Standardized serological tests were performed in the College of American Pathologists and Clinical Laboratory Improvement Amendments–certified OMRF Morris Reichlin Clinical Immunology Laboratory at the time of enrollment in the LFRR. ANAs were determined by indirect immunofluorescence with human epithelial type 2 cells and anti‐dsDNA autoantibodies by immunofluorescence against Crithidia (Inova Diagnostics). Precipitating levels of autoantibodies directed against Ro/SSA, La/SSB, Sm, RNP, and ribosomal P were detected by immunodiffusion (30 ). Samples were also tested for 11 autoantibody specificities using an xMAP multiplex assay (BioPlex 2200; Bio‐Rad Technologies). The BioPlex 2200 ANA kit uses fluorescently labeled magnetic beads for the simultaneous detection of 11 autoantibody specificities, including dsDNA, ribosomal P, chromatin, Ro/SSA, La/SSB, Sm, the Sm/RNP complex, RNP, Jo‐1, Scl‐70, and centromere B. Serum verification beads specific for factor XIII are included in each reaction as a positive control (Bio‐Rad Technologies). Levels of Ro52 autoantibodies were confirmed by enzyme‐linked immunosorbent assay (ELISA) (Inova Diagnostics).
Bioplex 2200
Manufactured by Bio-Rad
Sourced in United States, Canada
The BioPlex 2200 is an automated multiplex assay system designed for the quantitative analysis of multiple analytes in a single sample. It utilizes magnetic bead-based technology to perform simultaneous measurements of various biomarkers, proteins, and other molecules. The system offers high-throughput capabilities and is suitable for a wide range of applications in clinical diagnostics, research, and drug development.
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Lab products found in correlation
Elisa, by Inova Diagnostics (2 mentions)
Bio plex 200, by Bio-Rad (1 mentions)
Multiplex assay, by R&D Systems (1 mentions)
Line blot lia, by EUROIMMUN (1 mentions)
Multiplex bead technology, by DiaSorin (1 mentions)
Centaur xp, by Siemens (1 mentions)
Immulite 2000, by Siemens (1 mentions)
Elisa, by R&D Systems (1 mentions)
Hep 2 cells, by Bio-Rad (1 mentions)
Human igg elisa quantitation set e80 104, by Fortis Life Sciences (1 mentions)
Euroline systemic sclerosis nucleoli profile igg, by EUROIMMUN (1 mentions)
Hemosil silica clotting time, by Werfen (1 mentions)
Dilute russell s viper venom time, by Siemens (1 mentions)
29 protocols using bioplex 2200
1
Standardized Serological Tests for Autoantibody Screening
2
SARS-CoV-2 Spike Protein Antibody Assay
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Example 9
A population of fluorescently labeled beads to which an individual SARS-CoV-2 spike protein, spike protein variant or fragment thereof is immobilized is contacted with a patient sample, e.g., a biological sample from an individual suspected of having been exposed to SARS-CoV-2 or a subject immunized with a vaccine comprising SARS-CoV2 spike protein along with a sample diluent. After incubation unbound sample is washed away. A biotinylated fusion protein comprising human ACE2 receptor fused to an immunoglobulin Fc domain (a hACE2R-Fc fusion protein biotinylated on the Fc domain) is added to the washed beads and incubated. The reaction is incubated and then washed and streptavidin-PE is added to the washed population of beads and incubated. After incubation, the population of beads is washed prior to detection using a Bio-Plex 2200, Bio-Plex 200 or Luminex LX-200 platform. The sample fluorescence intensity is compared to the fluorescence intensity of a set of standards or calibrators to generate the relative fluorescence intensity (RFI) and, subsequently, a qualitative, semi-quantitative or quantitative result. A lack of signal or a reduced signal (relative to a set of standards or calibrators) indicates that a neutralizing antibody to that SARS-CoV-2 spike protein variant or fragment thereof is present in the sample.
