A two-site monoclonal antibody (MAB) quantitative enzyme-linked immunosorbent assay (ELISA) was developed that enables quantitation of toxic shock syndrome toxin-1 (TSST-1) down to 0.25 ng/ml and detection of TSST-1 to 0.06 ng/ml. Interference by Staphylococcus protein A was eliminated by incorporating normal rabbit serum into the test sample diluent. In the process of selecting an MAB pair for a two-site 'sandwich'-type ELISA, the MABs were screened for inhibition or common epitope binding. Some MABs that reacted with antigen that was adsorbed to a polystyrene well would not bind to antigen that was presented in a more natural configuration, as in the case of antigen immobilized by trapping antibody. Conversely, MABs that reacted with antigen that was immobilized by another antibody did not all function as trapping antibodies when adsorbed directly to a polystyrene surface. ELISAs that used polyclonal antibodies in the capture mode and MAB conjugate as the second antibody were generally more sensitive than were those that used polyclonal antibodies for both capture and indicator functions. MAB screening and selection schemes should be carefully designed to evaluate MABs in the mode in which they will be used in the final assay.