Research paper
Evaluation of heterophilic antibody blocking agents in reducing false positive interference in immunoassays for IL-17AA, IL-17FF, and IL-17AF

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Abstract

IL-17AA, IL-17FF, and IL-17AF are proinflammatory cytokines that have been implicated in the pathogenesis of autoimmune diseases such as rheumatoid arthritis (RA). In order to measure the levels of these cytokines in synovial fluid and serum samples from RA patients, immunoassays specific for IL-17AA, FF, and AF were developed. Although these assays could tolerate up to 50% pooled normal human serum, false positive reactivity was problematic in patient samples suggesting interference from heterophilic antibodies. We therefore evaluated the ability of several commercially available heterophilic antibody blocking agents to reduce false positive reactivity by testing them against samples that were confirmed as false positives in the IL-17AA, FF, and AF assays. Several of the blockers performed well, including HBR-1, HBR-9, HBR-11, HBR-Plus, Serum Cytokine Assay Diluent, and IIR. We chose to move forward using IIR blocker for sample analysis and verified that IIR had no effect on the assay standard curves and did not affect IL-17 quantitation in plasma from ex vivo stimulated human whole blood. IL-17FF and IL-17AF were below the limits of quantitation of the assays (12.3 and 10.5 pg/ml, respectively) in synovial fluid and serum samples from patients with RA and osteoarthritis (OA). For the more sensitive IL-17AA assay (1.6 pg/ml limit of quantitation), low levels of IL-17AA were measurable in 48% of RA synovial fluid samples (mean, 7.9 pg/ml; median, < 1.6 pg/ml; range, < 1.6–29.7 pg/ml; n = 23) but not in synovial fluid from patients with OA (n = 33). For serum samples, however, IL-17AA was below the limit of detection for both RA and OA patients. When these same serum samples were analyzed in the absence of a heterophilic antibody blocker, false positive reactivity yielded apparent mean IL-17AA levels of 43.3 pg/ml (28% positive; n = 50) and 14.8 pg/ml (12% positive; n = 50) for RA and OA patients, respectively, results that could potentially be interpreted as consistent with disease biology. These studies demonstrate the importance of ensuring that HAb interference is well controlled, particularly when measuring low concentrations of cytokines in samples from patients with autoimmune disease.

Introduction

Interleukin 17A (IL-17A) is a proinflammatory cytokine produced by Th17 cells (reviewed in Kolls and Linden, 2004). Upon signaling through IL-17RA and IL-17RC, IL-17A elicits the production of a number of inflammatory mediators including cytokines, chemokines, and matrix metalloproteinases. In addition, IL-17A synergizes with TNFα and IL-1 to stimulate stronger proinflammatory responses. IL-17A is the initiating member of the IL-17 family, which includes IL-17B, C, D, E, and F. With 50% sequence identity (Hymowitz et al., 2001), IL-17F is the most closely related to IL-17A and has similar but less potent proinflammatory activities. Both IL-17A and IL-17F form disulfide-bonded homodimers. Recently, they have also been shown to heterodimerize to form IL-17AF, which has activity intermediate between the IL-17AA and IL-17FF homodimers (Wright et al., 2007, Chang and Dong, 2007, Wright et al., 2008). IL-17A and F have been implicated in a number of autoimmune diseases including psoriasis, multiple sclerosis, and inflammatory bowel disease (IBD) (Ouyang et al., 2008). In addition, a role for IL-17 in rheumatoid arthritis (RA) is supported by compelling clinical and experimental evidence (Lubberts, 2003).

The ability to detect IL-17 in relevant patient samples is important in further elucidating its role in a disease and may also be useful in identifying patient groups that would derive the most benefit from a therapeutic agent targeting IL-17. Various ELISA kits and reagents are commercially available and have been widely used to detect circulating IL-17A. However, the levels of circulating IL-17A measured in normal subjects varies across reports, ranging from undetectable to 40 pg/ml (Arican et al., 2005; Fujino et al., 2003, Agarwal et al., 2008, Singh et al., 2007, Manukyan et al., 2008, Wong et al., 2000, Wong et al., 2008, Lei et al., 2008). Furthermore, despite evidence for a role of IL-17A in RA, the serum/plasma levels of IL-17A in RA patients varied over the same range as normal subjects (Singh et al., 2007, Kageyama et al., 2007a, Kageyama et al., 2007b). One publication, in contrast, detected considerably higher levels of IL-17A in RA sera using an ELISA developed in the investigators' laboratory (mean levels > 500 pg/ml; Ziolkowska et al., 2000). These inconsistencies in circulating IL-17A concentrations may arise from true variation in patient groups, differences in sample handling, or differences in the specific assay method used. Other possible sources of variation, however, include interference of serum factors in the assays and differences in the measures taken to address potential serum interference issues.

