Serum samples from patients with positive DSA were tested for C1q-binding DSA using commercially available kits (C1qScreen; One Lambda, Canoga Park, CA) according to the manufacturer’s specifications. In brief, 5 µL of HLA class I or II antigen-coated Luminex beads were incubated with 5 µL of heat inactivated patient sera for 20 minutes at room temperature with shaking. Phycoerythrin-conjugated anti-C1q (5 µl) was then added, and samples incubated an additional 20 minutes. As above, particle florescence was assessed by Luminex 100 IS (Luminex, Austin, TX). Normalized values >1000 MFI were considered positive.
C1qscreen
Manufactured by Thermo Fisher Scientific
Sourced in United States
The C1qScreen is a laboratory instrument designed for the detection and quantification of the C1q protein. C1q is a component of the complement system, which plays a crucial role in the immune response. The C1qScreen provides a reliable and efficient method for measuring C1q levels in biological samples, enabling researchers and clinicians to assess immune system function.
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Lab products found in correlation
Hla fusion software, by Thermo Fisher Scientific (2 mentions)
Disodium edta, by Merck Group (1 mentions)
Labscreen mixed kit, by Thermo Fisher Scientific (1 mentions)
Labscreen, by Thermo Fisher Scientific (1 mentions)
Flowpra, by Thermo Fisher Scientific (1 mentions)
Labscreen single antigen beads, by Thermo Fisher Scientific (1 mentions)
Hla fusion 2, by Thermo Fisher Scientific (1 mentions)
Labscreen single antigen, by Thermo Fisher Scientific (1 mentions)
11 protocols using c1qscreen
1
Detection of C1q-Binding Donor-Specific Antibodies
2
HLA Antibody Profiling for Transplant Evaluation
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HLA class I and class II typing was performed as previously described (22 (link)).
Anti-HLA class I and class II IgG antibodies were tested with a bead-based detection assay. We used the LABScreen Mixed kit (One Lambda Inc., Canoga Park, CA, USA), which simultaneously detects class I and class II antibodies and the SAB assays (Single Antigen kit, One Lambda) to identify HLA class I and class II specificities (22 (link)). Before testing, all sera were pre-treated with disodium EDTA (final concentration 10 mM, pH 7.4) (Sigma-Aldrich, Milan, Italy), in order to rule out underestimation of antibody MFI strength due to the prozone phenomenon. Screening assay results above a cut-off value of 3.0 ratio between sample and negative control were considered positive. Single antigen results above a MFI cut-off value of 1,000 were considered positive.
C1qScreen™ (One Lambda) was employed for identification of complement binding antibodies (22 (link)). Serum samples were analyzed in a blinded fashion for the presence of C3d-binding DSA with the Lifecodes C3d Detection kit according to the manufacturer’s protocol (Immucor Inc., Norcross, GA, USA) (22 (link)).
Anti-HLA class I and class II IgG antibodies were tested with a bead-based detection assay. We used the LABScreen Mixed kit (One Lambda Inc., Canoga Park, CA, USA), which simultaneously detects class I and class II antibodies and the SAB assays (Single Antigen kit, One Lambda) to identify HLA class I and class II specificities (22 (link)). Before testing, all sera were pre-treated with disodium EDTA (final concentration 10 mM, pH 7.4) (Sigma-Aldrich, Milan, Italy), in order to rule out underestimation of antibody MFI strength due to the prozone phenomenon. Screening assay results above a cut-off value of 3.0 ratio between sample and negative control were considered positive. Single antigen results above a MFI cut-off value of 1,000 were considered positive.
C1qScreen™ (One Lambda) was employed for identification of complement binding antibodies (22 (link)). Serum samples were analyzed in a blinded fashion for the presence of C3d-binding DSA with the Lifecodes C3d Detection kit according to the manufacturer’s protocol (Immucor Inc., Norcross, GA, USA) (22 (link)).
3
Comprehensive HLA Antibody Profiling
4
Detecting C1q-binding Anti-HLA Antibodies
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DSA-positive samples were tested for C1q-binding anti-HLA antibodies (SAB-C1q) using commercially available kits (C1qScreen; One Lambda). The serum samples were heat-treated (56°C for 30 minutes) to denature endogenous complement components and the test was performed per the manufacturer's protocol. The analyses of C1q results were performed by HLA FUSION software (One Lambda, Inc.) following the interpretation method published by Tyan et al.22
5
Evaluating HLA Antibodies with EDTA Treatment
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Patient sera were tested undiluted (neat), after ethylene diamine tetraacetic acid (EDTA) treatment and at 1 in 20 dilution using LabScreen single-antigen HLA class I–specific antibody detection beads (One Lambda, Canoga Park, CA). The addition of EDTA to sera used in the SAB assay acts as a chelating agent that sequesters calcium ions and abrogates C1q formation and obviates complement interference of FITC-labeled IgG detection reagent. Addition of EDTA to sera results in only 5% dilution (5 μL 6% EDTA solution to 95 μL test serum) and in validation tests by ourselves and others, does not alter antibody binding to SAB.16 (link) In parallel, undiluted sera were tested using the C1QScreen (One Lambda) according to standard procedures. IgG-SAB and C1q-SAB binding levels were expressed as normalized mean fluorescence intensity (MFI) using HLA Fusion software (v3.2.0; One Lambda). All tests were undertaken at the same time using the same kit batches to minimise technical and operator variability.
