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

Martins, Thomas B.; Burlingame, Rufus W.; Mühlen, Carlos Alberto von; Jaskowski, Troy D.; Litwin, Christine M.; Hill, Harry R.

doi:10.1128/cdli.11.6.1054-1059.2004

Cited by 70 publications

(49 citation statements)

References 28 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…For example, serum concentration can alter the standard curve, and some studies revealed that human sera may contain heterophilic antibodies that directly bind to beads causing immunoassay interference. 49,106,156 Methods including dilution of sera and inactivation of complement in a HLA BBMA prevented false-negative reactions caused through a "prozone" effect. 158 Nonspecific binding of antibodies to microspheres causing false-positive reactions was also noted in another study.…”

Section: Sample and Assay Optimizationmentioning

confidence: 99%

Opportunities for bead-based multiplex assays in veterinary diagnostic laboratories

et al. 2013

View full text Add to dashboard Cite

Abstract. Bead-based multiplex assays (BBMAs) are applicable for high throughput, simultaneous detection of multiple analytes in solution (from several to 50-500 analytes within a single, small sample volume). Currently, few assays are commercially available for veterinary applications, but they are available to identify and measure various cytokines, growth factors and their receptors, inflammatory proteins, kinases and inhibitors, neurobiology proteins, and pathogens and antibodies in human beings, nonhuman primates, and rodent species. In veterinary medicine, various nucleic acid and protein-coupled beads can be used in, or for the development of, antigen and antibody BBMAs, with the advantage that more data can be collected using approximately the same amount of labor as used for other antigen and antibody assays. Veterinary-related BBMAs could be used for detection of pathogens, genotyping, measurement of hormone levels, and in disease surveillance and vaccine assessment. It will be important to evaluate whether BBMAs are "fit for purpose," how costs and efficiencies compare between assays, which assays are published or commercially available for specific veterinary applications, and what procedures are involved in the development of the assays. It is expected that many veterinary-related BBMAs will be published and/or become commercially available in the next few years. The current review summarizes the BBMA technology and some of the currently available BBMAs developed for veterinary settings. Some of the human diagnostic BBMAs are also described, providing an example of possible templates for future development of new veterinary-related BBMAs.

show abstract

Section: Sample and Assay Optimizationmentioning

confidence: 99%

Opportunities for bead-based multiplex assays in veterinary diagnostic laboratories

et al. 2013

View full text Add to dashboard Cite

show abstract

“…Multiplex assays based on fluorescent microbead flow cytometry and microarray systems have proven to be powerful tools, supporting greater throughput analysis and more comprehensive testing of multiple patient samples (4)(5)(6)(7)(8)(9)(10)(11). The novel detection technology presented in this study employs fluorescence-coded microbeads detected by a multicolor fluorescence image-capture based system with novel pattern recognition algorithms for multiplex testing (12).…”

mentioning

confidence: 99%

Multiplex assessment of non‐organ‐specific autoantibodies with a novel microbead‐based immunoassay

Großmann

Roggenbuck

Schröder³

et al. 2011

Cytometry Pt A

View full text Add to dashboard Cite

Advances in immunofluorescence assay development paved the way for the simultaneous detection of several antibodies in one sample, for the serological diagnosis of systemic rheumatic diseases. Standardized automated screening of such antibodies can be achieved by HEp-2 cell-based indirect immunofluorescence (IIF) using a multicolor fluorescence imaging technical platform. To create a common platform for both screening and specific antibody assessment, multiplex measurement of antibodies using fluorescence-coded immobilized microbeads was employed on the same platform. The multicolor fluorescence detection system VideoScan (AKLIDES 1 ) was used for the fluorescence analysis of a multiplex microbead-based immunoassay (MIA). First, immunoglobulin G (IgG) was covalently coupled to one microbead population in duplicate and in three independent experiments. The coupled IgG was detected by a Cy TM 5-conjugated secondary antibody. Thus, intra-and interassay coefficients of variation (CV) were obtained. Second, a multiplex determination of antinuclear autoantibodies (ANA) to Scl-70, Sm, dsDNA, SS-A (Ro60), CENP-B, and La/SS-B by solid-phase MIA was investigated, using 72 sera from patients with autoimmune diseases such as systemic lupus erythematosus and systemic sclerosis (SS). The reproducibility study revealed intra-assay CVs ranging from 3.2% to 9.9%, and interassay CVs ranging from 9.6% to 14.7%. The detection of Scl-70-, Sm-, CENP-B-, and La/SS-B-ANA with MIA showed very good agreement with the ELISA results (kappa 5 1.0). The resulting relative sensitivities and specificities for Scl-70-, Sm-, CENP-B-, dsDNA-, and La/SS-B-ANA were 100%, respectively, with the exception of dsDNA (specificity 97%). Multiplex detection by immobilized fluorescence-coded microbeads using multicolor fluorescence is a reliable method for the assessment of rheumatic-disease-specific antibodies. Multicolor fluorescence analyses with pattern detection algorithms provide a common platform technique for both the screening of ANA by cell-based IIF and specific antibody assessment by multiplex detection. ' 2011 International Society for Advancement of Cytometry Key termsHEp-2 cell; multiplex; microbead-based immunoassay; autoantibodies ANTINUCLEAR antibodies (ANA) to extractable nuclear antigens (ENA) are hallmarks in the serological diagnosis of systemic rheumatic diseases (1). The detection of ANA by indirect immunofluorescence (IIF) employing HEp-2 cells as a multiple antigen source has been established as the ''gold standard'' in routine diagnostics (2). However, advances in assay development and recombinant technology have paved the way for the detection of ANA to individual ENA, such as double-stranded DNA (dsDNA), improving the diagnostic power of ANA testing. The growing variety of ANA found in different systemic rheumatic diseases has generated the need for innovative techniques to overcome the shortcomings of single ENA detection, with regard to cost and time taken to obtain results (3). Hence, multiplexed platforms have been ...

