Serum samples were first assayed for AMA and ANA by IIF on sections of rat kidney, stomach and liver, and on HEp2 cells, respectively (test kits from EUROIMMUN, Lübeck, Germany, and INOVA Diagnostics, San Diego, CA, USA) and by an ELISA screening test (PBC Screen, INOVA Diagnostics, and EUROIMMUN) using three coating antigens: recombinant pMIT3 and purified gp210 and sp100 with anti-IgG and anti-IgA dual conjugate. The manufacturer’s cut-off was established at 25 units. Samples were considered as negative (≤20.0 units), equivocal (20.1–24.9 units), or positive (≥25.0 units). A negative result indicated the absence of antibodies to MIT3, gp210, or sp100. A positive result indicated the presence of antibodies to one or more of the antigens included in the PBC Screen assay. Subsequently, sera from patients with PBC were tested on the individual MIT3, gp210 and sp100 IgG Quanta Lite ELISAs (INOVA Diagnostics) [19] .
Quanta lite elisa
Manufactured by Inova Diagnostics
Sourced in United States
The QUANTA Lite ELISA is a laboratory equipment used for enzyme-linked immunosorbent assay (ELISA) testing. It is designed to detect and quantify specific analytes, such as antibodies or antigens, in a sample. The QUANTA Lite ELISA provides a standardized and automated process for immunoassay analysis.
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Lab products found in correlation
Alkaline phosphatase conjugated goat anti mouse igg, by Southern Biotech (1 mentions)
Goat anti mouse immunoglobulin, by Jackson ImmunoResearch (1 mentions)
Horseradish peroxidase conjugated goat anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Bioplex 2200 system, by Bio-Rad (1 mentions)
Quanta lite, by Inova Diagnostics (1 mentions)
9 protocols using quanta lite elisa
1
Serological Profiling in Primary Biliary Cholangitis
2
Antiphospholipid Antibody Detection by ELISA and LA
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Serum IgG/IgM anti-cardiolipin antibodies (aCL) and IgG/IgM anti-β2glycoprotein I antibodies (aβ2GPI) were detected by enzyme-linked immunosorbent assay (ELISA) (QUANTA Lite® ELISAs, INOVA Diagnostics, San Diego, CA, USA). The cutoff values for positivity were set as 40 IgG phospholipid (GPL) units or 40 IgM phospholipid (MPL) units. LA was detected by a traditional three-step procedure based on the guidelines of the International Society on Thrombosis and Hemostasis [9 (link)]. LA test positivity was defined as a prolonged diluted Russell viper venom time (dRVVT) in the screening step, which was not reversed by mixing with normal plasma but reversed by the addition of excess phospholipids in the confirmation step [11 (link)].
3
Autoantibody Detection by ELISA
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Antibodies to dsDNA, chromatin, SSA (Ro52/tripartite motif (TRIM)21 and Ro60 mixture), SSB/ La, ribosomal P protein, C1Q, RNP and Sm were detected by the QUANTA Lite ELISAs (Inova Diagnostics) performed according to the manufacturer's instructions.
4
Autoantibody Profiling in Mouse Models
5
Antiphospholipid Antibody Evaluation in Patients
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Patients were studied for the presence of aPL, in accordance with the Sydney criteria: anticardiolipin (aCL) IgG and IgM, anti-beta-2-glycoprotein-I (aB2GPI) of IgG and IgM isotypes, and lupus anticoagulant (LA). aCL and aB2GPI IgG/IgM were evaluated using a Bioplex-2200 system (Bio-Rad, Hercules, CA, USA) with our laboratory cutoffs (99th percentile). LA was measured using two methods, HemosIL dRVVT (cutoff ratio 1.20) and HemosIL Silica Clotting Time (cutoff ratio 1.30) assays (Instrumentation Laboratory SpA, Milano, Italy). LA was performed in all patients without active anticoagulation therapy. Patients positive for aPL were studied for the presence of antinuclear antibodies (ANA) and anti-double stranded DNA (anti-dsDNA). ANA were tested by indirect immunofluorescence (IIF) on Hep2 cells and anti-dsDNA were studied using the Crithidia lucilae immunofluorescence test.
The QUANTA Lite ELISA (INOVA DIAGNOSTICS, San Diego, CA, USA) was used to evaluate aPS/PT IgG & IgM. We had previously described our calculated aPS/PT for our local population based on the 99th percentile, which resulted in 30 U/mL for IgG and 40 U/mL for IgM [21 (link)].
