Biotinyl-estradiol derivatives in enzyme immunoassays: structural requirements for optimal antibody binding.
The use of the avidin/biotin complex in immunoassays is well documented. No comprehensive studies, however, are available on the structural requirements of the linkage between biotin and small molecules to get an optimal antigen-antibody interaction. We have synthesized seven different biotinylated estradiol derivatives. They were evaluated in an antibody- and in an antigen-immobilized enzyme immunoassay system. All three derivatives lacking a spacer group were useless for use in immunoassays, demonstrating the importance of a long distance between the biotin- and estradiol-moiety. In addition, the chemical structure of the linkage at the site of attachment to the steroid skeleton is very important for the antibody recognition: it may either be rigid but identical to that one used in the immunogen (6-carboxymethyloxime), or must be structurally flexible as exemplified by a 6-amido-linkage. A rigid structure (hydrazone) different from that of the immunogen absolutely prevents antibody binding.
Tiefenauer
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Medical Bioanalytics Project, Paul Scherrer Institute, Switzerland.
Andres
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D000937:Antigen-Antibody Reactions; D001710:Biotin / Q000031:analogs & derivatives* / Q000276:immunology; D055598:Chemical Phenomena; D002621:Chemistry; D002851:Chromatography, High Pressure Liquid; D004958:Estradiol / Q000031:analogs & derivatives / Q000276:immunology*; D007124:Immunoenzyme Techniques; D013329:Structure-Activity Relationship
D004958:Estradiol; D001710:Biotin