Journal: Analytical biochemistry

Article Title: High affinity human Fc specific monoclonal antibodies for capture kinetic analyses of antibody-antigen interactions.

doi: 10.1016/j.ab.2021.114455

Figure Lengend Snippet: Fig. 1. Binding specificity of different AHC antibodies to purified immunoglobulin using ELISA. The ELISA plates were first coated overnight at 4◦C with 0.5 μg/mL of (A) hIgG1, (B) hIgG2, (C) hIgG4, (D) hFab_1, (E) hFab_4, (F) rabbit IgG, (G) cynomologous monkey IgG, or (H) rhesus monkey IgG. After three washes in wash buffer, the plates were blocked with blocking buffer for 1 h. The wells were later incubated with varying concentrations of AHC antibodies for 1 h and after washing thrice in wash buffer, the wells were incubated with HRP conjugated goat anti-mouse Fc antibody or bovine anti-goat IgG antibody and finally processed for enzymatic reaction for the detection of bound AHC antibodies. For each IgG coat, control antibody (black triangle) was used as positive control and the concentration dependent binding of goat AHC pAb-1 (magenta circle), goat AHC pAb-2 (blue circle), goat AHC pAb-3 (green circle), REGN2567 (orange circle), REGN2681 (black circle), REGN7779 (magenta triangle), REGN7941 (blue triangle), REGN7942 (green triangle), or REGN7943 (orange triangle) to different purified immunoglobulins are shown. Each data point represents the average of duplicate samples for each concentration ± standard deviation. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: Different AHC pAbs were purchased from Jackson ImmunoResearch (Catalog# 109-005-098), MP Biomedicals (Catalog# 0855071) and Southern Biotech (Catalog# 2048–01).

Techniques: Binding Assay, Purification, Enzyme-linked Immunosorbent Assay, Blocking Assay, Incubation, Control, Positive Control, Concentration Assay, Standard Deviation

Journal: Analytical biochemistry

Article Title: High affinity human Fc specific monoclonal antibodies for capture kinetic analyses of antibody-antigen interactions.

doi: 10.1016/j.ab.2021.114455

Figure Lengend Snippet: Fig. 2. Binding of different AHC antibodies to wild-type hIgG subclasses. Different goat AHC pAbs or mouse AHC mAbs were captured at a very low density (3–7 RU) using mouse anti-goat Fc mAb or rabbit anti- mouse Fc pAb, respectively. Different concentrations of (A) hIgG1, (B) hIgG2, or (C) hIgG4 (serially diluted by three-fold) were subsequently injected for 3 min followed by a 20 min dissociation time. The real time binding sensorgrams (shown in black) represent duplicate injections for each hIgG concentration, while the fits generated by globally fitting the data using 1:1 binding model with mass transport limitation are shown as red lines. The highest hIgG concentration used to fit the data is also provided and the results of the kinetics analyses is listed in Table 3. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: Different AHC pAbs were purchased from Jackson ImmunoResearch (Catalog# 109-005-098), MP Biomedicals (Catalog# 0855071) and Southern Biotech (Catalog# 2048–01).

Techniques: Binding Assay, Injection, Concentration Assay, Generated

Journal: Analytical biochemistry

Article Title: High affinity human Fc specific monoclonal antibodies for capture kinetic analyses of antibody-antigen interactions.

doi: 10.1016/j.ab.2021.114455

Figure Lengend Snippet: Fig. 3. Binding of different AHC antibodies to six representative hmAb variants. Different goat AHC pAbs or mouse AHC mAbs were captured at a very low density (3–7 RU) using mouse anti-goat Fc mAb or rabbit anti-mouse Fc pAb surfaces, respectively. Different concentrations of hIgG variants (serially diluted by three-fold) were later injected for 3 min followed by a 20 min dissociation time. Sensorgrams for seven of the 43 hIgG variants listed in Table 4 are shown: (A) mAb-1, (B) mAb-2, (C) mAb-6, (D) mAb-10, (E) mAb-11, (F) mAb-29, or (G) mAb-31. The mAbs are either expressed as a hIgG1 (A–E) or as a hIgG4 (F–G). The real time binding sensorgrams (shown in black) represent duplicate injections of each hIgG concentration, while the fits generated by globally fitting the binding sensorgrams using 1:1 binding model with mass transport limitation is shown as red lines. The highest hIgG concentration used to fit the data is provided and the results of the kinetic analyses are listed in Table 3. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: Different AHC pAbs were purchased from Jackson ImmunoResearch (Catalog# 109-005-098), MP Biomedicals (Catalog# 0855071) and Southern Biotech (Catalog# 2048–01).

Techniques: Binding Assay, Injection, Concentration Assay, Generated

Journal: Analytical biochemistry

Article Title: High affinity human Fc specific monoclonal antibodies for capture kinetic analyses of antibody-antigen interactions.

doi: 10.1016/j.ab.2021.114455

Figure Lengend Snippet: Fig. 4. Comparison of binding kinetic data performed on six mouse AHC mAbs and three goat AHC pAbs for binding to three wild-type hIgG and 43 different hIgG variants. The scatter plot shows (A) ka, (B) kd, (C) KD, (D) t½ values of different AHC antibodies binding to wild-type hIgG1 ( ), hIgG2 ( ), hIgG4 ( ), or different variants of hIgG1 ( ) or hIgG4 ( ).