The QUANTA Lite ELISA (INOVA DIAGNOSTICS, San Diego, CA, USA) was used to evaluate aPS/PT IgG & IgM. We had previously described our calculated aPS/PT for our local population based on the 99th percentile, which resulted in 30 U/mL for IgG and 40 U/mL for IgM [21 (link)].
6
Quantification of Antiphospholipid Antibodies
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Quantitative values of anticardiolipin and anti-β2GPI immunoglobulin (Ig) G and IgM were measured with Cardiolipin IgM/IgG ELISA (IBL International IBL International GmbH) or IMTEC β2GPI antibodies IgG/IgM ELISA (Clindia Benelux BV) with a cutoff value for positivity on 12.0 GPL/mL for anticardiolipin IgG, 7.0 MPL/mL for anticardiolipin IgM, 7.0 GPL/mL for anti-β2GPI IgG, and 7.0 MPL/mL for β2GPI IgM, based on the 99th percentile of healthy reference individuals [24 (link)]. Quantitative values of antiphosphatidylserine/prothrombin (aPS/PT) IgG and IgM were measured with QUANTA Lite ELISA (Inova Diagnostics) with a cutoff value for positivity on 30 units/mL based on manufacturer’s recommendations.
7
Antiphospholipid Antibody Quantification
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Quantitative values of anticardiolipin and anti‐β2GPI IgG and IgM were measured with Cardiolipin IgM/IgG ELISA (IBL International GmbH) or IMTEC β2GPI antibodies IgG/IgM ELISA (Clindia Benelux BV) with a cutoff value for positivity on 12.0 GPL/ml for anticardiolipin IgG, 7.0 MPL/ml for anticardiolipin IgM, 7.0 GPL/ml for anti‐β2GPI IgG, and 7.0 MPL/ml for β2GPI IgM, based on a nonparametric 99th percentile of 120 reference individuals. Quantitative values of antiphosphatidylserine/prothrombin (aPS/PT) IgG and IgM were measured with QUANTA Lite ELISA (Inova Diagnostics) with a cutoff value for positivity on 30 units/ml based on the manufacturer's recommendations.
8
Evaluation of Autoantibodies in PBC
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Sera from a total of 487 patients with clinically documented PBC or PBC/AIH overlap diagnosed according to European Association for the Study of the Liver (EASL) guidelines were collected at five expert clinical sites (Barcelona, Spain; Salamanca, Spain; Calgary, Canada; Edmonton, Canada; Warsaw, Poland) and tested for the presence of autoantibodies to HK-1 and a KLHL12-derived immunodominant peptide, referred to as “KL-p,” using research use only ELISA kits (QUANTA Lite®, Inova Diagnostics, San Diego, CA). All sera were also tested for anti-M2 (MIT3) antibodies (subsequently referred to as anti-MIT3 for simplicity) by ELISA (QUANTA Lite® ELISA, Inova Diagnostics, San Diego, CA). Specificity was assessed by testing 127 sera from patients without PBC, including patients with primary sclerosis cholangitis (PSC, n = 41), autoimmune hepatitis (AIH, n = 20), AIH/PSC overlap (n = 12), various infectious diseases (n = 20), colorectal cancer (n = 14), and healthy controls (n = 20).
Differences between the five cohorts was assessed by one-way analysis of variance (ANOVA) using Krustal-Wallis test and between specific geographic chorts using Mann-Whitney non-parametric two-tailed t-test (Graphpad ver 5.03, San Diego, CA). P-value <0.05 was considered significant.
Differences between the five cohorts was assessed by one-way analysis of variance (ANOVA) using Krustal-Wallis test and between specific geographic chorts using Mann-Whitney non-parametric two-tailed t-test (Graphpad ver 5.03, San Diego, CA). P-value <0.05 was considered significant.
9
Confirming Ku Antigen Binding
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To confirm specific binding, the Ku antigen used in the CIA (Lot. 73B02, 0.8 mg/lane) was fractionated by sodium dodecyl sulphate polyacrylamide gel electrophoresis and transferred onto nitrocellulose membranes. Success of transfer as verified by protein staining with Ponceau S showed two bands corresponding to the two subunits of the Ku antigen. Sera containing anti-Ku antibodies as defined using the Ku CIA were diluted 1:100 in sample diluent (kit component of QUANTA Lite ELISA, Inova Diagnostics) and incubated for 1 h at room temperature. After washing away unbound compounds, the membrane was incubated with a goat anti-human IgG conjugated with alkaline phosphatase. All membrane strips were developed at the same time using BCIP substrate.
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