Thomas B. Martins

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.

Comparison of a Multiplex Flow Cytometric Assay with Enzyme-Linked Immunosorbent Assay for Quantitation of Antibodies to Tetanus, Diphtheria, and Haemophilus influenzae Type b

ABSTRACT 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 (≥0.1 IU/ml). For Dip, 81.5% of the samples had protective antibody levels (≥0.1 IU/ml) by ELISA and 80.2% had protective antibody levels by Luminex. Protective levels (≥1.0 μg/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 (R2) 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.

Development of Internal Controls for the Luminex Instrument as Part of a Multiplex Seven-Analyte Viral Respiratory Antibody Profile

ABSTRACT The ability of the Luminex system to simultaneously quantitate multiple analytes from a single sample source has proven to be a feasible and cost-effective technology for assay development. In previous studies, my colleagues and I introduced two multiplex profiles consisting of 20 individual assays into the clinical laboratory. With the Luminex instrument’s ability to classify up to 100 distinct microspheres, however, we have only begun to realize the enormous potential of this technology. By utilizing additional microspheres, it is now possible to add true internal controls to each individual sample. During the development of a seven-analyte serologic viral respiratory antibody profile, internal controls for detecting sample addition and interfering rheumatoid factor (RF) were investigated. To determine if the correct sample was added, distinct microspheres were developed for measuring the presence of sufficient quantities of immunoglobulin G (IgG) or IgM in the diluted patient sample. In a multiplex assay of 82 samples, the IgM verification control correctly identified 23 out of 23 samples with low levels (<20 mg/dl) of this antibody isotype. An internal control microsphere for RF detected 30 out of 30 samples with significant levels (>10 IU/ml) of IgM RF. Additionally, RF-positive samples causing false-positive adenovirus and influenza A virus IgM results were correctly identified. By exploiting the Luminex instrument’s multiplexing capabilities, I have developed true internal controls to ensure correct sample addition and identify interfering RF as part of a respiratory viral serologic profile that includes influenza A and B viruses, adenovirus, parainfluenza viruses 1, 2, and 3, and respiratory syncytial virus. Since these controls are not assay specific, they can be incorporated into any serologic multiplex assay.

Analysis of Proinflammatory and Anti-Inflammatory Cytokine Serum Concentrations in Patients With Multiple Sclerosis by Using a Multiplexed Immunoassay

We examined cytokines and other inflammatory markers in serum samples from 833 patients with multiple sclerosis and 117 healthy control subjects. A multiplexed immunoassay was used to assess the concentrations of 13 cytokines/inflammatory markers: interferon (IFN)-γ; interleukins (ILs)-1β, 2, 4, 5, 6, 8, 10, 12, and 13; tumor necrosis factor (TNF)-α; IL-2 receptor; and soluble CD40 ligand. Significant increases between patients and control subjects were found for IFN-γ (mean, 7.5 vs 0.4 pg/mL; P = .0002), IL-2 (mean 5.7 vs 1.0 pg/mL; P =.0002), IL-1β (mean, 23.0 vs 11.3 pg/mL; P ≤ .0001), TNF-α (mean, 4.1 vs 1.2 pg/mL; P = .01), IL-4 (mean, 1.4 vs 0.1 pg/mL; P ≤ .0001), IL-10 (mean, 16.8 vs 7.5 pg/mL; P = .03), and IL-13 (mean, 4.5 vs 0.8 pg/mL; P ≤ .0001). Profiling cytokines in multiple sclerosis may help to identify mechanisms involved in the pathogenesis of the disease, aid in monitoring the disease course and in evaluating responses to specific therapies, and, potentially, lead to new therapies directed at cytokines or their receptors.

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.

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 μl 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.

Evaluation of Multiplexed Fluorescent Microsphere Immunoassay for Detection of Autoantibodies to Nuclear Antigens

ABSTRACT Antibodies to extractable nuclear antigens (ENA) are found in a variety of collagen vascular diseases. Determining the individual specificities of these antibodies is extremely useful in establishing the disease diagnosis and in some cases the prognosis. With a multiplexed fluorescent microsphere immunoassay, reactivity to five of the most diagnostically useful ENA was measured in 249 serum samples, including samples from 56 patients previously documented to have systemic lupus erythematosus (SLE). Results of the multiplexed assay were compared to results from established ENA enzyme-linked immunosorbent assays (ELISAs), and the agreement, sensitivity, and specificity, respectively, for the five ENA evaluated were as follows: SSA, 99.1, 100.0, and 98.8%; SSB, 98.6, 88.9, and 99.5%; Sm, 97.6, 95.8, and 97.9%; RNP, 97.2, 92.7, and 98.8%; Scl-70, 93.6, 50.0, and 99.0%. In the 56 confirmed SLE patients, the frequency of significant concentrations of autoantibodies with the multiplexed assay was 21.4% for SSA, 7.1% for SSB, 10.7% for Sm, 32.1% for RNP, and 0% for Scl-70. The new flow cytometric bead-based multiplexed assay showed excellent correlation with the well-established single-analyte ELISA methods for four of five the ENA markers investigated in this study. The most notable discrepancies between the two assays were for the Scl-70 antigen, which was most often resolved in favor of the multiplexed assay. Our studies show that the multiplexed microsphere-based immunoassay is a sensitive and specific method for the detection and semiquantitation of ENA antibodies in human sera.