It is widely known that human serum contains immunoglobulins that affect the results of immunoassays by binding to reagent antibodies used in the assay (Levinson and Miller, 2002, Clinical and Laboratory Standards Institute, 2008). These immunoglobulins include polyspecific antibodies, consisting of poorly defined antibodies with multiple binding specificities that arise naturally out of the process of antibody generation; autoantibodies, such as rheumatoid factor (RF); and anti-animal antibodies arising from occupational or social exposures or from dietary intake. These types of interfering immunoglobulins are termed heterophilic antibodies (HAb). Although HAb can cause false negative results, a more frequent type of interference is a false positive signal. This occurs as a result of the HAb bridging the assay coat and detection antibodies to yield a signal even in the absence of analyte. HAb interference is well documented in the clinical chemistry literature, and various recommendations have been made to detect false positives and to reduce interference of HAbs in immunoassays (Bjerner et al., 2005a). Antibodies arising as a result of treatment with a therapeutic antibody, such as a human anti-mouse antibody (HAMA) response, are considered to be separate from HAb since they constitute a high affinity response targeting a specific antigen. However, for the purposes of the studies presented here, HAMA-type antibodies will be considered with HAb since both can interfere with immunoassay analysis regardless of the original antigen's identity (Kaplan and Levinson, 1999).

The goal of these studies was to measure IL-17 concentrations in RA synovial fluid and serum samples. Because no IL-17A ELISA kit clearly gave the most consistent results across literature reports and because of our interest in specifically measuring the concentrations of different forms of IL-17 in patient samples, we developed immunoassays for IL-17AA, IL-17FF, and IL-17AF.

In this report, we describe efforts to reduce or eliminate HAb interference in these IL-17 assays. We first identified samples with false positive reactivity in each assay and then used these samples to screen several commercially available HAb blocking agents. One blocker was chosen as optimal and was used during analysis of synovial fluid and serum samples from patients with RA and osteoarthritis (OA) as well as sera from patients with IBD. While IL-17AA was not detected in RA or OA sera, false positive reactivity was readily apparent when the same samples were analyzed in the absence of the HAb blocking agent.

Section snippets

Generation of monoclonal antibodies against human IL-17A and F

Recombinant human IL-17 A or F was resuspended in monophosphoryl lipid A/trehalose dicorynomycolate adjuvant (Corixa Corporation, Seattle, WA). BALB/c mice were immunized into each hind footpad (2 μg/animal) at 3 to 4 day intervals for a total of 12 boosts. Three days after the final boost, popliteal lymph nodes were fused with subcloned mouse myeloma cell line P3X63AgU.1 (ATCC CRL1597; American Type Culture Collection, Rockville, MD) using polyethylene glycol (PEG). Hybridomas were selected

Identification of false positive samples

In order to evaluate the ability of HAb blocking agents to reduce interference in the IL-17AA assay, it was first necessary to identify samples with false positive reactivity. Of the panel of serum samples screened in the IL-17AA assay, 10 samples (5 Scantibodies and 5 in-house) gave signal above baseline. Further characterization of only 8 of these samples is presented here (Fig. 1, Fig. 2; Samples 01–08, Table 2).

To demonstrate whether the reactivities were true positives or false positives,

Discussion

Since the initial description of HAb interference by Prince et al. (1973), the impact of HAbs on two-site immunoassays has been extensively reviewed in the clinical chemistry literature (Kricka, 1999, Levinson and Miller, 2002, Bjerner et al., 2005a, Clinical and Laboratory Standards Institute, 2008). Although the reported incidence of HAbs in the general population varies widely depending on the study, some estimates are as high as 80% (Kricka, 1999) to 100% (Hennig et al., 2000a), indicating

Acknowledgements

The authors gratefully acknowledge the contributions of Sherry Yeh, Michelle Simpson, Kristen Wolslegl, Cecile Holweg, and Mary Keir, and thank Judy Young and Wenjun Ouyang for helpful discussions.

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