Screening for HLA class 1 and class 2 antibodies was performed using a MAB assay (LABScreen® Mixed Kit, One Lambda, Canoga Park, CA, USA). All sera that tested positive and a random subset of negative sera were subject to SAB assays to identify antibody specificities (LABScreen Single Antigen HLA Class I kit and/or LABScreen Single Antigen HLA Class II kit, One Lambda). Both MAB and SAB were performed according to the manufacturer’s instructions. Briefly, following heat-inactivation at 56 °C for 30 min and clearance from debris (0.22 μm filter), 20 μl of undiluted serum was added to 3 μl of the LABScreen bead mix and incubated for 30 min in the dark at room temperature. After a washing step in 1x LABScreen wash buffer, the bead mix was incubated with 100 μl of a 1:100 dilution of the PE-conjugated goat anti-human IgG detection antibody for 30 min in the dark at room temperature. After a final washing step in 1x LABScreen wash buffer, data acquisition was performed using a FLEXMAP3D Analyser in combination with xPONENT software version 4.1 (Luminex Corporation, Texas, USA).
Labscreen mixed kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The LABScreen Mixed Kit is a laboratory equipment product designed for comprehensive human leukocyte antigen (HLA) antibody screening. It provides a reliable and efficient method for the detection and identification of HLA class I and II antibodies in a single test. The kit utilizes Luminex technology to offer a high-throughput solution for HLA antibody analysis.
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Lab products found in correlation
Labscreen single antigen kit, by Thermo Fisher Scientific (4 mentions)
Hla fusion 3, by Thermo Fisher Scientific (2 mentions)
Labscreen single antigen hla class 1 kit, by Thermo Fisher Scientific (1 mentions)
Disodium edta, by Merck Group (1 mentions)
C1qscreen, by Thermo Fisher Scientific (1 mentions)
Single antigen assays, by Thermo Fisher Scientific (1 mentions)
Single antigen beads, by Thermo Fisher Scientific (1 mentions)
9 protocols using labscreen mixed kit
1
HLA Antibody Screening and Identification
2
Screening for HLA Antibodies using Luminex
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Patient plasma/serum was screened for class I (i.e., HLA-A,-B,-C) and class II (i.e., HLA-DR) HLA antibodies with a LABScreen Mixed Kit (One Lambda, Canoga Park, CA, USA). The samples (7 uL) were incubated with mixed HLA class I- and class II-coated microspheres for 30 min in the dark under gentle agitation. The specimens were then washed before being incubated with anti-human immunoglobulin G-conjugated fluorescein isothiocyanate as described above for the first incubation. Next, the samples were analyzed with a Luminex 200 flow analyzer (Luminex, Austin, TX, USA), and the data were analyzed with the HLA Fusion 3.2 software (One Lambda). The MFI of anti-HLA antibodies was obtained from the output file generated by the flow analyzer and adjusted for the background signal using the formula: sample beads − negative control beads. The samples with a MFI >500 were further tested for the specificity of the antibody, using a LABScreen Single Antigen Kit (One Lambda). The MFI was adjusted for the background signal using the formula described above. The patients and donors underwent HLA allele typing of at least the A, B, and DRB1 loci routinely.
3
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)).
4
Anti-HLA Antibody Screening Protocol
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Before transplantation, all patients were screened for the presence of anti-HLA antibodies with the LABScreen SAB assay (One Lambda, Canoga Park, CA). Peripheral blood DSA analysis was performed on all index patients at or close to the time of the reference biopsy. Anti-HLA IgG reactivity was analyzed in each center with validated beadbased assays using the LABScreen mixed kit (One Lambda, Canoga Park, CA) and the single-antigen class I and class II kits. Analyses were performed using One Lambda software (HLA Fusion Version 2.0). In the mixed assay, results above a cutoff value of 3.0 (ratio) were considered positive, according to a beta test performed on each laboratory's samples. To identify HLA specificity, single-antigen assays (One Lambda) were performed, using mean fluorescence intensity (MFI) as a measure of antibody reactivity. We considered DSA-positive patients those with MFI values ≥1400. 17 For statistical analyses, in the cases with more than one DSA, the DSA with the highest MFI (immunodominant DSA) was selected. Only 1 immunologist (E.C.) was included in the panel, but he reviewed all immunological datasheets and discussed with the immunologists from the different centers, when needed.
