2166 DIABETES, VOL. 48, NOVEMBER 1999 Heterophile Anti-Mouse Immunoglobulin Antibodies May Interfere With Cytokine Measurements in Patients With HLA Alleles Protective for Type 1A Diabetes Maria J. Redondo, Peter A. Gottlieb, Teresa Motheral, Christine Mulgrew, Marian Rewers, Sunanda Babu, Elizabeth Stephens, Dale R. Wegmann, and George S. Eisenbarth Wilson and coworkers (Wilson SB, Kent SC, Patton KT, Orban T, Jackson RA, Exley M, Porcelli S, Schatz DA, Atkinson MA, Balk SP, Strominger JL, Hafler DA: Extreme Th1 bias of invariant V24J Q T-cells in type 1 diabetes. Nature 391:177–181, 1998) have recently reported raised serum levels of interleukin-4 (IL-4) in anti-islet autoantibody-positive first-degree relatives of patients with type 1A diabetes who did not progress to diabetes. Protection from diabetes has been noted for several human lymphocyte antigen (HLA) alleles, such as HLA DR2-DQA1*0102- DQB1*0602. We, therefore, wanted to determine whether this cytokine phenotype was associated with HLA genes protective for type 1A diabetes. We used a two-site fluoroimmunoassay with the same monoclonal antibodies as those reported by Wilson et al. Using this assay, we have found evidence for human heterophile antibodies mimicking serum IL-4: all serum IL-4 reac- tivity was lost if mouse serum or mouse immunoglobu- lin were added to the assay; serum IL-4 activity was bound and then eluted by protein A/G chromatography; and levels of anti-mouse antibodies correlated with apparent serum IL-4. This pseudo-IL-4 activity was found in a subset of control subjects, patients with type 1A diabetes, and their relatives and was primarily asso- ciated with specific HLA alleles protective for type 1A diabetes (e.g., DQB1*0602). After adjustment for HLA, positive levels of heterophile antibodies were not asso- ciated with protection from diabetes. The confounding effect of protective HLA alleles associated with het- erophile antibodies could explain the previously reported association between raised serum IL-4 and protection from type 1A diabetes. The mechanism by which specific DQ alleles protect from diabetes and are associated with increased heterophile antibodies is cur- rently unknown. Diabetes 48:2166–2170, 1999 R aised serum levels of interleukin-4 (IL-4) have been reported in anti-islet autoantibody-positive first-degree relatives of patients with type 1A dia- betes who did not progress to diabetes (1). Pro- tection from diabetes is also associated with specific human lymphocyte antigen (HLA) alleles, such as HLA DR2- DQA1*0102-DQB1*0602 (2,3), the strongest protective hap- lotype, and DQA1*0501-DQB1*0301 (4). The aim of the current study was to determine whether elevated serum IL-4 was associated with HLA alleles protective for type 1A diabetes. A sensitive time-resolved two-site fluoroimmunoassay with Europium with the same monoclonal antibodies as reported by Wilson et al. (1) was initially used to determine serum levels of IL-4. We examined a series of patients with type 1A diabetes, control subjects, and autoantibody-positive and -negative first-degree relatives of patients with type 1A dia- betes. We found that elevated serum IL-4 levels were corre- lated with the presence of HLA-DQA*0102/DQB*0602 or HLA-DQA*0501/DQB*0301. As we were conducting these studies, our colleague Dr. John Hutton raised a concern regarding a potential artifact of two-site antibody assays, namely false positives created by heterophile antibodies in a subset of individuals. Heterophile antibodies have been reported to both falsely raise and falsely lower immunoassay results (5–9). The current report documents the presence of heterophile antibodies producing false-positive IL-4 determinations. RESEARCH DESIGN AND METHODS Subjects. Serum samples from 182 patients with type 1A diabetes, 307 first- degree relatives of patients with type 1A diabetes, and 165 control subjects with- out a family history of type 1A diabetes were tested with a Europium-based IL-4 immunoassay. The control group included 109 3-year-old children from the gen- eral population screened as part of the Diabetes Autoimmunity Study in the Young (DAISY) study (10–11). Among the 307 first-degree relatives, 99 were pos- itive for at least one anti-islet autoantibody out of three measured (autoantibod- ies to insulin, GAD65, and ICA512/IA-2) (autoantibody-positive relatives). The remaining 208 relatives were negative for all three autoantibodies (autoantibody- negative relatives). All subjects donated their blood for the above studies, with Institutional Board Approval for the studies including signed consent. Europium cytokine assay. The assay used was modified from that kindly pro- vided by David Hafler and colleagues (1). Capture mouse anti-human IL-4 anti- body (cat. no. 18651D, clone 8D4-8; PharMingen, San Diego, CA) (4 μg/ml) or mouse anti-human -interferon (IFN-) antibody (cat. no. 18891D; PharMingen) (2 μg/ml) was adsorbed to the wells of 96-well modified flat-bottom enzyme-linked From the Barbara Davis Center for Childhood Diabetes, University of Col- orado Health Sciences Center, Denver, Colorado. Address correspondence and reprint requests to George S. Eisenbarth, MD, PhD, Barbara Davis Center for Childhood Diabetes, University of Col- orado Health Sciences Center, Box B140, 4200 E 9th Ave., Denver, Colorado 80262. E-mail: george.eisenbarth@uchsc.edu. Received for publication 10 May 1999 and accepted in revised form 30 July 1999. BSA, bovine serum albumin; IFN-, -interferon; IL-4, interleukin-4; PBS, phosphate-buffered saline.