 2 replicates each), or a minimum of 6 determinations at 100% of the test concentration [39].
The % CV for each sample is calculated by finding the standard deviation of multiplicate results dividing that by the multiplicate mean, and multiplying the result by 100 (Eq. (8)). The average of the individual CVs is reported as the intra-assay CV (CVintra-assay).
Usually, CV intra-assay of 10% or less is considered satisfactory [44].
Intermediate precision (sometimes called within-lab reproducibility) shows the reproducibility between assays done on different days, or different plates. Satisfactory inter-assay precision is typically <10% [44].
For example, to monitor plate-to-plate variation the same samples are analyzed in quadruplicate on ten different plates. The plate means are calculated and then used to calculate the overall mean, standard deviation, and % CV. Overall % CV is calculated by dividing the SD of the plate means with mean of the plate means and multiplying by 100 (Eq. (8)). The average of the all plates % CV represents the inter-assay CV (CVintermediate). In order to monitor daily variation quadruplicate samples are analyzed in ten different days and analyzed in the same way.
Reproducibility is assessed by means of an inter-laboratory trial. The outcome of the cross validation is critical in determining whether the obtained data are reliable and whether they can be compared and used. Reproducibility should be considered in case of the standardization of an analytical procedure, for instance, for inclusion of procedures in pharmacopeias.
Satisfactory value for CVinter-assay is 10–15% [43].
Analyzing the literature it can be seen that the term inter assay is sometimes used for precision assessment on different days or on different plates, and sometimes for testing in different laboratories. Acording to EMEA, the term inter assay precision describes precision of the measurement assessment in different laboratories. If it is to be used in a different context it shold be described.
Robustness testing involves monitoring the effects of small unintentional errors on the quantitative and qualitative characteristics of the method, where the errors relate to the internal parameters described in the method prescription. For example, buffer temperature, incubation temperature, sample incubation time, secondary antibody incubation time, number of washes before color development, color development time, and the like. This feature shows the reliability of the method despite minor deviations in performance.
There is also the notion of rigidity - as a sub-notion of robustness - which monitors the effects of changes in external parameters such as other lots of chemicals, other people working, other instruments used and the like.
Practically, this property is not measured or calculated in a certain way, but is established during the development of the method (optimization). Data on this can also be collected during operation.
This guideline describes full validation methodology. In case when method is already validated, when a smaller change to the protocol is instated, a full validation may not be necessary. It is possible to perform partial validation, and the nature of the modification will determine the extent of validation required. All modifications should be reported and the scope of revalidation or partial validation justified [34].
In our experience ELISA is an excellent analytical method which can be used for the detection and quantification of numerous biomolecules. No matter what this specific biomolecule is, the basis of ELISA is the antigen–antibody interaction. The existence of this specific interaction usually enables the construction of different ELISA protocols, dependent on your prior knowledge and imagination. After careful protocol optimization, determination of validation characteristics and the acquirement of an appropriate standard you can get a reliable and inexpensive analytical method useful in diagnostics, research or biomedicine in general.
Work presented in this paper was supported by the Institute of Virology, Vaccines and Sera “Torlak” funds and by Ministry of Education, Science and Technological Development, Republic of Serbia.
The authors declare that they have no known competing financial interest or personal relationship that could have appeared to influence the work reported in this paper.
