Journal: ImmunoTargets and Therapy
Article Title: Development of Anti- Yersinia pestis Human Antibodies with Features Required for Diagnostic and Therapeutic Applications
doi: 10.2147/ITT.S267077
Figure Lengend Snippet: Antibody specificity of binding to F1–positive Yersinia pestis . ( A ) Whole-cell ELISA. Plastic-bound whole Y. pestis or Y. pseudotuberculosis were used as “antigens”. Cells are indicated as YP ( Y. pestis) or YPS (Y. pseudotuberculosis) 37 or 23 (grown at 37°C or 23°C respectively), F1 + or F1 – (F1 positive or negative respectively). The binding of primary antibodies (anti-F1 IgG isotype 1: αF1Ig 1 [gray], αF1Ig 2 [red], αF1Ig 3 [green], αF1Ig 4 [purple], αF1Ig 6 [yellow], αF1Ig 8 (blue), or negative-control natural human IgG1 [brown]) to cells was detected using a goat anti-human-horseradish peroxidase (HRP) conjugate and HRP substrate TMB (whose acidified product causes absorbance at 450 nM (Abs 450 ). All αF1Ig types bound to F1 + Y. pestis , both live and fixed, but not to F1 – Y. pestis grown at 23°C, live or fixed, or to F1 – Y. pseudotuberculosis grown at 37°C, live or fixed. ( B ) Flow cytometry. Live Y. pestis grown at 37°C (left side) or at 23°C (right side) were incubated with phycoerythrin-conjugated antibodies (same color coding as for ELISA, αF1Ig 1 not included) and analyzed with flow cytometry. Commercial anti-F1 mouse antibody YPF19 (black) and natural human IgG1 (brown) were used as positive and negative controls, respectively. All αF1Ig clones bound to F1 + Y. pestis significantly more than the negative-control antibody (left), but did not bind to F1 – Y. pestis (right). F1 + Y. pestis treated with PE-labeled αF1 antibodies could be distinguished from its F1 - counterpart even by naked-eye observation of the cell pellets obtained after antibody treatment (insets, only αF1Ig 2–PE staining is shown, but other antibody conjugates behaved similarly).
Article Snippet: Y. pseudotuberculosis ATCC 27,802 and Pseudomonas fluorescens ATCC 13,475 were obtained from the American Tissue Culture Collection.
Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, Negative Control, Flow Cytometry, Incubation, Clone Assay, Labeling, Staining
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![Antibody specificity of binding to F1–positive Yersinia pestis . ( A ) Whole-cell ELISA. Plastic-bound whole Y. pestis or Y. <t>pseudotuberculosis</t> were used as “antigens”. Cells are indicated as YP ( Y. pestis) or YPS (Y. pseudotuberculosis) 37 or 23 (grown at 37°C or 23°C respectively), F1 + or F1 – (F1 positive or negative respectively). The binding of primary antibodies (anti-F1 IgG isotype 1: αF1Ig 1 [gray], αF1Ig 2 [red], αF1Ig 3 [green], αF1Ig 4 [purple], αF1Ig 6 [yellow], αF1Ig 8 (blue), or negative-control natural human IgG1 [brown]) to cells was detected using a goat anti-human-horseradish peroxidase (HRP) conjugate and HRP substrate TMB (whose acidified product causes absorbance at 450 nM (Abs 450 ). All αF1Ig types bound to F1 + Y. pestis , both live and fixed, but not to F1 – Y. pestis grown at 23°C, live or fixed, or to F1 – Y. pseudotuberculosis grown at 37°C, live or fixed. ( B ) Flow cytometry. Live Y. pestis grown at 37°C (left side) or at 23°C (right side) were incubated with phycoerythrin-conjugated antibodies (same color coding as for ELISA, αF1Ig 1 not included) and analyzed with flow cytometry. Commercial anti-F1 mouse antibody YPF19 (black) and natural human IgG1 (brown) were used as positive and negative controls, respectively. All αF1Ig clones bound to F1 + Y. pestis significantly more than the negative-control antibody (left), but did not bind to F1 – Y. pestis (right). F1 + Y. pestis treated with PE-labeled αF1 antibodies could be distinguished from its F1 - counterpart even by naked-eye observation of the cell pellets obtained after antibody treatment (insets, only αF1Ig 2–PE staining is shown, but other antibody conjugates behaved similarly).](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_6875/pmc07716875/pmc07716875__ITT-9-299-g0001.jpg)
![αF1Ig 2 and αF1Ig 8 recognize Y. pestis (YP) in a mixed bacterial community. ( A ) A mixed bacterial community of Y. pseudotuberculosis , Pseudomonas fluorescens , Escherichia coli , and Bacillus anthracis Sterne (represented equally at 22.5% each) was spiked with YP (10% representation) grown either at 37°C (YP 37, F1 + ) or 23°C (YP 23, F1 – ). The resulting cell suspensions were stained with αF1Ig 2–phycoerythrin (PE). After one wash, the bacterial pellet containing YP 37 (F1 + ) appeared lightly pink, whereas the pellet containing YP 23 (F1 – ) appeared white. ( B ) The mixed community spiked with YP 37 (F1 + ), stained with either αF1Ig 2–PE or αF1Ig 8–PE was analyzed by flow cytometry. Proportionally, 13.9% (αF1Ig 2–PE staining) and 15.7% (αF1Ig 8–PE staining) of the total events were fluorescent (gated events detected by side scatter, SSC, and PE fluorescence). ( C ) Single cells sorted during the flow experiment depicted in B were lysed. The resulting DNA was used as template for PCR amplification of either the 16S rRNA–encoding region (16S RNA, present in any bacteria, 1,500 bp in length) or a fragment (~200 bp) unique to the genome of Y. pestis (putative “helix-turn-helix” protein [HTH]). While flow-sorted stained and unstained single events tested positive for the 16S rRNA (showing that the detected events were actually bacteria), only the fluorescently labeled bacteria tested positive for HTH and were thus confirmed to be Y. pestis . The discrepancy between the intended YP representation and the representation measured by flow was likely due to inaccuracy in the relationship between cell density (OD 600 ) and cell number, which for the various bacteria used in this experiment was assumed to be OD 600 = 0.2 → ×10 8 cells/mL.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_6875/pmc07716875/pmc07716875__ITT-9-299-g0004.jpg)
