Demonstration and minimization of serum interference in flow cytometric two-site immunoassays

The ability of serum factors to cross-link labeled mouse monoclonal antibody (mAb) of irrelevant specificity (mAb FN61, subclass IgG1) to different particle types coated with sheep IgG, bovine gamma-globulin, or 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 with bovine gamma-globulin. These were designated type 1 sera. Seven sera, designated type 2, displayed a dominant interference with the mAb FN61-coated particles. The interference reaction in the two serum types was characterized by different kinetics, dependence on particle concentration, and response to blocking agents. The interference was minimized by addition of 500 micrograms of bovine gamma-globulin and 50 micrograms of mAb HH1 (IgG1) of irrelevant specificity per 10 microL of serum sample in a final assay volume of 100 microL.