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.

[1]  G. Hughes,et al.  Serological markers in progressive systemic sclerosis: clinical correlations. , 1983, Annals of the rheumatic diseases.

[2]  A. Hood,et al.  Neonatal lupus erythematosus: new serologic findings. , 1981, The Journal of investigative dermatology.

[3]  J F Fries,et al.  The 1982 revised criteria for the classification of systemic lupus erythematosus. , 1982, Arthritis and rheumatism.

[4]  Y. Moroi,et al.  Antinuclear antibodies in the relatives of patients with systemic sclerosis , 1985, The British journal of dermatology.

[5]  T. Martins.  Development of Internal Controls for the Luminex Instrument as Part of a Multiplex Seven-Analyte Viral Respiratory Antibody Profile , 2002, Clinical and Vaccine Immunology.

[6]  F. Arnett,et al.  Ro(SSA) and La(SSB) antibodies in the clinical spectrum of Sjögren's syndrome. , 1982, The Journal of rheumatology.

[7]  Anne-Marie Rouquette,et al.  Evaluation of the new multiplexed immunoassay, FIDIS, for simultaneous quantitative determination of antinuclear antibodies and comparison with conventional methods. , 2003, American journal of clinical pathology.

[8]  K. Matsumoto,et al.  Clinical significance of IgG anti-Sm antibodies in patients with systemic lupus erythematosus. , 1990, The Journal of rheumatology.

[9]  E. Tan,et al.  Autoantibodies to nuclear antigens (ANA): their immunobiology and medicine. , 1982, Advances in immunology.

[10]  J. W. Pickering,et al.  A multiplexed fluorescent microsphere immunoassay for antibodies to pneumococcal capsular polysaccharides. , 2002, American journal of clinical pathology.

[11]  R J Fulton,et al.  Advanced multiplexed analysis with the FlowMetrix system. , 1997, Clinical chemistry.

[12]  Daniel P. Stites,et al.  Basic and Clinical Immunology , 1976 .

[13]  J. Sánchez-Guerrero,et al.  Utility of anti-Sm, anti-RNP, anti-Ro/SS-A, and anti-La/SS-B (extractable nuclear antigens) detected by enzyme-linked immunosorbent assay for the diagnosis of systemic lupus erythematosus. , 1996, Arthritis and rheumatism.

[14]  S. Orton,et al.  Practical Evaluation of Methods for Detection and Specificity of Autoantibodies to Extractable Nuclear Antigens , 2004, Clinical Diagnostic Laboratory Immunology.

[15]  T. Medsger,et al.  Clinical correlations and prognosis based on serum autoantibodies in patients with systemic sclerosis. , 1988, Arthritis and rheumatism.

[16]  A. Ahmed,et al.  Antibodies to cytoplasmic antigens in lupus erythematosus , 1977 .

[17]  E. Beutner,et al.  Scl 70 antibody—a specific marker of systemic sclerosis , 1986, The British journal of dermatology.

[18]  H. L. V. van Duijnhoven,et al.  A comparison of ELISA assays as routine diagnostic test for detection of autoantibodies against extractable nuclear antigens. , 1999, Clinical biochemistry.

[19]  S. Adelstein,et al.  Autoantibodies to Extractable Nuclear Antigens: Making Detection and Interpretation More Meaningful , 2002, Clinical and Vaccine Immunology.

[20]  R. Sinico,et al.  The use of laboratory tests in diagnosis and monitoring of systemic lupus erythematosus. , 2002, Journal of nephrology.

[21]  M. Reichlin,et al.  Patterns of clinical disease associated with antibodies to nuclear ribonucleoprotein. , 1978, The Journal of rheumatology.

[22]  R. Jonsson,et al.  Current concepts on diagnosis, autoantibodies and therapy in Sjögren's syndrome. , 2000, Scandinavian journal of rheumatology.

[23]  M. Mattioli,et al.  Correlation of a precipitin reaction to an RNAprotein antigen and a low prevalence of nephritis in patients with systemic lupus erythematosus. , 1972, The New England journal of medicine.

[24]  E. Tan,et al.  Antinuclear antibodies: diagnostic markers for autoimmune diseases and probes for cell biology. , 1989, Advances in immunology.

[25]  T. Jaskowski,et al.  Screening for antinuclear antibodies by enzyme immunoassay. , 1996, American journal of clinical pathology.

[26]  M. Satoh,et al.  Recombinant 52 kDa Ro(SSA) ELISA detects autoantibodies in Sjögren's syndrome sera that go undetected by conventional serologic assays. , 1997, The Journal of rheumatology.

[27]  J. W. Pickering,et al.  Comparison of a Multiplex Flow Cytometric Assay with Enzyme-Linked Immunosorbent Assay for Quantitation of Antibodies to Tetanus, Diphtheria, and Haemophilus influenzae Type b , 2002, Clinical and Vaccine Immunology.

[28]  M. Reichlin,et al.  Serological findings in patients with "ANA-negative" systemic lupus erythematosus. , 1981, Medicine.

[29]  E. Scopelitis,et al.  Anti-SS-A antibody and other antinuclear antibodies in systemic lupus erythematosus. , 1980, Arthritis and rheumatism.

[30]  C. K. Osterland,et al.  Laboratory diagnosis and monitoring in chronic systemic autoimmune diseases. , 1994, Clinical chemistry.

[31]  J. W. Pickering,et al.  Determination of cytokine responses using a multiplexed fluorescent microsphere immunoassay. , 2002, American journal of clinical pathology.

[32]  K. Kellar,et al.  Multiplexed fluorescent bead-based immunoassays for quantitation of human cytokines in serum and culture supernatants. , 2001, Cytometry.