Comparison of different luminex single antigen bead kits for memory B cell-derived HLA antibody detection.
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ABSTRACT:
SUBMITTER: Karahan GE
PROVIDER: S-EPMC8456970 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Previously, a distinct MHC class II Luminex-single antigen bead (SAB) pattern was described and attributed to antibodies targeting denatured antigens. In this study, we describe a distinct MHC class I reactivity pattern observed in 1.8% (105/5992) of samples resulted in 2017. The pattern displays reactivity to the following Luminex-SABs: HLA-A*33:03, A*36:01, A*80:01, B*54:01, B*53:01, C*06:02, C*07:02, C*18:02, C*14:02, C*03:03, C*03:04, and C*15:02. This pattern was identified in patients with no sensitization history, negative FlowPRA results, and antibody to self-antigen(s). Epitope analysis failed to reveal a common determinant(s) to explain this pattern of reactivity. Additionally, we found this pattern to be prevalent in female patients (62%) and also those with systemic lupus erythematosus (62%). Given these findings, we speculate this pattern likely represents false-positive reactivity, possibly due to antibody targeting denatured antigens or a specific peptide, molecular mimicry, autoimmunity, or a combination thereof.
| S-EPMC7357571 | biostudies-literature
Project description:Objectives. Since the outbreak of SARS-CoV-2 in late 2019, nearly 12.2 billion doses of the COVID-19 vaccine have been administered worldwide; however, the humoral immune responses induced by different types of vaccines are yet to be fully validated. Methods. We analyzed antibody levels in 100 serum samples after vaccination with different types of COVID-19 vaccines and their reactivity against the RBD antigen of Delta and Omicron variants using a bead-based microarray. Results. Elevated levels of anti-wild-type (WT)-RBD IgG and anti-WT-NP IgG were detected in participants who received two doses of the inactivated vaccines (CoronaVac or BBIBP-CorV) and three doses of the recombinant spike protein vaccine (ZF2001), indicating that antibody responses to SARS-CoV-2 were generated regardless of the vaccine administered. We found highly correlated levels of serum anti-RBD IgG and anti-NP IgG (r = 0.432, p < 0.001). We observed that the antibodies produced in vivo after COVID-19 vaccination still reacted with variants of SARS-CoV-2 (p < 0.0001). Conclusions. Our results show that high levels of specific antibodies can be produced after completion of COVID-19 vaccination (two doses of the inactivated vaccines or three doses of ZF2001), with some degree of cross-reactivity to the RBD antigen of Delta and Omicron variants, and provide an accessible and practical experimental method for post-vaccination antibody detection.
| S-EPMC9964277 | biostudies-literature
Project description:BACKGROUND:Single antigen beads (SAB) are used for monitoring HLA antibodies in pretransplant and posttransplant patients despite the discrepancy between virtual and actual crossmatch results and transplant outcomes. This discrepancy can be attributed to the presence of conformational variants of HLA-I on SAB, assessment of which would increase the concordance between SAB and flow cytometry crossmatch (FCXM) results, thus enabling improved organ accessibility for the waiting list patients and a better prediction of antibody-mediated rejection. METHODS:The conformational variants were examined on HLA-I beads, iBeads, acid-/alkali-treated beads, and T cells using HLA-I monoclonal antibodies (W6/32, TFL-006, and heavy chain (HC)-10). RESULTS:The affinity of the monoclonal antibodies against HLA-I beads confirmed the presence and heterogeneous density of peptide-associated ?2-microglobulin-associated HLA HC (pepA-?2aHC), peptide-free-?2aHC (pepF-?2aHC), and ?2-free HC (?2fHC) on every single antigen-coated bead. In contrast, iBeads harbor a high density of pepA-?2aHC, low density of pepF-?2aHC, and are lacking ?2fHC. The FCXM analyses confirmed the prevalence of pepA-?2aHC, but not pepF-?2aHC or ?2fHC on resting T cells. CONCLUSIONS:The strength of a donor-specific antibody should be assessed with a bead-specific mean fluorescence intensity cutoff based on TFL-006 reactivity against HLA-I beads, and HC-10 against iBeads, where the ?2fHC or pepF-?2aHC normalized donor-specific antibody level would reveal the true anti-pepA-?2aHC reactivity associated with positive FCXM.
