In our 14-valent Luminex assay for pneumococcal antibodies, we identified two groups of sera that caused false-positive results. The first group bound nonspecifically to the Luminex microspheres. The second group reacted specifically with bovine serum albumin (BSA). We describe here methods that eliminated the falsepositive reactivity of both groups.We previously described a multiplexed pneumococcal antibody assay based on the Luminex xMAP technology (11). Several other laboratories subsequently also described multiplexed Luminex assays for detecting these antibodies (1,6,15). In addition, the Luminex xMAP technology has been used for multiplexed assays to measure antibody responses to vaccines for other infectious diseases, including those caused by Neisseria meningitidis, Haemophilus influenza type b, Bordetella pertussis, Clostridium tetani, Corynebacterium diphtheriae, Bacillus anthracis, and papilloma virus (3,4,10,12,13,17). Waterboer et al. (18) documented an intrinsic problem with the use of the Luminex technology for serological assays. They found that some human sera bind directly to the carboxylated MicroPlex (formally MultiAnalyte) microspheres, causing a very high level of nonspecific background. These workers also found that SeroMAP microspheres, introduced by Luminex Corporation specifically for use in serological assays, reduced but did not eliminate the nonspecific-binding problem.Using our original protocol (11) for the 14-valent pneumococcal antibody assay with lot B MicroPlex microspheres, we encountered serum samples with very high-level false-positive results that were near or above the analytical measurement range (AMR) of the assay (Table 1). Approximately 15 of every 1,000 sera tested for pneumococcal antibodies exhibited this behavior. We termed these samples "polyspecific," although they did not react specifically to pneumococcal polysaccharides (PnPs). We tested a panel of 33 of these polyspecific sera and 1 control serum sample not showing polyspecific reactivity against an unconjugated MicroPlex microsphere and an unconjugated SeroMAP microsphere. The serum samples used in this study were submitted to ARUP Laboratories for pneumococcal antibody testing. All samples were deidentified according to protocols approved by the University of Utah Institutional Review Board (no. 7275). Serum samples were diluted 1:25 in phosphate-buffered saline (PBS), pH 7.2, with 5 g/ml pneumococcal C-polysaccharide (C-Ps) (Staten Serum Institut, Copenhagen, Denmark), 5 g/ml pneumococcal polysaccharide 22F (American Type Culture Collection, Manassas, VA), and 0.0015% bromcresol purple (BCP) (Sigma-Aldrich, St. Louis, MO). A MicroPlex (region 7) (Luminex Corporation, Austin, TX) microsphere and a SeroMAP (region 8) (Luminex Corporation, Austin, TX) microsphere were pelleted by centrifugation and resuspended in blocking/storage (B/S) buffer consisting of PBS with 0.1% bovine serum albumin (BSA) (Sigma-Aldrich, St. Louis, MO) or in BSA-free StabliGuard immunoassay stabilizer (SG01) (SurModics, Inc., Eden P...
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