show abstract

“…Very elevated titers of anti-Ro antibodies may be useful in the assessment of disease activity especially in patients that do not present with elevated titers of antidsDNA autoantibodies. In another recent study, 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 SLE (Martins et al 2004). Significant concentrations of 5 autoantibodies measured by the multiplexed assay in 56 SLE patients were similar to ours, except for RNP, where we had a lower frequency (Table I).…”

Section: Discussionmentioning

confidence: 99%

“…The multiplex bead array system has recently been utilized for autoantibody detection in various autoimmune diseases (Martins et al 2004). Thus, in our previous study, we compared the specificity and sensitivity of the recognition of ANA and 9 autoantibodies by the AtheNA system to the established ELISA method.…”

Section: Discussionmentioning

confidence: 99%

The Homogeneous Multiplexed System‐a New Method for Autoantibody Profile in Systemic Lupus Erythematosus

Zandman‐Goddard

Gilburd

Shovman

et al. 2005

Journal of Immunology Research

View full text Add to dashboard Cite

Systemic lupus erythematosus (SLE) is a multi-systemic autoimmune disease leading to immunological aberrations and excessive multiple autoantibody production. The aim of this study was to investigate the prevalence of multiple autoantibodies in SLE patients utilizing the multiplex system method.We analyzed the presence of elevated titers of anti-Ro, anti-La, anti-RNP, anti-Sm, anti-Jo1, anti-centromere, anti-Scl-70, anti-histone, and anti-dsDNA antibodies in 199 serum samples (113 SLE patients, 86 healthy donors). We compared the type, level and number of autoantibodies and the correlation between the autoantibody profile and disease severity utilizing the SLEDAI score.Elevated titers of at least one autoantibody were detected in 48% of 42 SLE patients. Elevated titers of anti-Ro antibodies were most commonly detected. The distribution of specific autoantibodies was: anti-Ro-23.8%, anti-dsDNA-19%, antihistone-19%, anti-RNP-14.2%, anti-La antibodies-11.9%, anti-Sm-7.1%, anti-Scl 70-4.7%, and anti-centromere-2.4%. Utilizing ROC analysis, the sensitivity and specificity of anti-DNA antibodies at a cutoff value of 34 IU/ml were 87.1% and 79.4% respectively. Elevated titers of anti-Jo1 antibody were not detected. There was a correlation with the titer of anti-Ro antibodies and disease activity by the SLEDAI score. Seven patients harbored one autoantibody only, 15 patients harbored 2-3 autoantibodies, 3 patients harbored 4-5 autoantibodies, and one patient harbored 6 autoantibodies. A correlation between the number of autoantibodies per patient and disease severity was found. One patient with a multitude of autoantibodies had severe lupus and a myriad of clinical manifestations.In conclusion, the multiplex system is specific and sensitive, provides an autoantibody profile in a single test, and may be useful as a diagnostic test for SLE. Elevated anti-Ro antibodies are associated with severe disease. An autoantibody load may be indicative of more severe disease.

show abstract

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

Cited by 70 publications

References 28 publications

Opportunities for bead-based multiplex assays in veterinary diagnostic laboratories

Opportunities for bead-based multiplex assays in veterinary diagnostic laboratories

Multiplex assessment of non‐organ‐specific autoantibodies with a novel microbead‐based immunoassay

The Homogeneous Multiplexed System‐a New Method for Autoantibody Profile in Systemic Lupus Erythematosus

Contact Info

Product

Resources

About