| S-EPMC7228605 | biostudies-literature
Project description:The present study was conducted to compare two stool antigen detection kits with PCR for the diagnosis of Entamoeba histolytica infections by using fecal specimens submitted to the Department of Microbiology at St. Vincent's Hospital, Sydney, and the Institute of Medical and Veterinary Science, Adelaide, Australia. A total of 279 stool samples containing the E complex (E. histolytica, Entamoeba dispar, and Entamoeba moshkovskii) were included in this study. The stool specimens were tested by using two commercially produced enzyme immunoassays (the Entamoeba CELISA PATH and TechLab E. histolytica II kits) to detect antigens of E. histolytica. DNA was extracted from all of the samples with a Qiagen DNA stool mini kit (Qiagen, Hilden, Germany), and a PCR targeting the small-subunit ribosomal DNA was performed on all of the samples. When PCR was used as a reference standard, the CELISA PATH kit showed 28% sensitivity and 100% specificity. The TechLab ELISA (enzyme-linked immunosorbent assay) kit did not prove to be useful in detecting E. histolytica, as it failed to identify any of the E. histolytica samples which were positive by PCR. With the TechLab kit, cross-reactivity was observed for three specimens, one of which was positive for both E. dispar and E. moshkovskii while the other two samples contained E. moshkovskii. Quantitative assessment of the PCR and ELISA results obtained showed that the ELISA kits were 1,000 to 10,000 times less sensitive, and our results show that the CELISA PATH kit and the TechLab ELISA are not useful for the detection of E. histolytica in stool samples from patients in geographical regions where this parasite is not endemic.
| S-EPMC2395085 | biostudies-literature
Project description:BackgroundNo single marker of bladder cancer (BC) exists in urine samples with sufficient accuracy for disease diagnosis and treatment monitoring. The multiplex Oncuria BC assay noninvasively quantifies the concentration of 10 protein analytes in voided urine samples to quickly generate a unique molecular profile with proven BC diagnostic and treatment-tracking utility. Test adoption by diagnostic and research laboratories mandates reliably reproducible assay performance across a variety of instrumentation platforms used in different laboratories.MethodsWe compared the performance of the clinically validated Oncuria BC multiplex immunoassay when data output was generated on three different analyzer systems. Voided urine samples from 36 subjects (18 with BC and 18 Controls) were reacted with Oncuria test reagents in three 96-well microtiter plates on Day 1, and consecutively evaluated on the LED/image-based MagPix, and laser/flow-based Luminex 200 and FlexMap 3D (all xMAP instruments from Luminex Corp., Austin, TX) on Day 2. The BC assay uses magnetic bead-based fluorescence technology (xMAP, Multi-analyte profiling; Luminex) to simultaneously quantify 10 protein analytes in urine specimens [i.e., angiogenin (ANG), apolipoprotein E (ApoE), carbonic anhydrase IX (CA9), CXCL8/interleukin-8 (IL-8), matrix metalloproteinase-9 (MMP-9), matrix metalloproteinase-10 (MMP-10), serpin A1/alpha-1 anti-trypsin (A1AT), serpin E1/plasminogen activator inhibitor-1 (PAI-1), CD138/syndecan-1 (SDC1), and vascular endothelial growth factor-A (VEGF-A)]. All three analyzers quantify fluorescence signals generated by the Oncuria assay.ResultsAll three platforms categorized all 10 analytes in identical samples at nearly identical concentrations, with variance across systems typically < 5%. While the most contemporary instrument, the FlexMap 3D, output higher raw fluorescence values than the two comparator systems, standard curve slopes and analyte concentrations determined in urine samples were concordant across all three units. Forty-four percent of BC samples registered ≥ 1 analyte above the highest standard concentration, i.e., A1AT (n = 7/18), IL-8 (n = 5), and/or ANG (n = 2), while only one control sample registered an analyte (A1AT) above the highest standard concentration.ConclusionMultiplex BC assays generate detailed molecular signatures useful for identifying BC, predicting treatment responsiveness, and tracking disease progression and recurrence. The similar performance of the Oncuria assay across three different analyzer systems supports test adaptation by clinical and research laboratories using existing xMAP platforms.Trial registrationThis study was registered at ClinicalTrials.gov as NCT04564781, NCT03193528, NCT03193541, and NCT03193515.