IntechOpen publishes different types of publications
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Shohel"}],equalEditorOne:null,equalEditorTwo:null,equalEditorThree:null,productType:{id:"1",chapterContentType:"chapter",authoredCaption:"Edited by"}}]},subject:{topic:{id:"974",title:"Signal Processing",slug:"applied-mathematics-signal-processing",parent:{id:"163",title:"Applied Mathematics",slug:"applied-mathematics"},numberOfBooks:2,numberOfSeries:0,numberOfAuthorsAndEditors:68,numberOfWosCitations:36,numberOfCrossrefCitations:33,numberOfDimensionsCitations:62,videoUrl:null,fallbackUrl:null,description:null},booksByTopicFilter:{topicId:"974",sort:"-publishedDate",limit:12,offset:0},booksByTopicCollection:[{type:"book",id:"10065",title:"Wavelet Theory",subtitle:null,isOpenForSubmission:!1,hash:"d8868e332169597ba2182d9b004d60de",slug:"wavelet-theory",bookSignature:"Somayeh Mohammady",coverURL:"https://cdn.intechopen.com/books/images_new/10065.jpg",editedByType:"Edited by",editors:[{id:"109280",title:"Dr.",name:"Somayeh",middleName:null,surname:"Mohammady",slug:"somayeh-mohammady",fullName:"Somayeh Mohammady"}],equalEditorOne:null,equalEditorTwo:null,equalEditorThree:null,productType:{id:"1",chapterContentType:"chapter",authoredCaption:"Edited by"}},{type:"book",id:"5411",title:"Fourier Transforms",subtitle:"High-tech Application and Current Trends",isOpenForSubmission:!1,hash:"5c45d1a91daef66093a42a82448a70f0",slug:"fourier-transforms-high-tech-application-and-current-trends",bookSignature:"Goran S. 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The hydration processes of biopolymers have been extensively studied in the past 20 years with reference to a considerable variety of models and concepts. In all recent works, a distinction is made between intracellular water that maintains the ordinary liquid state (bulk water) and water ordered in extended hydrogen‐bonded lattices at the surface and structured in the internal grooves of macromolecules (hydration water) in dependence on the chemical properties of the macromolecule surface. FTIR spectroscopy has been implemented in this field both for the sensitivity in the conformational analysis of biological macromolecules and the reliability in the investigation of the water network. A perturbation technique such as dehydration‐rehydration treatment modifies the macromolecule structure and water distribution. It was applied to two structurally different proteins: lysozyme, a globular (α + β) protein and collagen, a fibrous protein characterized by the triple helix structure. Submitted to the treatment both of them display irreversible conformational changes.",book:{id:"5411",slug:"fourier-transforms-high-tech-application-and-current-trends",title:"Fourier Transforms",fullTitle:"Fourier Transforms - High-tech Application and Current Trends"},signatures:"Maria Grazia Bridelli",authors:[{id:"108760",title:"Dr.",name:"Maria Grazia",middleName:null,surname:"Bridelli",slug:"maria-grazia-bridelli",fullName:"Maria Grazia Bridelli"}]},{id:"74096",doi:"10.5772/intechopen.94521",title:"Time Frequency Analysis of Wavelet and Fourier Transform",slug:"time-frequency-analysis-of-wavelet-and-fourier-transform",totalDownloads:1162,totalCrossrefCites:6,totalDimensionsCites:8,abstract:"Signal processing has long been dominated by the Fourier transform. However, there is an alternate transform that has gained popularity recently and that is the wavelet transform. The wavelet transform has a long history starting in 1910 when Alfred Haar created it as an alternative to the Fourier transform. In 1940 Norman Ricker created the first continuous wavelet and proposed the term wavelet. Work in the field has proceeded in fits and starts across many different disciplines, until the 1990’s when the discrete wavelet transform was developed by Ingrid Daubechies. While the Fourier transform creates a representation of the signal in the frequency domain, the wavelet transform creates a representation of the signal in both the time and frequency domain, thereby allowing efficient access of localized information about the signal.",book:{id:"10065",slug:"wavelet-theory",title:"Wavelet Theory",fullTitle:"Wavelet Theory"},signatures:"Karlton