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

Journal: Cancers

Article Title: CD46-ADC Reduces the Engraftment of Multiple Myeloma Patient-Derived Xenografts.

doi: 10.3390/cancers15225335

Figure Lengend Snippet: Figure 2. Samples from newly diagnosed MM patients engraft better than do those from patients with relapsed/refractory MM. (A) NSG mice harboring human fetal bone grafts injected with patient samples, and clonal human free light chain detected in serum of engraftment mice via ELISA for 6 weeks followed by endpoint analysis. (B) Serum ELISA resulting in mice showing detectable levels of human light chain that matched the kappa or lambda restriction of the patient sample of origin. (C) Detection of CD38+, CD138+ (left panel) and light chain-restricted (right panel) human myeloma cells via flow cytometry of cells isolated from bone graphs with a detectable serum-free light chain. (D) Myeloma engraftment in 8/19 (42%) and samples selected from newly diagnosed (Dx) patients being engrafted more frequently than samples from relapsed/refractory (RR) patients. Error bars—standard deviation.

Article Snippet: After washing, secondary antibody mouse anti-human kappa/lambda AP (SouthernBiotech, Birmingham, AL, USA) was applied and incubated for 2 h at 37 ◦C.

Techniques: Injection, Enzyme-linked Immunosorbent Assay, Cytometry, Isolation, Standard Deviation

Journal: Molecular Therapy

Article Title: Expression of Immunomodulatory Neutrophil-activating Protein of Helicobacter pylori Enhances the Antitumor Activity of Oncolytic Measles Virus

doi: 10.1038/mt.2012.4

Figure Lengend Snippet: Schematic representation of the recombinant measles virus (MV) strains used in the experiments: MV-lambda, MV-lambda-NAP, MV-s-NAP and MV-NIS. NAP, neutrophil-activating protein.

Article Snippet: Proteins were transferred to polyvinylidene fluoride membrane (Bio-Rad) and probed with the NAP-specific monoclonal antibody 16F4 or antibody against human lambda light chain as previously described.

Techniques: Recombinant, Virus

Journal: Molecular Therapy

Article Title: Expression of Immunomodulatory Neutrophil-activating Protein of Helicobacter pylori Enhances the Antitumor Activity of Oncolytic Measles Virus

doi: 10.1038/mt.2012.4

Figure Lengend Snippet: In vitro antitumor activity of engineered measles virus (MV) strains against breast cancer cells. (a) MCF-7 cell line and the highly tumorigenic MDA231-lu-P4 in vivo derivative of (b) MDA-MB-231 cells were infected with MV strains at an multiplicity of infection (MOI) = 1.0 and cell viability was determined by MTT assay. Both MV-lambda-NAP and MV-lambda viruses propagated rapidly causing complete destruction of the breast cancer monolayers 72 hours postinfection. MV-s-NAP and MV-NIS showed similar tumor cell-killing kinetics with complete eradication of MCF-7 cells or ~80% reduction of cell viability of MDA231-lu-P4 cells. (c) Vero cells were used as control. The data are presented as percent of untreated control cells ± SD. (d) NAP transgene induced significant interleukin (IL)-8 expression in MV vector-infected THP-1 cells. THP-1 monocytic cells were infected with MV-s-NAP or control MV-lambda virus at a MOI = 0.1 and incubated for 72 hours. Supernatants were collected and IL-8 concentration was measured by enzyme-linked immunosorbent assay (ELISA) specific for human IL-8. The effect of TNF-α (one of the known NAP-triggered inflammatory cytokines) on proliferation of MV-infected or uninfected MDA231-lu-P4 cells was examined by MTT assay (e,f). MDA231-lu-P4 cells were plated at 104 cell/well density and inoculated at MOI = 0.5 of MV-s-NAP in the presence or absence of 250 U/ml recombinant human TNF-α (e). The same experiment was repeated with lower cell density (2.5 × 103 per well) and MV-s-NAP at MOI of 0.25 (f).

Article Snippet: Proteins were transferred to polyvinylidene fluoride membrane (Bio-Rad) and probed with the NAP-specific monoclonal antibody 16F4 or antibody against human lambda light chain as previously described.

