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 l 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.

Effects of serum and plasma matrices on multiplex immunoassays

“…Previous studies have reported inhibition of detection for specific cytokines in the context of serum [6–10]. In recent years, vendors of immunoassay kits have in fact taken into account this phenomenon by diluting cytokine standards in a buffer that mimics the serum matrix.…”

“…This makes it difficult to detect small changes in their abundance that may accompany a disease state. Furthermore, previous studies have shown that serum and plasma themselves inhibit the detection of many cytokines, as shown by spike recovery experiments [6–10]. This so-called matrix inhibition can be due to specific and non-specific factors and varies by cytokine.…”

Circulating Levels of the Innate and Humoral Immune Regulators CD14 and CD23 Are Associated with Adult Glioma

“…The serological Luminex assays were developed using a standard sandwich capture format (27, 28). Briefly, monoclonal antibodies to human sCD14 (clone 55-3, BD Pharmingen, San Diego, CA) or sCD23 (clone 138633, R&D, Minneapolis, MN) were coupled to carboxylated Luminex microspheres by using a two-step carbodiimide reaction.…”

The Integration of Emotional, Physiologic, and Communication Responses to Medical Oncology Surveillance Appointments During Breast Cancer Survivorship

“…Cytokine levels were measured using a multi-analytic fluorescent detection method in contrast to the more familiar enzyme linked immunosorbent assays (ELISA) 38 . This procedure allows the simultaneous detection of multiple serum cytokines as opposed to ELISA assays that detect one cytokine per assay.…”