Wirsing",authors:[{id:"325178",title:"Dr.",name:"Karlton",middleName:null,surname:"Wirsing",slug:"karlton-wirsing",fullName:"Karlton Wirsing"}]},{id:"52810",doi:"10.5772/65776",title:"Study of Green Nanoparticles and Biocomplexes Based on Exopolysaccharide by Modern Fourier Transform Spectroscopy",slug:"study-of-green-nanoparticles-and-biocomplexes-based-on-exopolysaccharide-by-modern-fourier-transform",totalDownloads:2e3,totalCrossrefCites:2,totalDimensionsCites:6,abstract:"The intention of this chapter is to contribute in clarification of nanoparticle synthesis and biocomplexes based on exopolysaccharide, green synthetic method development, their physico‐chemical characterization by modern spectroscopy, as well as testing of their antimicrobial activity. Silver nanoparticles of polysaccharide type have scientific interest, but practical importance too, because of their application in pharmaceutical and cosmetic product development due to proven antimicrobial and antioxidant activities. On the other hand, the biocomplexes based on exopolysaccharides are important in treatment of biometal deficiency in human and veterinary medicine, as well as in metal ion transporting in organism. Despite a number of studies of this kind of complexes, the investigations of effect of their structure to pharmaco‐biological activity are still interesting. It is important that question of interaction between reducing and stabilizing agents with metal ions is still opened. In this respect, the presented chapter offers further progress in the examination of silver nanoparticles and cobalt biocomplex synthesis with dextran oligosaccharides and its derivatives (such as dextran sulfate and carboxymethyl dextran). The complex structure, spectroscopic characterization, and the spectra‐structure correlation have been analyzed by different Fourier transform infrared (FTIR) spectroscopic techniques combined with energy‐dispersive X‐ray (EDX), X‐ray diffraction (XRD), scanning electron microscopy (SEM), and surface plasmon resonance UV‐Vis methods.",book:{id:"5411",slug:"fourier-transforms-high-tech-application-and-current-trends",title:"Fourier Transforms",fullTitle:"Fourier Transforms - High-tech Application and Current Trends"},signatures:"Goran S. Nikolić, Milorad D. Cakić, Slobodan Glišić, Dragan J.\nCvetković, Žarko J. Mitić and Dragana Z. Marković",authors:[{id:"23261",title:"Prof.",name:"Goran",middleName:"S.",surname:"Nikolic",slug:"goran-nikolic",fullName:"Goran Nikolic"},{id:"195519",title:"Dr.",name:"Milorad",middleName:null,surname:"Cakic",slug:"milorad-cakic",fullName:"Milorad Cakic"},{id:"195520",title:"MSc.",name:"Slobodan",middleName:null,surname:"Glišić",slug:"slobodan-glisic",fullName:"Slobodan Glišić"},{id:"195521",title:"Prof.",name:"Dragan",middleName:"J.",surname:"Cvetkovic",slug:"dragan-cvetkovic",fullName:"Dragan Cvetkovic"},{id:"195522",title:"Dr.",name:"Žarko",middleName:null,surname:"Mitić",slug:"zarko-mitic",fullName:"Žarko Mitić"},{id:"195523",title:"MSc.",name:"Dragana",middleName:null,surname:"Marković-Nikolić",slug:"dragana-markovic-nikolic",fullName:"Dragana Marković-Nikolić"}]},{id:"53388",doi:"10.5772/66107",title:"Fourier Transform Hyperspectral Imaging for Cultural Heritage",slug:"fourier-transform-hyperspectral-imaging-for-cultural-heritage",totalDownloads:1772,totalCrossrefCites:1,totalDimensionsCites:6,abstract:"Hyperspectral imaging is a technique of analysis that associates to each pixel of the image the spectral content of the radiation coming from the scene. This content can be helpful to recognize the chemical nature of the materials within the scene or to calculate their colours under particular conditions. Different solutions of hyperspectral imager have been realized with different spatial resolution, spectral resolution and range in the electromagnetic spectrum. In particular, improving the spectral resolution allows discriminating smaller features in the spectrum and the unambiguous detection of the absorption bands characteristic of superficial materials. Hyperspectral imagers based on interferometers have the advantage of having a spectral resolution that can be varied according to the needs by changing the optical path delay of the interferometer. A spectrum for each pixel is obtained