Antibodies Applications and New Developments

This chapter deals with the discovery and application of the antibody-antigen reaction in the form of inoculation, variolation and vaccination to induce immunity - at first to smallpox, later to other diseases and common afflictions such as rabies, anthrax and cholera, caused by microorganisms. </p><p> The work of the pioneers in this field and their discoveries is briefly described here. </p><p> The in vitro applications of the antibody-antigen reaction - mainly in the form of radioimmunoassays - is covered and includes the early development of this technique both in assay design and data reduction, which allowed the rapid acceptance of radioimmunoassay in clinical diagnostic procedures, especially in the fields of endocrinology, microbiology and pharmacology. </p><p> The successors to radioimmunoassay which use non-radioisotopic labels have also been described in detail as well as listing the pioneers in immunoassay development. </p><p> The above topics have been considered in the context of antigen-antibody reactions and some of the persons who can be termed pioneers in this area of predominantly medically-related research.

The second chapter continues the history of immunoassay and mainly deals with the commercialization and automation of immunoassays, the impact of monoclonal antibodies and their potential role in standardization and the continued development towards microformats and multianalyte immunoassays. </p><p> This chapter also includes the terminology used in immunoassays and schematic representation of assay design in the form of diagrams, as well as a brief look into the possible future development of immunoassay techniques and their application and impact in clinical in vitro diagnostic procedures.

Fundamental immunological principles and factors influencing production strategies for antibodies used as reagents in analytical and diagnostic methods are highlighted. A role of the peptideprotein design for an antibody formation, covering particularly epitope selection, peptide and protein chemistry, carrier-protein conjugation including multiple antigen protein approach are described. Special attention is paid to hapten design and production of antibodies against molecules having lowmolecular character. Critical stages in polyclonal and monoclonal antibody development as well as practical hints for their production are discussed.

While immunoassays are now used in a variety of fields including medical, food technology and environmental protection because of their high sensitivity, high specificity and ease of automation to provide highly cost effective analyses, it has been in medical testing that their standardization has been most intensely developed. The use of internationally agreed clinical protocols, common reference intervals or decision limits and even electronic health records across health-care institutions are all dependent on medical testing laboratories performing assays that are traceable to internationally recognized reference measurement systems. The application of metrological principles to achieve traceability and standardization for immunoassays is being pursued to this end. Comprehensive measurement systems are available for the total serum hapten assays currently measured in the clinical laboratory by immunoassay. Current research is investigating the usefulness of developing defined systems for the measurement of non-bound fractions or "free" hormone levels of these types of analytes. One strategy that has been successfully applied for the standardization of assays for large molecular weight polypeptide measurands has been to localise the biological activity to a small molecular weight moiety of the meausurand and to establish a reference measurement procedure for this moiety as a surrogate for the total molecule of clinical interest. The standardization or harmonization of assays for heterogeneous polypeptide hormones is also is another area of current research for standardization of clinical immunoassays. The accreditation of medical testing laboratories to ISO15189 standard requires that all testing methods including immunoassays are validated or verified to be fit for purpose including the accuracy of the assay and the estimation of measurement uncertainty as assessed by traceability to certified reference materials through a documented unbroken chain of calibrations.

Immunochemical methods, methods that employ antibodies as analytical reagents, have revolutionized laboratory medicine. Although the noncovalent bound between analyte in biological specimen and complementary antibody in reagent is specific, positive or negative interferences are possible. Interfering substances can be present in both specimen and reagents, respectively. Most interfering substances are inherent in patients’ blood under physiological and pathophysiological conditions, especially under diagnostic and therapeutic procedures. Some interferences are similar to those in chemical analyses and some are typical only for immunoassays (hook effect; cross-reactivity with structurally similar or identical epitopes; heterophile antibodies; anti-animal antibodies; autoantibodies; the matrix effect). The main characteristic of all immunoassays is that the reagent which discovers or quantifies the target analyte contains the specific antibody. Results higher than real values are recorded due to a low antibody specificity. Results can also be influenced by pre-analytical factors or assay formulations. One should suspect interferences a) after obtaining an unacceptable result; b) if there is non-linearity during dilution; c) if there is no agreement with other test results or clinical data; d) if different immunoassays in determination of the same analyte provide significantly different results. It is necessary to think about present predictable and always possible unpredictable and unrecognizable interferences. Unawareness and non-recognition of interferences could lead to diagnostic errors, unnecessary laboratory tests, inadequate and unnecessary treatment and therapy. There is no simple and practical way to identify interferences in specimens before analysis. Whenever interference is suspected, there are a few possibilities to solve them, e.g. serial dilution, pre-treatment with blocking reagent, use of more specific methods. At the same time a proper information upon pre-analytical diagnostic and therapeutic procedures is needed for accurate interpretation of the results. In this process, consistent communication and compliance between laboratory professionals and clinicians are required.

With new developments in agriculture and food production in the past two decades, bioanalytical detection of toxins and allergens in foods became a challenging task. In addition, efficient validation and labelling of food products demand reliable technical approaches. Complex, heterogeneous matrices and low amounts of specific analytes complicate detection and characterization of harmful substances in food. A highly relevant tool to achieve this goal is represented by several immunochemical approaches, based on antibody technology. Different types of antibodies have been developed, which are characterized by high affinity and specificity. Although, alternative methods have been introduced into food analysis (e.g., mass-spectrometry and PCR), antibodies still play a key role, especially in detection and characterization of allergens. In this chapter, I discuss basic immunochemical methods of food analysis based on antibody technologies. The increasing importance of establishing suitable detection schemes for several allergens (e.g., allergens from peanut and soy) in an immunoanalytical approach, will be particularly emphasized.

The food chain is threatened by various hazards and the presence of residues of antibiotics, used during cattle-breeding, is one of the serious risks for consumers. In food and feed analysis for antibiotics, screening technologies are powerful tools that provide a rapid screen of large numbers of samples when conventional analytical methods are too cumbersome. Due to their simplicity and/or high-throughput capacity, immunoassays are applicable for screening at critical control points in the food chain and in control laboratories. However, in general, they are very specific, and only suitable for the detection of one or two antibiotics, which seriously limits their application. New antibodies and new assay formats with multiplex capacity might give new possibilities for control agencies and food industries for increased and more efficient controls on antibiotics and other food contaminants. Multiplex applications and future perspectives of poly- and monoclonal as well as recombinant antibodies for the compound- or group-specific detection of antibiotics, using different immunoassay formats (e.g. ELISAs, dip sticks, biosensors and flow cytometers), are described and discussed.