DEMONSTRATION AND MINIMIZATION OF SERUM INTERFERENCE IN FLOW CYTOMETRIC 2-SITE IMMUNOASSAYS

The ability of serum factors to cross-link labeled mouse monoclonal an tibody (mAb) of irrelevant specificity (mAb FN61, subclass IgG1) to di fferent particle types coated with sheep IgG, bovine gamma-globulin, o r mAb FN61 was measured simultaneously by flow cytometry. Significant interference with mAb FN61-coated particles was detected in 53 of 101 sera. Of the 30 sera showing the most pronounced interference, 23 were characterized by an even stronger cross-linking to particles coated w ith bovine gamma-globulin. These were designated type 1 sera. Seven se ra, designated type 2, displayed a dominant interference with the mAb FN61-coated particles. The interference reaction in the two serum type s was characterized by different kinetics, dependence on particle conc entration, and response to blocking agents. The interference was minim ized by addition of 500 mug of bovine gamma-globulin and 50 mug of mAb HH1 (IgG1) of irrelevant specificity per 10 muL of serum sample in a final assay volume of 100 muL.