Severity of symptom flare after moderate exercise is linked to cytokine activity in chronic fatigue syndrome

Chronic fatigue syndrome (CFS) patients often report symptom flare (SF) for >24 h after moderate exercise (post-ex). We hypothesized that SF is linked to increases in circulating cytokines and CD40 Ligand (CD40L). In 19 CFS patients and 17 controls, mental and physical fatigue and pain symptom ratings were obtained together with serum for 11 cytokines and CD40L before and at 0.5, 8, 24, and 48 h post-ex. Before exercise, CFS had lower CD40L (p<.05) but similar cytokines versus controls. In subgroups based on SF at 48 h, high SF patients (n=11) increased in IL-1beta, IL-12, IL-6, IL-8, IL-10, and IL-13 (p<.05) 8 h post-ex. Low SF patients (n=8) showed post-ex decreases in IL-10, IL-13, and CD40L, and controls decreased in IL-10, CD40L, and TNFalpha (p<.05). Thus, in CFS, cytokine activity may vary directly with SF, which may explain prior inconsistent findings.

Risk Factor Analysis of Plasma Cytokines in Patients With Coronary Artery Disease by a Multiplexed Fluorescent Immunoassay

Coronary artery disease (CAD) is the leading cause of death in the United States. Increasing evidence suggests involvement of inflammation in the atherosclerotic process. We examined cytokines and other inflammatory markers in 865 patients with chest pain in whom coronary angiography revealed no evidence of CAD or CAD with or without concomitant myocardial infarction (MI). We developed a multiplexed immunoassay to simultaneously assess the plasma concentrations of 8 cytokines (interferon gamma, interleukin (IL)-2, IL-4, IL-6, IL-8, IL-10, IL-12, and tumor necrosis factor alpha), IL-2r, and soluble CD40 ligand in the patient groups. Concentrations of C-reactive protein (CRP) and IL-18 also were determined. Significant differences (P < .05) between no CAD and combined CAD groups were found for IL-2, IL-4, IL-6, IL-12, and IL-18. When the no CAD group was compared with the group with CAD with subsequent MI, significant differences were found for proinflammatory markers IL-6 (P pound .001), IL-8 (P = .017), and CRP (P pound .001). Cytokine profiles may have a role in differentiating patients with CAD with MI from those with chest pain due to other disorders and in deciphering the role of inflammation in the pathogenesis of CAD.

Higher Serum 25-Hydroxyvitamin D Concentrations Associate with a Faster Recovery of Skeletal Muscle Strength after Muscular Injury

The primary purpose of this study was to identify if serum 25-hydroxyvitamin D (25(OH)D) concentrations predict muscular weakness after intense exercise. We hypothesized that pre-exercise serum 25(OH)D concentrations inversely predict exercise-induced muscular weakness. Fourteen recreationally active adults participated in this study. Each subject had one leg randomly assigned as a control. The other leg performed an intense exercise protocol. Single-leg peak isometric force and blood 25(OH)D, aspartate and alanine aminotransferases, albumin, interferon (IFN)-γ, and interleukin-4 were measured prior to and following intense exercise. Following exercise, serum 25(OH)D concentrations increased (p < 0.05) immediately, but within minutes, subsequently decreased (p < 0.05). Circulating albumin increases predicted (p < 0.005) serum 25(OH)D increases, while IFN-γ increases predicted (p < 0.001) serum 25(OH)D decreases. Muscular weakness persisted within the exercise leg (p < 0.05) and compared to the control leg (p < 0.05) after the exercise protocol. Serum 25(OH)D concentrations inversely predicted (p < 0.05) muscular weakness (i.e., control leg vs. exercise leg peak isometric force) immediately and days (i.e., 48-h and 72-h) after exercise, suggesting the attenuation of exercise-induced muscular weakness with increasing serum 25(OH)D prior to exercise. Based on these data, we conclude that pre-exercise serum 25(OH)D concentrations could influence the recovery of skeletal muscle strength after an acute bout of intense exercise.