Multiplex analysis of heterophil antibodies in patients with indeterminateHIV immunoassay results

We hypothesized that heterophil antibodies reactive with animal proteins us ed in blot preparation caused nonspecific staining (NSS) on HIV Western blo t (WB) studies, causing indeterminate results. We analyzed samples showing NSS on HIV WB using a multiplexed immunoassay to simultaneously measure IgG antibodies to animal IgG (bovine, goat, sheep, mouse) and bovine serum alb umin. Heterophil antibodies reactive with IgG from several animal species w ere detected in 23 (49%) of 47 samples showing NSS on HIV WB; 15 positive s amples demonstrated antibodies to all 5 antigens. Similar IgG heterophil an tibodies were detected in only 2 (8%) of 24 control samples. Of the HIV WB samples with a positive HIV-1 enzyme-linked immunosorbent assay (ELISA) res ult at the time of WB testing (11/47), heterophil antibodies were found in 8 (73%) of 11. Preabsorption with bovine, goat, and sheep IgG removed heter ophil antibodies detected by the multiplexed assay and, in some cases, elim inated reactivity in ELISA and WB testing. Heterophil antibodies are associ ated with indeterminate HIV immunoassay results and are an important cause of false-positive HIV ELISA results. Multiplexed immunoassays provide a pow erful tool for screening patients for heterophil antibodies and resolving p ossible false-positive results.