Antibodies for Immunoassays
What is an immunoassay without an antibody? Clearly the name provides the answer to this question; without antibodies there would be no immunoassays. An immunoassay is an analytical technique, quantitative or qualitative, that relies absolutely on the spe
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14 Antibodies for Immunoassays David J. Newman 1. Introduction 1.1. What Can Antibodies Do for Us in Immunoassays? What is an immunoassay without an antibody? Clearly the name provides the answer to this question; without antibodies there would be no immunoassays. An immunoassay is an analytical technique, quantitative or qualitative, that relies absolutely on the specificity and affinity of the interaction between epitope and paratope for generation of a detectable response. The actual detection of this binding interaction can be via one of literally hundreds of different signal transduction mechanisms, e.g., fluorimetry, chemiluminescence, agglutination (turbidimetry or nephelometry) enzyme reactions, and so forth (1–4), but these are simply transducing systems for the primary binding interaction. Antibodies thus provide us with an exquisitely sensitive and specific analytical technology for detecting and quantifying epitopic structures. These structures include amino-acid derivatives, e.g., thyroid hormones, peptides, e.g., vasopressin, proteins, e.g., cytokines, as well as carbohydrate structures, e.g., CA-125. Immunoassay technology has developed to such an extent that it is probably the most versatile analytical tool available able to identify and quantify epitopic structures across the milli- to zeptomolar concentration ranges (2). The structure of the epitope can be sequence-specific or conformational, and it can be closely related to the biological function of the complete molecule or completely unrelated. It is particularly important to discover which of the first is the case when selecting the format for an assay. For instance, if it is a conformational epitope that is recognized by an antibody then subtle differences in purification technique or method of conjugation to detection transducers or solid phases may alter the conformation of the epitope in such a way that it is no longer bound with the same affinity as the native molecule. This can From: Methods in Molecular Medicine, Vol. 40: Diagnostic and Therapeutic Antibodies Edited by: A. J. T. George and C. E. Urch © Humana Press Inc., Totowa, NJ
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Table 1 Advantages of Immunoassays vs PCR Immunoassay Fully quantitative Provides proof of protein synthesis Provides evidence of posttranslational modifications Detects conformation as well as sequence Can be used for nonprotein structures, e.g., carbohydrates Can be used for haptenic molecules, e.g., steroid and thyroid hormones Virtually infinite assay formats and detection systems
PCR Semiquantitative Provides proof of existence of mRNA Provides evidence of genetic differences Detects sequences only Not applicable Not applicable Restricted formats currently but potential for many more
significantly alter the specificity of any immunoassay developed using such an antibody. Regarding the biological activity of the structure of interest, because an antibody recognizes a structural determinant the only means of evaluating whether an antibody recognizes a determinant closely related
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