ABSTRACT
OBJECTIVES: To determine the clinical utility and performance characteristics of a laboratory-adapted flow cytometric method for the detection of acetylcholine receptor (AChR) modulating antibodies in myasthenia gravis (MG). METHODS: Serum samples from 120 healthy donors and 100 patients with suspected MG were assessed for the ability to reduce surface AChR concentrations (antigenic modulation) in RD (TE671) or DB40 human muscle cell lines by flow cytometry. Reference ranges were established by receiver operating characteristic curve analysis, and results were then compared with those of the current radioimmunoassay (RIA). RESULTS: Flow cytometric results from the RD cell line had an interpretive threshold of 46% modulation or greater and correlated best (98% sensitivity, 99% specificity) with those of the current RIA. CONCLUSIONS: The new flow cytometric method using the RD cell platform provided higher quality clinical results, a more robust and efficient assay format, a significant cost savings, and less environmental burden.
Subject(s)
Autoantibodies/blood , Flow Cytometry/methods , Myasthenia Gravis/diagnosis , Receptors, Cholinergic/immunology , Adult , Antibodies, Monoclonal , Area Under Curve , Autoantigens/immunology , Cell Line , Humans , Myasthenia Gravis/blood , Myasthenia Gravis/immunology , ROC Curve , Reference Values , Sensitivity and SpecificityABSTRACT
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.
Subject(s)
Antibodies, Bacterial/blood , Immunoglobulin G/blood , Luminescent Measurements/methods , Pneumococcal Vaccines/immunology , Protein Array Analysis/methods , Streptococcus pneumoniae/immunology , Enzyme-Linked Immunosorbent Assay , HumansABSTRACT
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.