| S-EPMC10763405 | biostudies-literature
Project description:BackgroundNo single marker of bladder cancer (BC) exists in urine samples with sufficient accuracy for disease diagnosis and treatment monitoring. The multiplex Oncuria BC assay noninvasively quantifies the concentration of 10 protein analytes in voided urine samples to quickly generate a unique molecular profile with proven BC diagnostic and treatment-tracking utility. Test adoption by diagnostic and research laboratories mandates reliably reproducible assay performance across a variety of instrumentation platforms used in different laboratories.MethodsWe compared the performance of the clinically validated Oncuria BC multiplex immunoassay when data output was generated on three different analyzer systems. Voided urine samples from 36 subjects (18 with BC and 18 Controls) were reacted with Oncuria test reagents in three 96-well microtiter plates on Day 1, and consecutively evaluated on the LED/image-based MagPix, and laser/flow based Luminex 200 and FlexMap 3D (all xMAP instruments from Luminex Corp., Austin, TX) on Day 2. The BC assay uses magnetic bead-based fluorescence technology (xMAP, Multi-analyte profiling; Luminex) to simultaneously quantify 10 protein analytes in urine specimens [i.e., angiogenin (ANG), apolipoprotein E (ApoE), carbonic anhydrase IX (CA9), CXCL8/interleukin-8 (IL-8), matrix metalloproteinase-9 (MMP-9), matrix metalloproteinase-10 (MMP-10), serpin A1/alpha-1 anti-trypsin (A1AT), serpin E1/plasminogen activator inhibitor-1 (PAI-1), CD138/syndecan-1 (SDC1), and vascular endothelial growth factor-A (VEGF-A)].ResultsAll three platforms categorized all 10 analytes in identical samples at nearly identical concentrations, with variance across systems typically <5%. While the most contemporary instrument, the FlexMap 3D, output higher raw fluorescence values than the two comparator systems, standard curve slopes and analyte concentrations determined in urine samples were concordant across all three units. Forty-four percent of BC samples registered ≥1 analyte above the highest standard concentration, i.e., A1AT (n=7/18), IL-8 (n=5), and/or ANG (n=2). In Controls, A1AT was higher in one sample.ConclusionMultiplex BC assays generate detailed molecular signatures useful for identifying BC, predicting treatment esponsiveness, and tracking disease progression and recurrence. The similar performance of the Oncuria assay across three different analyzer systems supports test adaptation by clinical and research laboratories using existing xMAP platforms.Trial registrationThis study was registered at ClinicalTrials.gov as NCT04564781, NCT03193528, NCT03193541, and NCT03193515.
| S-EPMC10690323 | biostudies-literature
Project description:This article describes the procedures used to isolate pure B-cell populations from whole blood using various Miltenyi magnetic-activated cell sorting (MACS) bead Isolation kits. Such populations are vital for studies investigating the functional capacity of B-cells, as the presence of other cell types may have indirect effects on B-cell function through cell-cell interactions or by secretion of several soluble molecules. B-cells can be isolated by two main approaches: 1) Negative selection-in which B-cells remain "untouched" in their native state; this is advantageous as it is likely that B-cells remain functionally unaltered by this process. 2) Positive selection-in which B-cells are labelled and actively removed from the sample. We used three Negative B-cell isolation kits as well as the Positive B-cell isolation kit from Miltenyi and compared the purity of each of the resulting B-cells fractions. Contamination of isolated B-cell fractions with platelets was the conclusive finding for all of the isolation techniques tested. These results illustrate the inefficiency of current available MACS B-cell isolation kits to produce pure B-cell populations, from which concrete findings can be made. As such we suggest cell sorting as the preferred method for isolating pure B-cells to be used for downstream functional assays.
