For autoimmune biomarker analysis, frozen plasma was shipped on dry ice to Exagen, Inc., which has a clinical laboratory accredited by the College of American Pathologists and certified under the Clinical Laboratory Improvement Amendments. Thawed plasma was aliquoted and distributed for the following tests: antinuclear antibodies (ANA) were measured using enzyme-linked immunosorbent assays (ELISA) (QUANTA Lite; Inova Diagnostics) and indirect immunofluorescence (IFA) (NOVA Lite; Inova Diagnostics); anti-dsDNA antibodies were also measured by ELISA and were confirmed by IFA with Crithidia luciliae; extractable nuclear antigen autoantibodies (anti-Sm, anti-SS-B/La IgG, anti-Scl-70 IgG, anti-U1RNP IgG, anti-RNP70 IgG, anti-CENP IgG, anti-Jo-1 IgG and anti-CCP IgG) as well as RF IgA and IgM were measured using the EliA test on the Phadia 250 platform (ThermoFisher Scientific); IgG, IgM and IgA isotypes of anticardiolipin and anti-β2‐glycoprotein, as well as anti-Ro52, anti-Ro60, anti-GBM, anti-PR3 and anti-MPO, were measured using a chemiluminescence immunoassay (BIO-FLASH; Inova Diagnostics); anti-CarP, anti-RNA-pol-III, and the IgG and IgM isotypes of anti-PS/PT were measured by ELISA (QUANTA Lite; Inova Diagnostics), whereas C-ANCA and P-ANCA were measured by IFA (NOVA Lite; Inova Diagnostics). All assays were performed following the manufacturer’s instructions.
Quanta lite
Manufactured by Inova Diagnostics
Sourced in United States
The QUANTA Lite is an automated enzyme-linked immunosorbent assay (ELISA) system designed for the detection and quantification of various analytes in a laboratory setting. It provides a reliable and efficient platform for conducting immunoassays.
Automatically generated - may contain errors
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26 protocols using quanta lite
1
Comprehensive Autoimmune Biomarker Panel Analysis
2
IgG, IgM, IgA Autoantibody Quantification
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IgG and IgM aPS/PT determination was performed by enzyme-linked immunosorbent assay (ELISA) (QUANTA Lite®, Inova Diagnostics, CA). The cut-off recommended by the manufacturer is 30 U/ml. IgA aCL and aB2GPI were quantified by ELISA (QUANTA Lite®, Inova Diagnostics, CA). The cut-off recommended by the manufacturer is 20 U/ml.
3
Antiphospholipid Antibodies and Thrombophilia
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Blood samples were collected in 3.8% trisodium citrate and centrifuged at 2000 g for 15 min to obtain platelet-poor plasma, which was frozen and stored in small aliquots at −70°C until tested. Antiphospholipid antibodies—LA and IgG, IgM aCL (QUANTA Lite, INOVA Diagnostics, San Diego, California, USA)—antithrombin and protein C (Berichrom Antithrombin and Protein C amidolytic assays, Behring, Germany) and free protein S (IMUCLONE Protein S ELISA, American Diagnostica, Stamford, Connecticut, USA) were determined in all patients. A confirmed presence of antiphospholipid antibodies was defined according to SSC criteria,15 (link) or the concurrent presence of more than one. DNA was extracted from peripheral blood leucocytes according to standard protocols.
FVL and PTm genotyping was performed by a TaqMan (Applied Biosystems, Foster City, California, USA) probe-based real-time PCR technique.16 (link)
FVL and PTm genotyping was performed by a TaqMan (Applied Biosystems, Foster City, California, USA) probe-based real-time PCR technique.16 (link)
4
ASCA ELISA for Antibody Detection
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QUANTA Lite (INOVA Diagnostics, Inc., San Diego, CA, United States) ASCA IgG and IgA enzyme-linked immunosorbent assays (ELISA) were used for determination of serum anti-Saccharomyces cerevisiae antibodies (ASCAs) levels as described by the manufacturer’s instructions. Briefly, 100 microliters of patient’s serum at a dilution of 1:100 were added to 96-well polystyrene microwell plates adhered with partially purified and disrupted Saccharomyces cerevisiae antigen. Bound ASCAs were detected by incubation with horseradish peroxidase IgG or IgA conjugate (goat anti-human). The absorbance (optical density, OD) was read at 450 nm using a SpectraMax MiniMax Imaging Cytometer. On each plate a high and a low positive, as well as a negative control were included. ASCA reactivity was determined by the formula: sample OD⁄low positive OD × 25. The positive cut off values for both ASCA IgA and IgG as set by the manufacturer were 25 Units (U)/mL.
5
Anti-dsDNA IgG Antibody Purification and Cell Stimulation
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Anti-dsDNA IgG antibodies from the pooled sera of 7 SLE patients (characterised
by high titres of anti-dsDNA) were purified using a commercial kit (Quanta
Lite-INOVA Diagnostics, San Diego, CA) through the ELISA-elution assay method
(Referencia ATVB). Monocytes, neutrophils, and ECs, were then incubated with
10 μg/ml LPS, synthetic human IgG (40 μg/ml) or
purified Anti-dsDNA IgG from the pooled sera of 5 SLE patients
(40 μg/ml) for 6 h at 37 °C.
by high titres of anti-dsDNA) were purified using a commercial kit (Quanta
Lite-INOVA Diagnostics, San Diego, CA) through the ELISA-elution assay method
(Referencia ATVB). Monocytes, neutrophils, and ECs, were then incubated with
10 μg/ml LPS, synthetic human IgG (40 μg/ml) or
purified Anti-dsDNA IgG from the pooled sera of 5 SLE patients
(40 μg/ml) for 6 h at 37 °C.