5
Detecting Donor-Specific HLA Antibodies
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Serum samples collected before surgery and at yearly intervals after transplantation from MSC recipients and control patients given basiliximab/low-dose RATG induction therapy were tested for class I and class II anti-HLA antibodies using LABScreen Mixed kit (One Lambda, Canoga Park, CA, USA) (27 (link)). Positive tests were quantified by Single-Antigen Beads (One Lambda). A mean fluorescence intensity (MFI) threshold of 2,000 was used to identify a donor-specific anti-HLA antibody (DSA). The tests were carried out according to the manufacturer’s instructions and the analysis was performed with One Lambda software (HLA Visual Version 2.2).
6
HLA Antibody Screening and Identification
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The patients and donors underwent HLA allele typing of at least the A, B, and DRB1 loci routinely. The examination was performed as previously [15 (link)]. In brief, patient plasma/serum was screened for class I and class II HLA antibodies with a LABScreen Mixed Kit (One Lambda, Canoga Park, CA, USA). The samples were incubated with mixed HLA class I- and class II-coated microspheres for 30 min in the dark and then washed before being incubated with anti-human immunoglobulin G-conjugated fluorescein isothiocyanate as described above for the first incubation. Finally, the samples were examined by a Luminex 200 flow analyzer (Luminex, Austin, TX, USA), and the data were analyzed with the HLA Fusion 3.2 software (One Lambda). The MFI of anti-HLA antibodies was obtained using the formula: sample beads − negative control beads. The samples with a MFI > 500 were further tested for the specificity of the antibody (DSA), using a LABScreen Single Antigen Kit (One Lambda).
7
Anti-HLA Antibody Detection in Transplant Patients
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Patient serum was collected before transplantation and was screened for the presence of class I and class II anti-HLA antibodies (HLA abs) of the immunoglobulin G type with a LABScreen Mixed Kit (One Lambda, Canoga Park, CA, USA) according to the manufacturer’s instructions and as previously reported9 (link). The mean fluorescence intensity (MFI) of the anti-HLA antibodies was adjusted for the background signal using the formula described previously. Samples with an MFI of 500 or more considered to be positive.
8
Plasma HLA Antibody Detection and Analysis
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A plasma sample was obtained by centrifugation of a peripheral blood tube with ethylenediaminetetraacetic acid (Vacutainer® spry-coated K2EDTA tubes, BD™), collected from all patients, at baseline, 12 and 52 weeks after control or ASC administration. HLA Abs were detected in a Luminex platform using a LabscreenMixed™ kit (One Lambda Inc.® Canoga Park, CA, US) according to manufacturer instructions. All samples with a signal >800 units of median fluorescence intensity (MFI) were considered positive, and donor specificities for HLA Abs were determined using Labscreen Single Antigen™ kit (One Lambda Inc.® Canoga Park, CA, US). All signals were normalized according to Quantiplex™ beads fluorescence and specificities > 20,000 units of standard fluorescent intensity were considered relevant. Qualitatively, we defined the HLA antibody titer as the resulting MFI sum of all the determinant beads of the HLA class I molecules included in the Labscreen Mixed kit. We will refer to pre-existing HLA Abs detected in patients before ASC administration as HLA Abs whereas donor ASC-induced HLA Abs will be referred as DSA.
9
Screening for Donor-Specific Antibodies Before Transplant
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Patients were tested for the presence of donor-specific antibodies (DSAs) including class I (i.e., HLA-A, -B, -C) and class II (i.e., HLA-DR) HLA antibodies. Immunoglobulin anti-HLA reactivity in the serum was tested with a bead-based screening assay. Briefly, we used the LABScreen Mixed kit (One Lambda, Canoga Park, CA, USA), which simultaneously detects class I and class II antibodies with microbeads coated with purified class I and class II HLA antigens. For HLA antibody-positive samples with a median fluorescent intensity (MFI) >500, DSAs were further tested using a LABScreen Single Antigen Kit (One Lambda). Above a cut-off value of MFI ≥2000 was considered positive. Patients with positive DSA received rituximab before transplantation, and the co-infusion of umbilical cord blood (26 (link)).
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