with an algorithm based on the Fourier transform of the calibrated interferogram. We present the results of the application of a hyperspectral imager based on Fabry‐Perot interferometers to the field of cultural heritage. On different artworks, the hyperspectral imager has been used for pigment recognition, for colour rendering elaborations of the image with different light sources or standard illuminants and for calculating the chromatic coordinates useful for specific purposes.",book:{id:"5411",slug:"fourier-transforms-high-tech-application-and-current-trends",title:"Fourier Transforms",fullTitle:"Fourier Transforms - High-tech Application and Current Trends"},signatures:"Massimo Zucco, Marco Pisani and Tiziana Cavaleri",authors:[{id:"20909",title:"Dr.",name:"Marco Q.",middleName:null,surname:"Pisani",slug:"marco-q.-pisani",fullName:"Marco Q. Pisani"},{id:"20910",title:"Dr.",name:"Massimo E.",middleName:null,surname:"Zucco",slug:"massimo-e.-zucco",fullName:"Massimo E. Zucco"},{id:"194761",title:"Dr.",name:"Tiziana",middleName:null,surname:"Cavaleri",slug:"tiziana-cavaleri",fullName:"Tiziana Cavaleri"}]},{id:"53524",doi:"10.5772/66733",title:"Fourier Analysis for Harmonic Signals in Electrical Power Systems",slug:"fourier-analysis-for-harmonic-signals-in-electrical-power-systems",totalDownloads:4493,totalCrossrefCites:3,totalDimensionsCites:4,abstract:"The harmonic content in electrical power systems is an increasingly worrying issue since the proliferation of nonlinear loads results in power quality problems as the harmonics is more apparent. In this paper, we analyze the behavior of the harmonics in the electrical power systems such as cables, transmission lines, capacitors, transformers, and rotating machines, the induction machine being the object of our study when it is excited to nonsinusoidal operating conditions in the stator winding. For this, a model is proposed for the harmonic analysis of the induction machine in steady‐state regimen applying the Fourier transform. The results of the proposed model are validated by experimental tests which gave good results for each case study concluding in a model proper for harmonic and nonharmonic analysis of the induction machine and for “harmonic” analysis in an electrical power system.",book:{id:"5411",slug:"fourier-transforms-high-tech-application-and-current-trends",title:"Fourier Transforms",fullTitle:"Fourier Transforms - High-tech Application and Current Trends"},signatures:"Emmanuel Hernández Mayoral, Miguel Angel Hernández López,\nEdwin Román Hernández, Hugo Jorge Cortina Marrero, José\nRafael Dorrego Portela and Victor Ivan Moreno Oliva",authors:[{id:"187793",title:"Dr.",name:"Emmanuel",middleName:null,surname:"Hernández",slug:"emmanuel-hernandez",fullName:"Emmanuel Hernández"},{id:"202757",title:"Dr.",name:"Miguel Angel",middleName:null,surname:"Hernández López",slug:"miguel-angel-hernandez-lopez",fullName:"Miguel Angel Hernández López"},{id:"202758",title:"Dr.",name:"Hugo Jorge",middleName:null,surname:"Cortina Marrero",slug:"hugo-jorge-cortina-marrero",fullName:"Hugo Jorge Cortina Marrero"},{id:"202759",title:"Dr.",name:"Edwin Román",middleName:null,surname:"Hernández",slug:"edwin-roman-hernandez",fullName:"Edwin Román Hernández"},{id:"202760",title:"Dr.",name:"Victor Iván Moreno",middleName:null,surname:"Oliva",slug:"victor-ivan-moreno-oliva",fullName:"Victor Iván Moreno Oliva"},{id:"202761",title:"Dr.",name:"José Rafael Dorrego",middleName:null,surname:"Portela",slug:"jose-rafael-dorrego-portela",fullName:"José Rafael Dorrego Portela"}]}],mostDownloadedChaptersLast30Days:[{id:"74096",title:"Time Frequency Analysis of Wavelet and Fourier Transform",slug:"time-frequency-analysis-of-wavelet-and-fourier-transform",totalDownloads:1162,totalCrossrefCites:6,totalDimensionsCites:8,abstract:"Signal processing has long been dominated by the Fourier transform. However, there is an alternate transform that has gained popularity recently and that is the wavelet transform. The wavelet transform has a long history starting in 1910 when Alfred Haar created it as an alternative to the Fourier transform. In 1940 Norman Ricker created the first continuous wavelet and proposed the term wavelet. Work in the field has proceeded in fits