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Abstract
Publisher Summary This chapter describes the methods for the preparation of enzyme–antibody conjugates for use in enzyme immunoassay. Enzyme-linked immunoadsorbent assay type assays are becoming established as the method of choice for screening for antigens derived from, and antibodies directed against, microorganisms. The effect of the cross-linking reaction on the antibody and enzyme activity can be determined by measuring both the activities before and after the conjugation reaction. Methods that have been used to measure the antibody activity include: radial immunodiffusion, reverse-rocket immunoelectrophoresis, and radioimmunoassay. The efficiency of the coupling reaction can be determined by separating the conjugated from the nonconjugated material and measuring the proportion of antibody and enzyme activity in the conjugate. The one-step glutaraldehyde method is a widely used and technically undemanding procedure and many enzyme immunoassays have been successfully developed, using labels prepared by it. Nonlabeled antibody should be removed from the conjugate, as it lowers the sensitivity of the assay. It is found that for those assays, where the enzyme activity is measured on the solid phase, removal of free enzyme from the conjugate is not essential.
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