Multilaboratory assessment of threshold versus fold-change algorithms for minimizing analytical variability in multiplexed pneumococcal IgG measurements.

Pneumococcal vaccination is frequently used to assess a patient's humoral immune function. The comparison of pre- and postvaccination levels of antipneumococcal antibodies is widely held to be the gold standard for documenting a response. However, many of the published criteria for defining an adequate response are based on assays that are no longer widely available. We compared the clinical classification of patient response by multiplex pneumococcal assays currently performed at three large reference laboratories using a variety of published criteria for defining responses in adults. The classification of responders agreed for 79% of the patients when using a threshold-based algorithm compared to 57 to 96% of the patients when using various fold-change-based algorithms. The highest rate of discordance was seen when the most stringent criteria for response were used (4-fold increase postvaccination in 70% of serotypes). The discordant samples tended to show similar patterns of response across all three assays, with small variations in the final number of serotypes converting postvaccination. We conclude that the use of published cut points for documenting response to pneumococcal vaccination can be affected by interlaboratory differences in pneumococcal assays, particularly for algorithms that require large fold changes for a response to be documented. However, the overall patterns of response were similar in virtually all samples, regardless of the assay used.

Measurement of antibodies to pneumococcal polysaccharides with Luminex xMAP microsphere-based liquid arrays.

The 23 valent pneumococcal polysaccharide vaccine (PPV) is often used to assess an individual's ability to produce antibodies to polysaccharides. The Luminex xMAP microsphere-based liquid array system, when applied to the determination of antibodies to pneumococcal polysaccharides (PnPs), allows for the antibody response to the 23 serotypes to be determined simultaneously. Multiplexing saves considerable time and expense over the traditional method of testing each PnPs serotype individually by the enzyme-linked immunosorbent assay (ELISA). This chapter describes methods for (a) conjugation of poly-L: -lysine (PLL) to PnPs, (b) coupling of PnPs-PLL conjugates to Luminex microspheres, and (c) a multiplex Luminex assay for the measurement of serotype-specific IgG concentrations to pneumococcal serotypes.

Elimination of false-positive results in a luminex assay for pneumococcal antibodies.

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 false-positive reactivity of both groups.

A 22-plex chemiluminescent microarray for pneumococcal antibodies.

We developed a chemiluminescent multiplexed microarray that simultaneously determines IgG antibody concentrations to 22 pneumococcal polysaccharide (PnPs) serotypes (1, 2, 3, 4, 5, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 23F, and 33F). We compared the microarray with an enzyme-linked immunosorbent assay (ELISA) for 9 of the 22 serotypes (1, 4, 5, 6B, 9V, 14, 18C, 19F, and 23F). Correlation coefficients (r2) for the comparison of the microarray with ELISA ranged from 0.91 to 0.97 for the 9 serotypes. The microarray detected more than 4-fold increases in antibody concentrations in serum samples from before and 1 month after administration of pneumococcal vaccine for all 22 serotypes tested. The mean interassay and intra-assay coefficients of variation for 12 serum samples for the 22 serotypes were 7.6% and 6.0%, respectively. Inhibition-of-binding studies showed more than 90% inhibition by homologous serotypes and, with few exceptions, less than 25% inhibition by heterologous serotypes. The microarray multiplexing technology is an attractive alternative to ELISA for antibody responses to 23-valent PnPs vaccines.

Evaluation of a (1->3)-beta-D-glucan assay for diagnosis of invasive fungal infections.

The Fungitell assay (Associates of Cape Cod, Inc.) is a commercial test that detects (1-3)-beta-D-glucan (BG) and is intended for diagnosis of invasive fungal infections. To evaluate the Fungitell assay, we tested serum and plasma samples from healthy blood donors and from patients with blood cultures positive for yeast or bacteria. All 36 blood donors were BG negative, and 13 of 15 candidemic patients were BG positive. Of 25 bacteremic patients, 14 (10 with gram-positive bacteremia) were BG positive. One of the latter patients with Staphylococcus aureus bacteremia also had invasive candidiasis, based on histological findings in a tissue biopsy; therefore, the BG result was a true positive. The sensitivity, specificity, and positive and negative predictive values of the Fungitell assay, by patient, for these three groups were 93.3%, 77.2%, 51.9%, and 97.8%, respectively. We also performed the Fungitell assay on sera that had been tested for Aspergillus galactomannan or Histoplasma antigen. All six Histoplasma antigen-positive patients and 31 of 32 Aspergillus galactomannan-positive patients were also BG positive. BG results for the 10 Histoplasma antigen-negative and the 32 Aspergillus galactomannan-negative patients varied, but we were unable to confirm many of the results. Between-run coefficients of variance (CVs) for the assay ranged from 3.2% to 16.8%; within-run CVs were < or =4.8%. The correlation coefficient for an interlaboratory reproducibility study was 0.9892. Concentrations of hemoglobulin, bilirubin, and triglycerides that caused 20% interference were 588, 72, and 466 mg/dl, respectively. Our results suggest that the Fungitel assay may be most useful for excluding invasive fungal infection.

Flow cytometric assay for genotyping cytochrome p450 2C9 and 2C19: comparison with a microelectronic DNA array.

