Heterophile antibody interference in a multiplexed fluorescent microsphere immunoassay for quantitation of cytokines in human serum

Abstract

While modern immunoassays provide sensitive and specific means for the quantitation of cytokines in biological fluids, heterophile antibodies are still a well-recognized cause of interference in the measurement of cytokines in these assays. We have developed a multiplexed fluorescent microsphere immunoassay for the simultaneous quantification of 10 cytokines in only 75 microl of serum. During the development of this multiplexed assay, the amount of assay interference due to heterophile antibodies was also determined, and methods for detecting heterophile interference and minimizing its effect were incorporated into the assay. Heterophile antibodies resulted in significantly elevated cytokine values compared to those of normal blood bank samples. These falsely elevated values, and thus the components of the assay the heterophile antibodies were binding to, were identified through the use of internal controls. This information was then used to design assay-specific blockers and absorbents that were shown to significantly reduce falsely elevated cytokine values while not affecting the standard and control values. The fluorescent multiplexed microsphere-based immunoassay can be used to quantitate multiple cytokines from a single sample and should be a useful tool in furthering our understanding of the role of cytokines in disease processes.

FIG. 1.

Mean MFI values for 25 blood bank serum samples (gray bars) and 10 heterophile serum samples (black bars) with mouse and rat protein-coupled microspheres. Reactivities of heterophile samples to mouse IgG, mouse serum, and rat IgG were significant (with P values of 0.0007, 0.001, and 0.0004, respectively) compared with those of blood bank samples. Error bars indicate standard deviations.

FIG. 2.

Comparison of the mean concentrations or MFI values of 10 heterophile antibody-containing serum samples in a multiplexed cytokine immunoassay. Most reductions in falsely elevated cytokine levels in the optimized serum diluent (gray bars) compared with the levels in PBST (black bars) were significant (IFN-γ, P = 0.006; IL-10, P = 0.035; IL-12, P = 0.024; TNF-α, P = 0.040; IL-1β, P = 0.062 [not significant]; IL-2, P = 0.006; IL-2r, P = 0.064 [not significant]; IL-4, P = 0.021; IL-6, P = 0.023; IL-8, P = 0.035; mouse IgG, P = 0.013; mouse serum, P = 0.026; rat IgG, P = 0.006). Error bars indicate standard deviations.