3
Multiplex Autoantibody Screening Protocol
4
Measles Immunity Assessment via BioPlex and PRNT
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All testing was performed at the National Microbiology Laboratory (NML) in Winnipeg, Canada. Samples were first tested using the BioPlex 2200 measles, mumps, rubella and varicella-zoster virus (MMRV) IgG assay, which was previously validated by our Network for measles sero-epidemiology work [23] . In our validation study, we determined the cut-offs that would result in a 100% sensitivity (95% CI = 93.5–100%) and 100% specificity (95% CI = 83.2–100%), which were then used in our study [23] . Samples with a BioPlex result ≥ 1.1 AU/mL were considered positive, those 0.13≤ and <1.1 AU/mL were equivocal, and <0.13 AU/mL were negative [23] . We retested samples with negative or equivocal BioPlex results using a Plaque Reduction Neutralization Test (PRNT), which is the gold-standard test for measuring measles immunity [24] . The PRNT was performed at the NML as previously described [25] (link). Samples with a PRNT result ≥ 192 mIU/mL were considered to be positive, 112≤ and <192 mIU/mL were equivocal, and <112 mIU/mL were considered non-immune [23] . We used PRNT results to determine the final immunity status of the BioPlex negative and equivocal samples.
5
Multiplex Cytokine Quantification
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Colon concentrations of interleukin-1β (IL-1 β), IL-13 and tumor necrosis factor alpha (TNF-α) were measured in duplicate by multiplex assay (R&D Systems, Abington, UK). This assay relied on use of polystyrene beads, each with a unique signature mix of fluorescent dyes that couldbe discriminated by a laser-based detection instrument (Bioplex 2200). Each bead type in the multiplex assay was coated with a specific antibody pair so as not to crossreact with other analytes in the panel.
6
Autoimmune Biomarkers in Rheumatic Diseases
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All individuals gave informed written consent and research was carried out in compliance with the Helsinki Declaration. The patients’ blood samples used for this study were collected under ethical approval, REC 10/H1306/88, National Research Ethics Committee Yorkshire and Humber–Leeds East, and healthy control participants’ peripheral blood was collected under the study number 04/Q1206/107. All experiments were performed in accordance with relevant guidelines and regulations.
SLE patients met ACR/SLICC 2012 criteria. All RA patients met ACR/EULAR 2010 criteria (ACPA-positive but ANA-negative)31 (link). Disease activity was assessed using BILAG-200432 (link). Undifferentiated connective tissue disease (UCTD) patients were individuals with positive ANA but did not meet criteria for CTD. Healthy controls (HC) had no autoimmune diseases or other infectious or inflammatory disease at the time of sampling. Absolute lymphocyte count was obtained from a routine diagnostic laboratory and used to calculate absolute counts for flow cytometry subsets. Number of reactivates against dsDNA, Ro52, Ro60, La, Sm, RNP, Sm/RNP and chromatin was measured using Bioplex 2200 and expressed as “ANA count”.
SLE patients met ACR/SLICC 2012 criteria. All RA patients met ACR/EULAR 2010 criteria (ACPA-positive but ANA-negative)31 (link). Disease activity was assessed using BILAG-200432 (link). Undifferentiated connective tissue disease (UCTD) patients were individuals with positive ANA but did not meet criteria for CTD. Healthy controls (HC) had no autoimmune diseases or other infectious or inflammatory disease at the time of sampling. Absolute lymphocyte count was obtained from a routine diagnostic laboratory and used to calculate absolute counts for flow cytometry subsets. Number of reactivates against dsDNA, Ro52, Ro60, La, Sm, RNP, Sm/RNP and chromatin was measured using Bioplex 2200 and expressed as “ANA count”.
