Use of avidin-biotin complex in an ELISA system: a quantitative comparison with two other immunoperoxidase detection...

Three systems--the indirect, peroxidase-antiperoxidase (PAP), and avidin-biotin peroxidase complex (ABC) detection system--were compared in quantitative ELISA titration and inhibition assays and tissue labeling with keratin antisera. The ABC system was found to be the most sensitive of the three in ELISA titration assays, being approximately 4-fold more sensitive than the indirect method and 2-fold more sensitive than the PAP method. In addition, the ABC method demonstrated increased sensitivity when compared on tissue sections. Furthermore, the secondary and tertiary components of the PAP and ABC systems were titrated and optimal concentration ranges determined for use in ELISA inhibition assays. In ELISA inhibition assays the broadest usable inhibitor range and maximal sensitivity were obtained using the ABC system as compared with indirect and PAP detection systems. Finally, the effects of varying microtiter plate-coating concentrations on range and sensitivity were examined, and possible explanations are discussed including the possibility of surface-bound antigen being a competitor for the antibody of the solution phase antibody-antigen complex.