and starts across many different disciplines, until the 1990’s when the discrete wavelet transform was developed by Ingrid Daubechies. While the Fourier transform creates a representation of the signal in the frequency domain, the wavelet transform creates a representation of the signal in both the time and frequency domain, thereby allowing efficient access of localized information about the signal.",book:{id:"10065",slug:"wavelet-theory",title:"Wavelet Theory",fullTitle:"Wavelet Theory"},signatures:"Karlton Wirsing",authors:[{id:"325178",title:"Dr.",name:"Karlton",middleName:null,surname:"Wirsing",slug:"karlton-wirsing",fullName:"Karlton Wirsing"}]},{id:"74032",title:"Wavelets for EEG Analysis",slug:"wavelets-for-eeg-analysis",totalDownloads:1157,totalCrossrefCites:2,totalDimensionsCites:2,abstract:"This chapter introduces the applications of wavelet for Electroencephalogram (EEG) signal analysis. First, the overview of EEG signal is discussed to the recording of raw EEG and widely used frequency bands in EEG studies. The chapter then progresses to discuss the common artefacts that contaminate EEG signal while recording. With a short overview of wavelet analysis techniques, namely; Continues Wavelet Transform (CWT), Discrete Wavelet Transform (DWT), and Wavelet Packet Decomposition (WPD), the chapter demonstrates the richness of CWT over conventional time-frequency analysis technique e.g. Short-Time Fourier Transform. Lastly, artefact removal algorithms based on Independent Component Analysis (ICA) and wavelet are discussed and a comparative analysis is demonstrated. The techniques covered in this chapter show that wavelet analysis is well-suited for EEG signals for describing time-localised event. Due to similar nature, wavelet analysis is also suitable for other biomedical signals such as Electrocardiogram and Electromyogram.",book:{id:"10065",slug:"wavelet-theory",title:"Wavelet Theory",fullTitle:"Wavelet Theory"},signatures:"Nikesh Bajaj",authors:[{id:"326400",title:"Dr.",name:"Nikesh",middleName:null,surname:"Bajaj",slug:"nikesh-bajaj",fullName:"Nikesh Bajaj"}]},{id:"53524",title:"Fourier Analysis for Harmonic Signals in Electrical Power Systems",slug:"fourier-analysis-for-harmonic-signals-in-electrical-power-systems",totalDownloads:4493,totalCrossrefCites:3,totalDimensionsCites:4,abstract:"The harmonic content in electrical power systems is an increasingly worrying issue since the proliferation of nonlinear loads results in power quality problems as the harmonics is more apparent. In this paper, we analyze the behavior of the harmonics in the electrical power systems such as cables, transmission lines, capacitors, transformers, and rotating machines, the induction machine being the object of our study when it is excited to nonsinusoidal operating conditions in the stator winding. For this, a model is proposed for the harmonic analysis of the induction machine in steady‐state regimen applying the Fourier transform. The results of the proposed model are validated by experimental tests which gave good results for each case study concluding in a model proper for harmonic and nonharmonic analysis of the induction machine and for “harmonic” analysis in an electrical power system.",book:{id:"5411",slug:"fourier-transforms-high-tech-application-and-current-trends",title:"Fourier Transforms",fullTitle:"Fourier Transforms - High-tech Application and Current Trends"},signatures:"Emmanuel Hernández Mayoral, Miguel Angel Hernández López,\nEdwin Román Hernández, Hugo Jorge Cortina Marrero, José\nRafael Dorrego Portela and Victor Ivan Moreno Oliva",authors:[{id:"187793",title:"Dr.",name:"Emmanuel",middleName:null,surname:"Hernández",slug:"emmanuel-hernandez",fullName:"Emmanuel Hernández"},{id:"202757",title:"Dr.",name:"Miguel Angel",middleName:null,surname:"Hernández López",slug:"miguel-angel-hernandez-lopez",fullName:"Miguel Angel Hernández López"},{id:"202758",title:"Dr.",name:"Hugo Jorge",middleName:null,surname:"Cortina Marrero",slug:"hugo-jorge-cortina-marrero",fullName:"Hugo Jorge Cortina Marrero"},{id:"202759",title:"Dr.",name:"Edwin Román",middleName:null,surname:"Hernández",slug:"edwin-roman-hernandez",fullName:"Edwin Román Hernández"},{id:"202760",title:"Dr.",name:"Victor Iván