The immunochemistry of sandwich ELISAs--VI. Greater than 90% of monoclonal and 75% of polyclonal anti-fluorescyl capture antibodies (CAbs) are denatured by passive adsorption.

Quantitative data are presented showing that the method most commonly used to immobilize antibodies in microtiter immunoassays functionally inactivates most of the antibodies. These results were collected using five affinity purified polyclonal antibodies (pAbs) and six monoclonal antibodies (mAbs) specific for fluorescein (FLU) as capture antibodies (CAbs). These CAbs were tested for their ability to capture FLU4.2-BSA after immobilization by passive adsorption, the Protein-Avidin-Biotin-Capture (PABC) system or using previously adsorbed anti-globulins. Results indicate that under optimal conditions, < 10% of monoclonal capture antibody equivalents (CAbeqv) and congruent to 22% of polyclonal CAbeqv remain functional after passive adsorption. Immobilization via the PABC system improved the performance of mAbs by more than five-fold but had less than a two-fold effect on pAbs. Many CAbs immobilized using an anti-globulin retained full activity including the ability to bind two molecules of FLU4.2-BSA/molecule of CAb. The latter result is not necessarily a recommendation for the use of anti-globulin immobilization, since the number of functional CAbeqv per well is not significantly greater than that which can be achieved using passive adsorption.