Antibodies: Applications and New Developments

329 Pages·2012·9.27 MB·English

by  Eline P. Meulenberg

Antibodies
Applications and New Developments
Edited By
Eline P. Meulenberg
Ambachtsweg 5, 6581 AX Malden
The Netherlands
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CONTENTS
Foreword i
Preface vi
List of Contributors vii
CHAPTERS
1. The Discovery and Development of the Antigen-Antibody Reaction
Part 1: A Brief History 3
Part 2: Commercial Immunoassay Development and Applications 14
William Graham Wood
2. Production of Antibodies for Immunoanalytical Methods 29
Ludek Eyer and Milan Franek
3. Standardization of Immunoassays 48
H.A. Morris
4. Interferences in Immunochemical Methods 58
Slavica Dodig
5. Antibodies as Bioanalytical Tools in Food Analysis 68
Georg Mahlknecht
6. Immunoassays for Antibiotics in the Food Chain 76
Willem Haasnoot
7. Immunoassays for Detecting Prohibited Growth Promoters in the Food Chain 93
Ph. Delahaut
8. Application of Antibodies in the Field of Environmental Analysis – An Overview 112
Eline P. Meulenberg
9. Multi-Analyte Immunoassays for Water Monitoring 146
G. Proll
10. Principles and Applications of Immunoaffinity Chromatography 156
Abby J. Jackson, Matthew R. Sobansky and David S. Hage
11. Lateral Flow Assays 175
Geertruida A. Posthuma-Trumpie and Aart van Amerongen
12. Trends and Perspectives in Immunosensors 184
Caroline Viguier, Carol Crean and Richard O’Kennedy
13. Immunoelectrochemistry for the Detection of Clinical Biomarkers 209
Sonia Centi and Marco Mascini
14. Antibody Fragment Engineering and Applications in Diagnosis and Therapeutics 225
Xuemei Xie, Gabrielle Richard, and John Christopher Hall
15. Therapeutic Antibodies: State of the Art and Promises 280
Patrick Chames and Daniel Baty
16. Aptamers the Chemical Antibodies 300
Frans P. Houwen and Andreas Kage
Index 315
i
FOREWORD
This book is intended to familiarise the reader with the history, development, terminology and theoretical
considerations associated with the development, production and use of Immunoassays. The reader will be
led through the theoretical and practical considerations associated with antibody, production and then into
the practical applications and complications of antibody’s use in assays. Finally there are a series of
chapters on specific applications and future technologies, ending with a chapter on Aptamers which may
represent an alternative to antibodies and the future of assay development.
This foreword will recount the theories and discoveries which have led to the explosion in commercial
immunoassays and to their development as powerful research tools.
The chapters provide details of the development of immunoassays establishing an “evolutionary tree”, the
path of events and the stages which have been reached and possible directions for future research and
developments. The relationship between immunoassays and other analytical techniques is presented clearly
and there are indications as to potential future developments.
Modern immunoassays have arisen from the desire to detect and quantify complex biological molecules
under conditions for which chemical and physical analytical techniques were either unsuitable or not
available. Current methodologies are based firmly in classical immunology. The theoretical high sensitivity
and high specificity of the antibody-antigen interaction attracted the attention of workers wishing to exploit
these properties in this search for improved analytical techniques. Indeed, modern trends are towards the
development of increasingly sensitive assays; expanding the range of analytes being detected: to an
increasing variety of substances which can be analysed in new and developing fields; to increasing rapidity
of results and to simplicity of use in terms of the intervention needed and the skills of the operator in
carrying out and interpreting the results.
The driving force for these developments has changed as assays have been shown to be successful and the
methods have become more established. Originally immunoassays were developed to facilitate the study of
the immune system, particularly the role of antibody-antigen interactions as a defence against disease. The
original assays were therefore used in clinical research and areas are closely related to medicine (Chapter
1).
As an increasing usefulness of the assays was demonstrated the demand for greater sensitivity and accuracy
increased. This has generated a greater knowledge of these type of assay outside the clinical field and their
use in a wider variety of fields began to increase.