Moreno",middleName:null,surname:"Oliva",slug:"victor-ivan-moreno-oliva",fullName:"Victor Iván Moreno Oliva"},{id:"202761",title:"Dr.",name:"José Rafael Dorrego",middleName:null,surname:"Portela",slug:"jose-rafael-dorrego-portela",fullName:"José Rafael Dorrego Portela"}]},{id:"53366",title:"New Spectral Applications of the Fourier Transforms in Medicine, Biological and Biomedical Fields",slug:"new-spectral-applications-of-the-fourier-transforms-in-medicine-biological-and-biomedical-fields",totalDownloads:2340,totalCrossrefCites:1,totalDimensionsCites:1,abstract:"This chapter reviews some recent spectral applications of the Fourier transform techniques as they are applied in spectroscopy. An overview about Fourier transform spectroscopy (FTS) used like a powerful and sensitive tool in medical, biological, and biomedical analysis is provided. The advanced spectroscopic techniques of FTS, such as Fourier transform visible spectroscopy (FTVS), Fourier transform infrared-attenuated total reflectance (FTIR-ATR), Fourier transform infrared-photoacoustic spectroscopy (FTIR-PAS), Fourier transform infrared imaging spectroscopy (FTIR imaging), and their biomedical applications are described. A special attention has been paid to the description of the FTVS method of commercial quantum dots like an innovative and reliable technique used in the field of nanobiotechnology.",book:{id:"5411",slug:"fourier-transforms-high-tech-application-and-current-trends",title:"Fourier Transforms",fullTitle:"Fourier Transforms - High-tech Application and Current Trends"},signatures:"Anca Armăşelu",authors:[{id:"189080",title:"Dr.",name:"Anca",middleName:null,surname:"Armăşelu",slug:"anca-armaselu",fullName:"Anca Armăşelu"}]},{id:"53419",title:"Fourier Transform Infrared Spectroscopy in the Study of Hydrated Biological Macromolecules",slug:"fourier-transform-infrared-spectroscopy-in-the-study-of-hydrated-biological-macromolecules",totalDownloads:2442,totalCrossrefCites:3,totalDimensionsCites:15,abstract:"The interaction between biological macromolecules (proteins, nucleic acids, lipids and other biomolecules in the cell) and environmental water is an important determining factor in their conformational properties, stability and function. The hydration processes of biopolymers have been extensively studied in the past 20 years with reference to a considerable variety of models and concepts. In all recent works, a distinction is made between intracellular water that maintains the ordinary liquid state (bulk water) and water ordered in extended hydrogen‐bonded lattices at the surface and structured in the internal grooves of macromolecules (hydration water) in dependence on the chemical properties of the macromolecule surface. FTIR spectroscopy has been implemented in this field both for the sensitivity in the conformational analysis of biological macromolecules and the reliability in the investigation of the water network. A perturbation technique such as dehydration‐rehydration treatment modifies the macromolecule structure and water distribution. It was applied to two structurally different proteins: lysozyme, a globular (α + β) protein and collagen, a fibrous protein characterized by the triple helix structure. Submitted to the treatment both of them display irreversible conformational changes.",book:{id:"5411",slug:"fourier-transforms-high-tech-application-and-current-trends",title:"Fourier Transforms",fullTitle:"Fourier Transforms - High-tech Application and Current Trends"},signatures:"Maria Grazia Bridelli",authors:[{id:"108760",title:"Dr.",name:"Maria Grazia",middleName:null,surname:"Bridelli",slug:"maria-grazia-bridelli",fullName:"Maria Grazia Bridelli"}]}],onlineFirstChaptersFilter:{topicId:"974",limit:6,offset:0},onlineFirstChaptersCollection:[],onlineFirstChaptersTotal:0},preDownload:{success:null,errors:{}},subscriptionForm:{success:null,errors:{}},aboutIntechopen:{},privacyPolicy:{},peerReviewing:{},howOpenAccessPublishingWithIntechopenWorks:{},sponsorshipBooks:{sponsorshipBooks:[],offset:8,limit:8,total:0},allSeries:{pteSeriesList:[{id:"14",title:"Artificial Intelligence",numberOfPublishedBooks:8,numberOfPublishedChapters:87,numberOfOpenTopics:6,numberOfUpcomingTopics:0,issn:"2633-1403",doi:"10.5772/intechopen.79920",isOpenForSubmission:!0},{id:"7",title:"Biomedical