6
Evaluation of Donor-Specific Antibodies in Transplant Recipients
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The serum from the recipients was screened for anti-HLA antibody with Flow PRA (One Lambda, Inc., Canoga Park, CA). The LTR was considered sensitized when the panel-reactive assay (PRA) was positive (greater than 0%) and regarded unsensitized when PRA was negative. The sensitized recipients were further tested for donor-specific antibody (DSA) with LABScreen single-antigen beads (One Lambda, Inc.). The positive DSA was defined by mean fluorescence intensity (MFI) units greater than 1000 or more. The complement-binding function in DSA was measured with C1q Screen (One Lambda Inc.). The test for anti-HLA antibody was done in ReproCELL Japan Inc. Yokohama.
7
Detecting HLA, MICA Antibodies in Serum
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IgG antibodies against HLA class I (A, B, and C) and class II (DR, DQ, and DP) were detected using a single antigen bead array on a Luminex platform (Gen Probe) according to the protocol suggested by the manufacturer. MICA antibody testing was performed on patient serum samples using single antigen beads conjugated with recombinant MICA*001, *002, *004, *007, *008, *009, *012, *016, *017, *018, *019, and *045. This kit was prepared in our laboratory and validated using the reference sera obtained from the 15th International Histocompatibility and Immunogenetics MICA workshop [11 (link)]. Antibody specificity was based on normalized mean fluorescence intensity (MFI) greater than 2000. DSAs were identified based on the reaction of patient sera to the mismatched antigens for a given donor. Some serum samples were also tested for antibody-C1q binding using a commercially available kit (C1qScreen, One Lambda).
8
C1q Binding Assay Protocol
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C1q testing was performed using the commercially available kit (C1qScreen, One Lambda, Inc.) according to the manufacturer's instructions. Antibody specificity was analyzed manually using baseline mean fluorescent intensity (MFI) values. Positive MFI thresholds were defined on the basis of >500 MFI when a DSA was noted for a donor mismatched HLA antigen.
9
Detecting Complement-Binding Donor-Specific Antibodies
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Experiments were performed by researchers blinded to patient information at the Clinical Transplantation Laboratory, Viapath, Guy’s Hospital, London, in a single run and using assays from the same batch. Patients were defined as ‘complement positive’ if at least one DSA showed complement fixing.
C1q-binding DSA were identified using C1qScreen™ (One Lambda) according to the manufacturer’s protocol [5 (link)]. Sera were pre-treated with heat inactivation of the complement system as part of the protocol. Analysis was performed using the HLA Fusion 2.0 software (One Lambda). Complement positivity was assigned at >1000 MFI based on the negative control sera and internal negative control beads.
C3d-binding DSA were identified using Lifecodes C3d and Single Antigen assay (Immucor, London, UK) according to the manufacturer’s protocol [6 (link)]. In addition, sera were tested for pan-IgG using Lifecodes Single Antigen kits (Immucor). Results were analysed using the same manufacturer’s MatchIt software, and complement positivity was defined as per the software algorithm.
C1q-binding DSA were identified using C1qScreen™ (One Lambda) according to the manufacturer’s protocol [5 (link)]. Sera were pre-treated with heat inactivation of the complement system as part of the protocol. Analysis was performed using the HLA Fusion 2.0 software (One Lambda). Complement positivity was assigned at >1000 MFI based on the negative control sera and internal negative control beads.
C3d-binding DSA were identified using Lifecodes C3d and Single Antigen assay (Immucor, London, UK) according to the manufacturer’s protocol [6 (link)]. In addition, sera were tested for pan-IgG using Lifecodes Single Antigen kits (Immucor). Results were analysed using the same manufacturer’s MatchIt software, and complement positivity was defined as per the software algorithm.
10
Detection of Donor-Specific HLA Antibodies
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DSHA were identified using a single antigen bead assay that utilized the multiplex flow-bead microarray method (Lifecodes LSA class I and II; Gen-Probe Transplant diagnostics, Inc., Stamford, CT, USA). The presence and antigen specificities of Abs to HLA-A, -B, -DR, and -DQ were determined. Results are expressed as mean fluorescence intensities (MFI). A normalized value of >1000 MFI was considered positive for DSHA. A C1q binding assay was performed on all available sera of recipients with DSHA (C1qScreen™, One Lambda, CA, USA). Anti-ABO antibody titers were measured using standard serological techniques.14 (link) Anti-angiotensin II type 1 receptor (AT1R) antibodies were retrospectively evaluated in cases of biopsyproven AMR without DSHA. Levels of anti-AT1R antibodies (U/mL) were quantified using AT1R assay kits (One Lambda, CA, USA), which utilize the enzyme-linked immunosorbent assay principle.
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