6
Complement Activation and Autoantibody Profiling
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Serum C3 and C4 were determined by immunoturbidimetry (The Binding Site, San Diego, California, USA)11 (link) and were considered low if below the manufacturer’s lower limits of normal (81.1 and 12.9 mg/dL for C3 and C4, respectively). Low complement status refers to low C3 and/or low C4.
Complement activation was determined using CB-CAPs measured by quantitative flow cytometry.11 (link) CB-CAPs were considered abnormal if levels of EC4d and/or BC4d were above the 99th percentile of a group of healthy individuals (>14 and >60 net mean fluorescence intensity, respectively).7 8 11 (link) Abnormal CB-CAPs status refers to abnormal EC4d and/or abnormal BC4d.
Anti-double-stranded DNA (dsDNA) antibodies were determined by ELISA (Quanta Lite, Inova Diagnostics, San Diego, California, USA). All serum samples above 301 IU/mL were further tested by indirect immunofluorescence assay (IFA) using the Crithidia luciliae assay (Nova-Lite, Inova Diagnostics). Anti-dsDNA antibodies were considered positive if confirmed by IFA.
Complement activation was determined using CB-CAPs measured by quantitative flow cytometry.11 (link) CB-CAPs were considered abnormal if levels of EC4d and/or BC4d were above the 99th percentile of a group of healthy individuals (>14 and >60 net mean fluorescence intensity, respectively).7 8 11 (link) Abnormal CB-CAPs status refers to abnormal EC4d and/or abnormal BC4d.
Anti-double-stranded DNA (dsDNA) antibodies were determined by ELISA (Quanta Lite, Inova Diagnostics, San Diego, California, USA). All serum samples above 301 IU/mL were further tested by indirect immunofluorescence assay (IFA) using the Crithidia luciliae assay (Nova-Lite, Inova Diagnostics). Anti-dsDNA antibodies were considered positive if confirmed by IFA.
7
HMGCR Autoantibody ELISA Protocol
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ELISA plates coated with recombinant HMGCR were incubated with diluted patient samples. Assay procedure followed standard protocol of QUANTA Lite assays (INOVA Diagnostics). A five-point calibration curve was used to convert optical density values into units. The cut-off was defined as the 99% percentile of level in a previous internal study based on disease controls.
8
Autoantibody Detection in Rheumatic Diseases
9
Detecting Antiphospholipid Antibodies in Postpartum Serum
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Ten ml of peripheral blood was collected at three months postpartum in a tube without anticoagulant. The serum was extracted by centrifugation at 3000 revolutions per minute during 10 min at room temperature.
This serum was used to determine the presence of aPLs by ELISA following manufacturer instructions. Specifically, IgG and IgM aCL and IgG and IgM aβ2GPI were measured using ORGENTEC detection kit (Diagnostika GmbH®, Straßberg, Germany) (ref ORG 515 y 521, respectively). Additionally, IgA aCL (ref 708635), IgA aβ2GPI (ref 708675), and IgG and IgM aPS/PT (ref 708835 and 708845, respectively) were detected using QUANTA Lite® (Inova Diagnostics, Inc., San Diego, CA, USA). The cut-off points established to determine the positivity of the different aPLs are depicted inSupplementary Table S3 .
This serum was used to determine the presence of aPLs by ELISA following manufacturer instructions. Specifically, IgG and IgM aCL and IgG and IgM aβ2GPI were measured using ORGENTEC detection kit (Diagnostika GmbH®, Straßberg, Germany) (ref ORG 515 y 521, respectively). Additionally, IgA aCL (ref 708635), IgA aβ2GPI (ref 708675), and IgG and IgM aPS/PT (ref 708835 and 708845, respectively) were detected using QUANTA Lite® (Inova Diagnostics, Inc., San Diego, CA, USA). The cut-off points established to determine the positivity of the different aPLs are depicted in
10
Serum Calprotectin Measurement in IMPROVED and RETRO Studies
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Patient serum was collected when patients were in remission and stored in − 80 °C until use. In the IMPROVED, circulating calprotectin was measured at baseline and 8 months (month of MTX stopping) using a fecal Calprotectin Extended Range kit adapted for serum (QUANTA Lite®, Inova Diagnostics, research use only for serum). In the RETRO, calprotectin was measured in baseline samples, which was at study inclusion directly preceding DMARD tapering, before any tapering or stopping changes were made to existing DMARDs, using an in-house sandwich ELISA developed by Johannes Roth (Muenster) for predicting flares in juvenile idiopathic arthritis as previously described [11 (link)]. Absorbance was converted to nanograms per milliliter using a standard curve of serial dilutions of a known concentration.
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