Techniques: In Vitro, Activity Assay, Virus, In Vivo, Infection, MTT Assay, Control, Expressing, Plasmid Preparation, Incubation, Concentration Assay, Enzyme-linked Immunosorbent Assay, Recombinant

Journal: Molecular Therapy

Article Title: Expression of Immunomodulatory Neutrophil-activating Protein of Helicobacter pylori Enhances the Antitumor Activity of Oncolytic Measles Virus

doi: 10.1038/mt.2012.4

Figure Lengend Snippet: Measles virus (MV) infection and neutrophil-activating protein (NAP) transgene expression in the malignant pleural effusion of mice-bearing MDA231-lu-P4 pleural xenografts. Mice were treated by a single transthoracic (t.t.). injection of MV-s-NAP or MV-lambda. NAP transgene expression in pleural fluid was demonstrated by immunoblotting using (a) NAP-specific monoclonal antibody (MAb) 16F4 or (b) lambda chain-specific antibody. The secretory form of the NAP transgene was detected in the pleural fluid of four of four MV-s-NAP-treated mice (lanes 1–4) but not in MV-lambda-injected (lanes 5, 6) control mice (a). Human lambda light chain was detected in three of four samples from MV-lambda-injected (lanes 3–6) mice but not in samples from MV-s-NAP-treated (lanes 1, 2) animals (b) using a lambda chain-specific detection antibody. MV-s-NAP (c) induced large multinucleated syncytia in infected tumor cells (Giemsa staining) in the pleural fluid of mice with MDA231-lu-P4 xenografts. Immunohistochemistry (IHC) staining for neutrophils in the pleural fluid of MV-s-NAP is shown in (d). MV-s-NAP was isolated from the pleural fluid by overlay on Vero cells. MV-s-NAP induced giant syncytia formation 24 hours after overlay (e). Uninfected control Vero cells (f).

Article Snippet: Proteins were transferred to polyvinylidene fluoride membrane (Bio-Rad) and probed with the NAP-specific monoclonal antibody 16F4 or antibody against human lambda light chain as previously described.

Techniques: Virus, Infection, Expressing, Injection, Western Blot, Control, Staining, Immunohistochemistry, Isolation

Journal: Molecular Therapy

Article Title: Expression of Immunomodulatory Neutrophil-activating Protein of Helicobacter pylori Enhances the Antitumor Activity of Oncolytic Measles Virus

doi: 10.1038/mt.2012.4

Figure Lengend Snippet: Therapeutic effect of neutrophil-activating protein (NAP)-expressing measles virus (MV) strains against lung metastatic breast cancer compared to control MV-NIS. (a) Engraftment of the systemically injected MDA231-lu-P3 or P4 cells was confirmed by bioluminescence imaging. (b,c) The animals with MDA231-lu-P3 lung tumors (8 per group) were treated with 10 repeat intravenous (i.v.) injections of 2 × 106 TCID50 of MV-lambda-NAP or heat-inactivated (HI-control) control. In a separate in vivo experiment MDA231-lu-P4 lung metastatic xenografts (9 mice per group) were treated by 7 i.v. injections of 106 TCID50 of MV-s-NAP, MV-NIS or the corresponding heat-inactivated controls (d–f). Both MV-lambda-NAP and MV-s-NAP improved the median survival with 10–12 days (P < 0.05) in this aggressive model of breast cancer metastatic to the lungs.

Article Snippet: Proteins were transferred to polyvinylidene fluoride membrane (Bio-Rad) and probed with the NAP-specific monoclonal antibody 16F4 or antibody against human lambda light chain as previously described.

Techniques: Expressing, Virus, Control, Injection, Imaging, In Vivo

Journal: Bmc Cancer [Electronic Resource]

Article Title: Upregulation of meiosis-specific genes in lymphoma cell lines following genotoxic insult and induction of mitotic catastrophe

doi: 10.1186/1471-2407-6-6

Figure Lengend Snippet: Antibody source and usage

Article Snippet: CREST serum against human kinetochore proteins (Acris Antibodies, Hiddenhausen, Germany) , Mouse anti-human IgG-FITC (Dr. MS Cragg).

Techniques: Plasmid Preparation