Journal: bioRxiv

Article Title: Profiling Allogeneic HLA-specific B-cell Responses Utilizing a 64-plex Single-HLA Reporter Cell Panel

doi: 10.64898/2026.01.19.700453

Figure Lengend Snippet: The HLA64-RC panel was labeled with fluorochrome-conjugated Abs or with unconjugated primary Abs followed by fluorochrome-conjugated secondary Abs. Following flow cytometry acquisition, data were analyzed using FlowJo ™ software. Live cell events were demultiplexed into 64 populations based on the six FP channels as in . Stacked histograms of the demultiplexed populations show fluorescence intensities corresponding to Ab labeling. Histograms are grouped by HLA-A (dark red), HLA-B (green), HLA-DQ (blue), and HLA-DR (purple) alleles, displayed upward in the order indicated on the far-right. Blue labels positioned in the upper-right (top row) or lower-right (bottom row) corners denote the reported specificities of the corresponding mAbs. hIgG1K and hIgG1L, human IgG1κ and IgG1λ isotype control Abs. BB7.2-hIgG1K, BB7.2 in human IgG1κ format. Anti-hIgG-PE and anti-mIgG-PE, PE-conjugated anti-human or anti-mouse IgG secondary Abs. SA-PE, PE-conjugated streptavidin.

Article Snippet: Following three additional washes, a cocktail of HRP-conjugated anti-human Igκ (Southern Biotech, 2061-05) and anti-human Igλ (Southern Biotech, 2071-05) Abs was added.

Techniques: Labeling, Flow Cytometry, Software, Fluorescence, Control

Journal: bioRxiv

Article Title: Profiling Allogeneic HLA-specific B-cell Responses Utilizing a 64-plex Single-HLA Reporter Cell Panel

doi: 10.64898/2026.01.19.700453

Figure Lengend Snippet: Using the HLA64-RC panel, ten serum samples (#01–#10) from sensitized transplant candidates were tested for HLA-binding activities at 1/3 serial dilutions from 1:2 to 1:486, followed by secondary labeling using PE-conjugated anti-human IgG at 2 µg/ml. Raw binding data are shown in Supplementary Document 1. The dilutions yielding the highest signal-to-background ratio for individual samples were selected and MFI values (Supplementary Data 3) were compared with those obtained from clinical SAB assays (Supplementary Data 4) using the same samples. a) Heatmaps comparing log 10 (MFI) values for shared alleles between the HLA64-RC and SAB assays. The selected dilutions for the HLA64-RC assay were indicated in parentheses. For the SAB assay, serum samples were tested neat. Alleles with MFI values below the binding threshold (see Methods) are shown in grey. Reactivities detected by the HLA64-RC assay but absent in the SAB assay are highlighted with blue boxes. b) Dot plots comparing log 10 (MFI) values for shared alleles between the HLA64-RC and SAB assays, as in ( a ). Data from multiple samples were pooled and plotted separately for HLA-A, -B, -DQ, and -DR loci, with color and shape indicating individual alleles within each locus. For consistency, only serum samples tested at 1:2 dilution in the HLA64-RC assay in ( a ) were included. Within each locus, Lin’s CCC with 95% bootstrap confidence interval (CI), Spearman correlation, and SMA regression with 95% bootstrap CI were calculated using log 10 (MFI) values from the two assays. Solid diagonal lines represent SMA regression fits; dotted lines indicate the 95% CI.

Article Snippet: Following three additional washes, a cocktail of HRP-conjugated anti-human Igκ (Southern Biotech, 2061-05) and anti-human Igλ (Southern Biotech, 2071-05) Abs was added.

Techniques: Binding Assay, Labeling

Journal: bioRxiv

Article Title: Profiling Allogeneic HLA-specific B-cell Responses Utilizing a 64-plex Single-HLA Reporter Cell Panel

doi: 10.64898/2026.01.19.700453

Figure Lengend Snippet: HLA monomer-binding single B-cell culture supernatants were screened using the HLA64-RC assay. R952-X06 and R952-X08 to -X12 cultures were derived from phenotyping antibody Panel 1 mediated sorting, and the rest from Panel 2 sorting. Secondary labeling was performed using PE-conjugated anti-human IgG (2 μg/ml). Flow cytometry data were analyzed and visualized using the HLA64 R package, and MFI values were exported (Supplementary Data 5). Demultiplexed HLA alleles corresponding to individual reporter cell populations are indicated on the right. Data are shown for HLA-specific cultures, and only HLA-A and -B loci are plotted. Human IgG1κ (hIgG1K), W6/32 in human IgG1κ format (W6/32-hIgG1K), and a no-B-cell culture supernatant were included as labeling controls. Populations exceeding the binding threshold (see Methods) relative to the no-Bcell control were automatically highlighted in red.

Article Snippet: Following three additional washes, a cocktail of HRP-conjugated anti-human Igκ (Southern Biotech, 2061-05) and anti-human Igλ (Southern Biotech, 2071-05) Abs was added.

Techniques: Binding Assay, Cell Culture, Derivative Assay, Labeling, Flow Cytometry, Control