As part of this demand there has been the development of a greater understanding of the nature of
antibodies and their production. Despite earlier historical references to protection against disease by the
injection of live organisms from smallpox pustules into healthy patients, the generally accepted beginnings
of Immunology as a science are Jenner’s observations on and use of cowpox as a protection against
smallpox. This then was the first scientifically reported observation of an antibody-antigen reaction,
although not realised as being such at the time. What Jenner had observed was a clinically useful incidence
of cross-reactivity. However it was not until over a hundred years, later that in 1897, Rudolph Kraus in
Vienna reported that the interaction between a soluble antigen and the corresponding soluble antibody
resulted in the formation of a precipitate. Thus the use of antibodies in assays systems was initiated and the
production of these useful antibody molecules as a part of scientific study became a study in its own right
(Chapter 2).
Much of the development of immunoassays is dependent upon knowledge of the structure of antibodies.
The gradual development of information regarding the structure of antibodies has been the key to the
establishment of immunoassays. The fact that immunoglobulins are proteins initially allowed researchers to
measure the concentration of immunoprecipitates by simply determining the concentration of nitrogen in
ii
the dried precipitate and then calculating the concentrations of antibody and antigen in the precipitate. This,
obviously, was a time consuming and technically demanding procedure and hence the aim of research in
one important aspect as to develop alternative methods for determining antigen concentration. In the
aftermath of the development of radioisotope development and detection the application of such isotopes to
the monitoring of the antibody antigen reaction was one of many attempts to use such material peacefully.
However, despite considerable initial success the health and safety issues combined with the high costs of
using radioactivity drove the search for yet another alternative method of detection. Thus returning to the
structure of antibodies as a source of inspiration resulted in the use of protein biochemistry to carry out
reactions linking labels to the antibody. The additional knowledge that IgG has a Y-shaped structure with
the specific binding region at the ends of the arms, F , binding region and the stem, F, crystallisable,
ab c
forming a region on which chemistries can be conducted without interfering with the specific binding
reactions provided a basis for future developments, labelled antibodies which retained their specific binding
capabilities.
Simultaneously the methods of separating the bound and unbound enzyme labelled antibody were being
developed. A significant step was the description of the use of reagents, either antibody or antigen, bound
to solid phases, including glass, plastic, latex and metals, which allowed the rapid and easy separation of
the reactants in immunoassay systems. Thus the ELISA, Enzyme Linked Immunosorbent Assay was
developed, using the F region to bind the antibody to solid substrates being one of the alternative models
c
of immunoassay.
There are now numerous immunoassay types; however what all the immunoassayists have to understand is
that the systems in all their forms consist of a molecule with a highly specific binding capacity and a system
for detecting the reaction between this binding molecule and a specific analyte. Specifically for
immunoassays the molecules concerned are conventional antibodies and antigens or haptens. Provided the
reader remembers that all immunoassays are based on the measurement of antibody-antigen interactions
and the variations attempt is only to provide more sensitive, accurate, reliable or robust means of achieving
this, little misunderstanding will occur.
However it is essential to standardise these assays. Without consistency in the results obtained when using
any form of analytical method, the method is worthless. Thus having demonstrated the ability of techniques
based on immune reactions to monitor concentrations of particular analytes in a highly specific and
extremely sensitive format the immunoassayist must also consider reliability, reproducibility, simplicity of
assays and assay formats. These aspects are dealt with in detail in (Chapter 3).
The final important development in application of immunoassays to Food samples has been the
consideration of matrix effects on the assay results. In essence, immunoassays were developed by the
clinical researchers, who used the techniques as the means of rapid diagnosis. Such clinical studies
routinely detected antibody or antibody’s level in body fluids, particularly the serum, under physiological
conditions. This application of the assays in a single, or very limited number of matrices, with very similar
characteristics simplifies the importance of matrix effects. However once the assays began to be applied to
systems which are not simple, do not have physiological characteristics such as pH, ion balance and
temperature. The effects of the matrix becomes significant. Thus in food systems the matrix has an
important function and the variable effects on immunoassays. For the immunoassayist using the techniques
in food related applications it is important to consider the matrices in which such reactions may occur and
how the matrix may interfere with the reaction. In (Chapter 4) these matrix effects are discussed in detail
and the options available in order to provide standardised, reliable assays are suitable for routine usage
described.
Having established the essentials of producing antibodies, developing assays and standardising these assays
to provide reliable analytical tools, the second section of the text provides examples of the application of
immunoassays in the field of food analysis. This is a very wide field and at one end can include aspects
which might be more appropriately considered at environmental analysis and at other supportive clinical
diagnosis for allergy. The first step is to understand how and when antibodies can be used in the analysis of
iii
food and food materials. The myriad of formats, ELISA, Transverse Flow, Lateral Flow, etc. and the
varieties of arrangements, direct, indirect, competitive, non-competitive, sandwich etc. and the possibilities
for single and multiple tests and multi-arrays, all need to be described and discussed in order to provide the
analyst with information on which decisions will be based as to how to select a suitable assay for the
particular analyte and matrix (Chapter 5).