7
Multiplex SARS-CoV-2 IgG Antibody Assay
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The SARS-CoV-2 IgG panel is analyzed on BioPlex 2200 by means of Luminex xMAP technology, which uses Luminex magnetic beads for the semiquantitative and qualitative detection of four different IgG antibodies against the receptor-binding domain (RBD), spike 1 (S1), spike 2 (S2), and N structural proteins of SARS-CoV-2. The semi-quantitation amplitude of these four target antibodies ranges from 1 to 100 U/mL; the automatic dilution (1:8, 1:16 or 1:32) of the instrument enables the detection interval to be increased up to 3200 U/mL. Manual dilutions were performed to quantify antibody values above 3200 U/mL. As reported by the manufacturer, in the clinical scenario, this multiplex immuno-assay is characterized by ≥99.9% specificity and 96.3% sensitivity ≥15 days after symptom onset. Total antibody levels are calculated as U/mL. According to the manufacturer’s recommendations, IgG anti-RBD, -S1 and -S2 are positive above 10 U/mL; values of anti-N IgG above 24 U/mL are considered reactive for SARS-CoV-2 infection.
8
Autoimmune Antibody Profiling Techniques
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An immunodot assay (Line blot LIA, Euroimmun, Lübeck, Germany and/or dot blot D-Tek, BlueDiver, Mons, Belgium) was used to identify anti-histidyl-tRNA synthetase (anti-Jo1) Abs, anti-isoleucyl-tRNA synthetase (anti-OJ) Abs, anti-Mi2 Abs and anti-tripartite motif containing 21 (TRIM21) Abs. Anti-cyclic citrullinated peptide (CCP) Abs, anti-Jo1 Abs, anti-centromere B (CENB) Abs, anti-small ribonucleoprotein (SmRNP) Abs, anti-Sjögren’s syndrome related antigen (SSA) Abs, and anti-TRIM21 Abs were all detected using a Luminex technology (Bioplex 2200, Biorad, Marnes la Coquette, France). Anti-3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) Abs were identified using a chemiluminescent immunoassay (QUANTA Flash® HMGCR, Werfen, France). Finally, IIF staining on polynuclear cells allowed the detection of anti-neutrophil cytoplasm Abs (ANCANOVALite® ANCA, Werfen, France).
9
Multiplex Bead-Based ANA Screening
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BioPlex 2200 (Bio-Rad, Hercules, CA) system is an automated analyzer that uses multiplex bead technology (Luminex, Austin, TX, USA) to simultaneously detect antibodies to several antigens in a single tube. The BioPlex 2200 ANA Screen is intended for the qualitative screening of ANA, the quantitative detection of antibody to dsDNA, and the semiquantitative detection of ten separate antibodies (Chromatin, Ribosomal P, SS-A, SS-B, Sm, SmRNP, RNP, Scl-70, Jo-1, and Centromere B) [11 (link), 12 (link)] in human serum and/or EDTA or heparinized plasma. The test system is used as an aid in the diagnosis of SARD. Characteristics of the assay are summarized in Table 1 .
10
Syphilis IgG Testing Workflow
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Syphilis IgG testing was performed on a Bioplex 2200 analyzer (Bio-Rad Laboratories, Hercules, CA, USA) using the Syphilis IgG (T. pallidum) assay in the UIHC core clinical laboratory. Samples are run 24 h a day and results can auto-verify if quality criteria are met [14] (link). Following the information in the assay package insert, the following were the reference ranges for the syphilis IgG assay using units of antibody index (AI): 0.8 AI or lower, negative; 0.9–1.0 AI, equivocal; 1.1 AI or greater, positive. RPR and TP-PA tests were referred to a commercial reference laboratory (ARUP Laboratories, Salt Lake City, UT, USA). After UIHC switched to the reverse algorithm, stand-alone RPR testing was available to order only as a follow-up test to check for treatment response [termed “Syphilis treatment follow-up (RPR with reflex titer)” in the electronic order entry system], as the reflex confirmation following an equivocal or positive syphilis IgG result, or for specific protocols (e.g., some organ transplant donor evaluations) that do not allow the use of reverse algorithm. Screening recommendations for syphilis at UIHC did not change during the retrospective time periods. For obstetric patients, the goal was universal testing unless specifically refused by the mother.
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