| S-EPMC6426237 | biostudies-literature
Project description:BackgroundSolid-phase assays to distinguish complement binding from noncomplement binding HLA-specific antibodies have been introduced, but technical limitations may compromise their interpretation. We have examined the extent to which C1q-binding to HLA-class I single-antigen beads (SAB) is influenced by denatured HLA on SAB, antibody titre, and complement interference that causes a misleading low assessment of HLA-specific antibody levels.MethodsSera from 25 highly sensitized patients were tested using Luminex IgG-SAB and C1q-SAB assays. Sera were tested undiluted, at 1:20 dilution to detect high-level IgG, and after ethylene diamine tetraacetic acid treatment to obviate complement interference. Conformational HLA and denatured HLA protein levels on SAB were determined using W6/32 and HC-10 monoclonal antibodies, respectively. Denatured HLA was expressed as HC-10 binding to untreated SAB as a percentage of maximal binding to acid-treated SAB.ResultsFor undiluted sera, Luminex mean fluorescence intensity (MFI) values for IgG-SAB and C1q-SAB correlated poorly (r = 0.42). ethylene diamine tetraacetic acid and serum dilution improved the correlation (r = 0.57 and 0.77, respectively). Increasing levels of denatured HLA interfered with the detection of C1q binding. Consequently, the correlation between IgG-SAB MFI and C1q-SAB MFI was lowest using undiluted sera and SAB with greater than 30% denatured HLA (r = 0.40) and highest using diluted sera and SAB with 30% or less denatured HLA (r = 0.86).ConclusionsAntibody level, complement interference, and denatured HLA class I on SAB may all affect the clinical interpretation of the C1q-SAB assay. The C1q-SAB assay represents a substantial additional cost for routine clinical use, and we question its justification given the potential uncertainty about its interpretation.
| S-EPMC5457814 | biostudies-literature
Project description:AimsTissue transglutaminase (tTG) is a major autoantigen recognised by IgA anti-endomysial antibodies (IgA EMA). Enzyme linked immunosorbent assays (ELISA) for IgA anti-tissue transglutaminase antibodies (IgA tTG) have therefore been developed as an alternative serological screening test to IgA EMA for coeliac disease (CD). The use of human tTG (h-tTG), as opposed to guinea pig liver tTG (gpl-tTG), in these assays has been reported to produce superior results. This study compared 13 commercial IgA tTG ELISA kits to ascertain their performance characteristics in the diagnosis of CD in patients with biopsy confirmed disease compared with controls. All patients and controls were adults aged 21 years or older.MethodsSera from the following groups of patients were tested in each kit: (1) 49 patients with CD confirmed on small bowel biopsies (all IgA EMA positive); (2) 34 patients with small bowel biopsies that were not consistent with CD; and (3) 30 patients with biopsy confirmed inflammatory bowel disease. All controls were negative for IgA EMA and were not IgA deficient. Sensitivities and specificities were determined using both the manufacturers' recommended cut off points and receiver operating characteristic (ROC) analysis derived decision thresholds. The area under the curve (AUC) for each ROC plot was also calculated and compared between kits.ResultsIn general, the h-tTG based IgA tTG ELISA kits demonstrated superior performance (especially specificity) compared with the gpl-tTG based kits, although 100% sensitivity and specificity (comparable to the IgA EMA assay) was obtained in only one recombinant h-tTG based kit.ConclusionsThe use of h-tTG in IgA tTG ELISA kits is generally, but not universally, associated with superior performance. Factors other than antigen source are important in determining kit performance.
| S-EPMC1769686 | biostudies-literature
Project description:The transporter associated with antigen processing (TAP) translocates the cytosol-derived proteolytic peptides to the endoplasmic reticulum lumen where they complex with nascent human leukocyte antigen (HLA) class I molecules. Non-functional TAP complexes and viral or tumoral blocking of these transporters leads to reduced HLA class I surface expression and a drastic change in the available peptide repertoire. Using mass spectrometry to analyze complex human leukocyte antigen HLA-bound peptide pools isolated from large numbers of TAP-deficient cells, we identified 334 TAP-independent ligands naturally presented by four different HLA-A, -B, and -C class I molecules with very different TAP dependency from the same cell line. The repertoire of TAP-independent peptides examined favored increased peptide lengths and a lack of strict binding motifs for all four HLA class I molecules studied. The TAP-independent peptidome arose from 182 parental proteins, the majority of which yielded one HLA ligand. In contrast, TAP-independent antigen processing of very few cellular proteins generated multiple HLA ligands. Comparison between TAP-independent peptidome and proteome of several subcellular locations suggests that the secretory vesicle-like organelles could be a relevant source of parental proteins for TAP-independent HLA ligands. Finally, a predominant endoproteolytic peptidase specificity for Arg/Lys or Leu/Phe residues in the P(1) position of the scissile bond was found for the TAP-independent ligands. These data draw a new and intricate picture of TAP-independent pathways.
| S-EPMC3608615 | biostudies-literature