The adulterants and contaminants found in food can be considered myriad. Many bacterial and chemical
contaminants are found in food purely as a result of accident but in other cases these, chemical
contaminants may be found in the food as the consequence of the use of these in the environment or as
treatments in food animals. One specific group of food contaminants which may be found in food are the
antibiotics. These are used to treat diseases in animals but they and their residues may be found in the foods
derived from those animals and may have unforeseen consequences. Older readers will recall Rachel
Carson’s “The Silent Spring” as ground-breaking text defining the adverse consequences of the use of
pesticides and the transfer of these materials along with the food chain. There is a similar progression of
antibiotics along with the food chain which has potentially greater consequences for the human population.
Simple financial consequences may be demonstrated by the example of the use of antibiotics to treat
mastitis in cattle. This can if controls are ignored lead to the presence of antibiotics in milk; if this milk is
used in cheese making the antibiotic can prevent the growth of the starter culture and lead to significant
losses in terms of Cheese production. The presence of antibiotics in milk, it was at one time, considered to
be the cause of various maladies in people, especially young children. Hence its detection in the food chain
of antibiotics has a particular relevance for both human health and industrial profitability (Chapter 6).
In a similar vein the treatment of animals with growth promoters results in the transfer of these materials
and their residues along with the food chain as well as their release into the environment where they may,
in the case of oestrogen-like agents, cause changes such as feminisation of fish and the modification of
population ratios (Chapter 7).
Immunoassays have other applications and one opportunity is in environmental analysis. This type of
analysis develops simply and obviously from the detection of pesticides and other chemical agents, e.g.
growth promoters, in food matrices to the detection of the same pesticides and their residues in the
environmental samples, especially potable water (Chapter 8). A significant feature of antibody-based
immunoassays is the specificity of the ligand and the application of this specificity to the detection of a
single analyte; however in many instances the analyst requires a methodology which detects more than one
analyte. The development and application of immunoassays, which have multi-analyte detection
capabilities, is a major research aim. Having established the criteria for the single analyte assay in many
formats and in numerous matrices the option for the development of multi-analyte assays was to return to a
simple matrix and begins their development with the simplest of all matrices, water (Chapter 9).
This foreword opens the section of the book which provides descriptions of novel and alternative assay
methodologies and provides an indication as to the future development of immunoassays. There are many
different alternative routes along which immunoassays that may develop. These four chapters demonstrate
the lines of development which have been investigated. A specific chapter is dedicated to each because of
the impact of these techniques that it may have had on the field. The importance of the method of
separation of the reactants and non-reactants following the reaction of antibody with antigen was stated in
the initial stages of immunoassay development. The interaction of the two large molecules often results in
the formation of an insoluble precipitate which easily solves the problem of separation as unreacted
components remain in solution allowing filtration or centrifugation, physical methods, to be used to
separate the components. However when the analyte is a small molecule the reaction with antibody may not
produce a precipitate necessitating the use of other methods for separating the antibody-analyte product
from the unreacted antibody and analyte in the reaction mixture. Binding one or other of the components to
a solid phase that allows the simplified separation of the bound materials. Thus the use of systems such as
latex bound antibody allows the development of agglutination assays, provided the antigen was multivalent
and thus allowed cross-linking of the latex beads via the bound antibody. If alternatively the analyte is
monovalent and hence cross-linking is not possible and the alternatives are the use of magnetic beads,
iv
which can be separated from the solution very simply or the binding of one component of the reaction to
another solid matrix which allows easy separation. A problem of these options is the matrix effect which
can interfere with or totally prevent the reaction. Such problems can be reduced or eliminated by using a
method which combines a separation technique and an immunological reaction. An important example of
this type of technique is Immunoaffinity chromatography (Chapter 10). This technique is also important in
establishing the use of chromatography in immunoassay systems. In chemistry the separation of
components by chromatography began with the use of paper as the separation medium across which the
liquid solvent carried solutes at different rates, separating Indian ink into its component colours, for
example. To achieve greater and more complex separations chemists moved from paper to glass plates
coated with other inert substances through which liquid might diffuse. Eventually the concepts were
combined and plastic backed nitrocellulose “paper” sheets were developed. Immunoassayist eventually
took this development in the form of Mylar-backed nitrocellulose and used it to produce lateral flow
immunoassays, basis for both rapid diagnostic and simple “dipstick” immunoassay techniques (Chapter
11). Finally, the use of alternative methods of detecting the immune reaction has led to the development of
some interesting alternative assays. As it has been described. One of the major problems which
immunoassayists have been attempting to overcome is how to detect the reaction between the antibody and
the antigen. The detection methods have in many cases been by means of secondary reaction, i.e. those
reactions such as precipitation which occur as a result of the “primary” reaction, i.e. binding of the antibody
to the antigen or hapten, however the requirements of the assay are that the “primary” reaction be measured
directly. Two examples, amongst an increasing number, are immunosensors (Chapter 12) and
immunoelectrochemistry (Chapter 13).