INTRODUCTION: Cytochrome p450 (CYP) 2C9 and 2C19 metabolize a wide range of therapeutically important drugs. Genetic polymorphisms in the CYP2C9 and CYP2C19 genes result in variations in drug response. To correlate the dose required for therapeutic drug efficacy with genotype, accurate and reliable methods for detecting single nucleotide polymorphisms (SNPs) of CYP2C9 and CYP2C19 are required. STUDY DESIGN: We evaluated two technologies for genotyping CYP2C9 (*2 and *3 alleles) and CYP2C19 (*2 and *3 alleles). We developed a multiplexed flow cytometric assay based on the Luminex xMAP system and oligonucleotide-tagged Universal Array microspheres. The Luminex assay was compared with the eSensor DNA detection system, provided by Motorola Life Sciences. Genotypes determined by the two methods were confirmed by sequence analysis. RESULTS: Of the 101 whole-genome amplified DNA samples genotyped by the Luminex method, 15 (14.8%) were heterozygous and 1 was homozygous for the CYP2C9*2 polymorphism. For the CYP2C9*3 polymorphism, 13 (12.9%) were heterozygous and 1 was homozygous. Two samples had the CYP2C9*2/*3 genotype. For CYP2C19*2, 17 (16.8%) of the samples were heterozygous and one was homozygous. The CYP2C19*3 polymorphism was not found. Genotypes determined by the Luminex assay were in complete concordance with the eSensor SNP assay results. A dilution study showed that 1.5 ng of nucleic acid was adequate for PCR and subsequent detection of SNPs by the Luminex assay. The within run and between run coefficients of variance (CVs) for allelic ratios determined by the Luminex procedure were found to be

Determination of cytokine responses using a multiplexed fluorescent microsphere immunoassay.

We used a multiplexedfluorescent microsphere immunoassay to develop a sandwich capture assay to assess simultaneously the production of thymus helper (TH) 1- and TH2-type cytokines in tissue culture supernatant obtained from stimulated peripheral blood mononuclear cells. The assay then was used to assess the cytokine production of patients with hyperimmunoglobulinemia E syndrome and in cord blood from neonates. The multiplexed assay has a reportable range of less than 10 to 50,000 pg/mL. For linearity and recovery studies, R2 values for the 6 cytokines ranged from 0.988 to 0.999 for samples spiked with known concentrations of recombinant cytokine standards and for patient samples. The assay showed good specificity, with little cross-reactivity between cytokines. Results from supernatants of Staphylococcus aureus-stimulated peripheral blood mononuclear cells obtainedfrom 6 patients with hyperimmunoglobulinemia E syndrome showed significantly less interferon (IFN)-gamma production than cells from healthy control subjects. Cord blood cells from neonates produced significantly less interleukin 12 and IFN-gamma than cells from adults in group B streptococci-stimulated mononuclear cells. The fluorescent multiplexed microsphere immunoassay can be used to quantitate multiple cytokines from 1 sample and should be useful for further understanding of the cytokine role in disease.

Comparison of a multiplex flow cytometric assay with enzyme-linked immunosorbent assay for auantitation of antibodies to tetanus, diphtheria, and Haemophilus influenzae Type b.

We developed a multiplexed indirect immunofluorescence assay for antibodies to Haemophilus influenza type b (Hib) polysaccharide and the toxoids of Clostridium tetani (Tet) and Corynebacterium diphtheriae (Dip) based on the Luminex multiple-analyte profiling system. A pooled serum standard was calibrated against World Health Organization standards for Dip and Tet and an international standard for Hib. The multiplexed Luminex assay was compared to individual enzyme-linked immunosorbent assays (ELISAs) for the same analytes. By both methods, 75 (92.6%) of 81 of random serum samples had protective levels of antibody to Tet (> or = 0.1 IU/ml). For Dip, 81.5% of the samples had protective antibody levels (> or = 0.1 IU/ml) by ELISA and 80.2% had protective antibody levels by Luminex. Protective levels (> or = 1.0 microg/ml) of antibody to Hib were found in 45.0% of the samples tested by ELISA and in 39.0% of the samples tested by Luminex. The correlations (R(2)) between ELISA and Luminex of the 81 samples were 0.96, 0.96, and 0.91 for Tet, Dip, and Hib, respectively. There was also similar agreement between Luminex and ELISA for sera collected before and 1 month after Tet, Dip, and Hib vaccine administration. Both methods detected strong postvaccination responses. The Luminex method is an attractive alternative to ELISA since it reduces labor and reagent costs, as well as assay time.

A multiplexed fluorescent microsphere immunoassay for antibodies to pneumococcal capsular polysaccharides.

We developed a multiplexed indirect immunofluorescent assay for antibodies to pneumococcal polysaccharides (PnPs) based on the Luminex multiple analyte profiling system (Luminex, Austin, TX). The assay simultaneously determines serum IgG concentrations to 14 PnPs serotypes: 1, 3, 4, 5, 6B, 7F, 8, 9N, 9V, 12F; 14, 18C, 19F, and 23F. To assess the specificity of the multiplexed assay for each individual serotype, inhibition-of-binding studies were conducted using adult serum samples obtained after pneumococcal vaccination. Except for the closely related serotypes 9V and 9N, we demonstrated inhibition by homologous serotypes of more than 95% and inhibition by heterologous serotypes of less than 15% for all 14 PnPs serotypes. There was, however, high heterologous inhibition of 50% or greater with some serotypes. These cross-reacting antibodies could not be removed by preabsorption with pneumococcal C-polysaccharide but were removed by additional preabsorption with serotype 22F polysaccharide. The multiplexed Luminex assay showed good overall agreement with a well-established enzyme-linked immunosorbent assay that is currently recommended for evaluation of pneumococcal vaccine immunogenicity.