Engineering",numberOfPublishedBooks:12,numberOfPublishedChapters:98,numberOfOpenTopics:3,numberOfUpcomingTopics:0,issn:"2631-5343",doi:"10.5772/intechopen.71985",isOpenForSubmission:!0}],lsSeriesList:[{id:"11",title:"Biochemistry",numberOfPublishedBooks:27,numberOfPublishedChapters:286,numberOfOpenTopics:4,numberOfUpcomingTopics:0,issn:"2632-0983",doi:"10.5772/intechopen.72877",isOpenForSubmission:!0},{id:"25",title:"Environmental Sciences",numberOfPublishedBooks:1,numberOfPublishedChapters:9,numberOfOpenTopics:4,numberOfUpcomingTopics:0,issn:"2754-6713",doi:"10.5772/intechopen.100362",isOpenForSubmission:!0},{id:"10",title:"Physiology",numberOfPublishedBooks:11,numberOfPublishedChapters:139,numberOfOpenTopics:4,numberOfUpcomingTopics:0,issn:"2631-8261",doi:"10.5772/intechopen.72796",isOpenForSubmission:!0}],hsSeriesList:[{id:"3",title:"Dentistry",numberOfPublishedBooks:8,numberOfPublishedChapters:129,numberOfOpenTopics:0,numberOfUpcomingTopics:2,issn:"2631-6218",doi:"10.5772/intechopen.71199",isOpenForSubmission:!1},{id:"6",title:"Infectious Diseases",numberOfPublishedBooks:13,numberOfPublishedChapters:106,numberOfOpenTopics:3,numberOfUpcomingTopics:1,issn:"2631-6188",doi:"10.5772/intechopen.71852",isOpenForSubmission:!0},{id:"13",title:"Veterinary Medicine and Science",numberOfPublishedBooks:9,numberOfPublishedChapters:101,numberOfOpenTopics:3,numberOfUpcomingTopics:0,issn:"2632-0517",doi:"10.5772/intechopen.73681",isOpenForSubmission:!0}],sshSeriesList:[{id:"22",title:"Business, Management and Economics",numberOfPublishedBooks:1,numberOfPublishedChapters:11,numberOfOpenTopics:2,numberOfUpcomingTopics:1,issn:null,doi:"10.5772/intechopen.100359",isOpenForSubmission:!0},{id:"23",title:"Education and Human Development",numberOfPublishedBooks:0,numberOfPublishedChapters:0,numberOfOpenTopics:2,numberOfUpcomingTopics:0,issn:null,doi:"10.5772/intechopen.100360",isOpenForSubmission:!1},{id:"24",title:"Sustainable Development",numberOfPublishedBooks:0,numberOfPublishedChapters:9,numberOfOpenTopics:4,numberOfUpcomingTopics:1,issn:null,doi:"10.5772/intechopen.100361",isOpenForSubmission:!0}],testimonialsList:[{id:"13",text:"The collaboration with and support of the technical staff of IntechOpen is fantastic. The whole process of submitting an article and editing of the submitted article goes extremely smooth and fast, the number of reads and downloads of chapters is high, and the contributions are also frequently cited.",author:{id:"55578",name:"Antonio",surname:"Jurado-Navas",institutionString:null,profilePictureURL:"https://s3.us-east-1.amazonaws.com/intech-files/0030O00002bRisIQAS/Profile_Picture_1626166543950",slug:"antonio-jurado-navas",institution:{id:"720",name:"University of Malaga",country:{id:null,name:"Spain"}}}},{id:"6",text:"It is great to work with the IntechOpen to produce a worthwhile collection of research that also becomes a great educational resource and guide for future research endeavors.",author:{id:"259298",name:"Edward",surname:"Narayan",institutionString:null,profilePictureURL:"https://mts.intechopen.com/storage/users/259298/images/system/259298.jpeg",slug:"edward-narayan",institution:{id:"3",name:"University of Queensland",country:{id:null,name:"Australia"}}}}]},series:{item:{id:"25",title:"Environmental Sciences",doi:"10.5772/intechopen.100362",issn:"2754-6713",scope:"\r\n\tScientists have long researched to understand the environment and man’s place in it. The search for this knowledge grows in importance as rapid increases in population and economic development intensify humans’ stresses on ecosystems. Fortunately, rapid increases in multiple scientific areas are advancing our understanding of environmental sciences. Breakthroughs in computing, molecular biology, ecology, and sustainability science are enhancing our ability to utilize environmental sciences to address real-world problems.
\r\n\tThe four topics of this book series - Pollution; Environmental Resilience and Management; Ecosystems and Biodiversity; and Water Science - will address important areas of advancement in the environmental sciences. They will represent an excellent initial grouping of published works on these critical topics.