All of these chapters provide considerable areas for research and development, however if this is not
sufficient then in the final chapters the reader is given information as to other possibilities. The intention is
for this book to lead the reader through the story of immunoassays and to suggest fields of current
interesting research and potential future significant developments. In attempting to predict the future there
is considerable room for error and hence the last three chapters describe current research with considerable
potential rather than developing speculative ideas.
In completing the story of immunoassays the research has now moved to considering alternatives to the
antibody molecule. Antibodies are molecules which are normally developed in vivo by “challenging” the
immune system of an animal with a “foreign” molecule. Simplistically the immune system is a defence
mechanism which recognises “foreign” or “non-self” molecules and responds by producing antibodies
which bind to and assist in the removal of these “threatening” molecules. Scientists have taken advantage of
this natural defence mechanism to induce animals to produce antibodies to specific molecules. Again
simplistically, the injection of the “foreign” molecules into animals results in an immune response and thus
produces antibodies in the blood which will specifically bind to this molecule. By collecting the blood, it is
possible to recover the antibody molecules and have these available for use in assays. Such a natural system
of production results in a plethora of antibody molecules with a range of specificities and affinities. The
first steps in moving away from this methodology were the development of antibodies from single cell
clones, monoclonal antibodies. Monoclonal antibodies are identical antibody molecules produced from a
clone of cells which have artificially been created by fusing an antibody producing cell with an “immortal”
cell line to yield cells which can be grown in vitro and thus make the antibody more akin to a standard
chemical reagent. Monoclonal antibodies are only one development in the attempts to modify or replace the
conventional, animal-derived, polyclonal antibodies used in early immunoassays. One possibility is to use
only parts of the antibody molecule, the F , antigen binding fragment. These type of modifications are
ab
refered to as “antibody engineering” (Chapter 14). Such techniques may currently be confined to the field
of medical diagnostics, but it is possible that they may be increasingly useful to the food scientist.
Similarly the modification of the antibody molecule to provide reagents which can be used in specialist
situations as a matter of fact. Specifically antibodies which can be used as therapeutic reagents are being
generated. Whether such techniques can be moved from the field of medical diagnosis to food analysis may
simply depend upon the ingenuity of the researchers working in these fields (Chapter 15).
v
Finally there is a possibility that the antibody molecule may be removed completely and that assays will
use an alternative. Some may suggest that the use of molecules derived from animals or involving the use
of animals is ethically and morally questionable. Whether or not this is the case, science is moving on and
the discovery of short sections of nucleotides which bind specifically, with high affinity to proteins and
peptides, aptamers, has provided a potential alternative to the antibody molecule over a period of time. In
the last two decades, since their discovery aptamers have been shown to have the potential to be more
specific, have superb affinity and to function in a wider range of matrices than antibodies. Aptamers are
short nucleotide sequences which can be produced without the use of animal, thus obviating the moral and
ethical objections which are associated with antibodies. Further, Aptamers can be selected from a naïve
DNA library. Specificity and affinity relates not only to proteins but may extend to a variety of other
molecules. Significantly once the aptamer has been identified it can be reproduced very simply by cloning
into E. coli and with sufficient quantities available sequence can be identified and further amounts
produced by chemical synthesis. The final Chapter (Chapter 16) describes the capabilities and possibilities
of aptamers and thus provides a range of potential future applications.
For the student and the established research it is hoped that the text, prepared by authors of considerable
standing in the field, would provide significant interest and suggestions for future research.
Christopher Smith
Professor, Food Science
Co-Director, Manchester Food Research Centre
Manchester Metropolitan University
UK