Article Snippet: Different AHC pAbs were purchased from Jackson ImmunoResearch (Catalog# 109-005-098), MP Biomedicals (Catalog# 0855071) and Southern Biotech (Catalog# 2048–01).

Techniques: Comparison, Binding Assay

Journal: Analytical biochemistry

Article Title: High affinity human Fc specific monoclonal antibodies for capture kinetic analyses of antibody-antigen interactions.

doi: 10.1016/j.ab.2021.114455

Figure Lengend Snippet: Fig. 7. Binding capacity of REGN7942, REGN7943 and the three goat AHC pAbs immobilized on CM5, CM4 and C1 sensor chips. REGN7942, REGN7943 or the three goat AHC pAbs were individually immobilized on different surfaces of CM5, CM4 and C1 sensor chips at a density of 10000–12000 RU, 4000–5000 RU and 1000 RU, respectively. Varying concentrations of hIgG1, hIgG2, hIgG4 or hIgG4P (0.1, 0.5, 2, 10 μg/mL) were injected for 3 min with a 10 min dissociation phase. An aveage of four replicate injections for each hIgG concentration is plotted (±standard deviation).

Article Snippet: Different AHC pAbs were purchased from Jackson ImmunoResearch (Catalog# 109-005-098), MP Biomedicals (Catalog# 0855071) and Southern Biotech (Catalog# 2048–01).

Techniques: Binding Assay, Injection, Concentration Assay, Standard Deviation

Journal: Analytical biochemistry

Article Title: High affinity human Fc specific monoclonal antibodies for capture kinetic analyses of antibody-antigen interactions.

doi: 10.1016/j.ab.2021.114455

Figure Lengend Snippet: Fig. 8. Binding capacity of REGN7942, REGN7943 and the three goat AHC pAb surfaces on a C1 sensor chip. Approximately 1000 RU of REGN7942, REGN7943 or the three goat AHC pAbs were individually immobilized on different surfaces of a C1 sensor chip. Varying concentrations of (A) hIgG1, (B) hIgG2, (C) hIgG4, or (D) hIgG4P were injected for 3 min followed by a 10 min dissociation phase. Binding sensorgrams are color coded based on the hIgG concentrations and an overlay of four replicate injections for each hIgG concentration is shown. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: Different AHC pAbs were purchased from Jackson ImmunoResearch (Catalog# 109-005-098), MP Biomedicals (Catalog# 0855071) and Southern Biotech (Catalog# 2048–01).

Techniques: Binding Assay, Injection, Concentration Assay

Journal: Analytical biochemistry

Article Title: High affinity human Fc specific monoclonal antibodies for capture kinetic analyses of antibody-antigen interactions.

doi: 10.1016/j.ab.2021.114455

Figure Lengend Snippet: Fig. 9. Binding sensorgrams showing long term stability of hIgG1 or hIgG4P captured on a CM5, CM4 or C1 sensor chip immobilized with REGN7942, REGN7943 or the three goat AHC pAbs. CM5, CM4 or C1 sensor chips were individually immobilized with REGN7942, REGN7943 or each of the three goat AHC pAbs at a density of approximately 10000–12000 RU, 4000–5000 RU and 1000 RU, respectively. Around 500 RU (CM5 chip), 350–450 RU (CM4 chip) or 100–200 RU (C1 chip) of hIgG1 (A, C, E) or hIgG4P (B, D, F) were captured on different AHC antibody surfaces and their dissociation in HBS-ET running buffer was monitored for 10,000 s (2 h and 47 min). An overlay of at least triplicate injections of hIgG is shown.

Article Snippet: Different AHC pAbs were purchased from Jackson ImmunoResearch (Catalog# 109-005-098), MP Biomedicals (Catalog# 0855071) and Southern Biotech (Catalog# 2048–01).

Techniques: Binding Assay

Journal: Analytical biochemistry

Article Title: High affinity human Fc specific monoclonal antibodies for capture kinetic analyses of antibody-antigen interactions.

doi: 10.1016/j.ab.2021.114455

Figure Lengend Snippet: Fig. 10. Capture kinetic analyses performed on hmAb-Ag interactions expressed with canonical hIgG and selective hIgG Fc variantusing three goat AHC pAbs, REGN7942 or REGN7943 immobilized surfaces. Capture kinetic analyses was performed by capturing 90 RU or 35 RU of hmAb binding to (A–D) 21.9kD Ag or (E, F) 74.7kD Ag, respectively. The hmAbs, expressed as (A) hIgG1, (B) hIgG2, (C) hIgG4, (D, E), hIgG4P or (F) hIgG1 with M252Y/V308P/N434Y variant were captured over three goat AHC pAbs, REGN7942 or REGN7943 immobilized CM5 sensor surfaces. Different concentrations of respective Ags (serially diluted by three- fold) were later injected for 3 min followed by a 15 min of dissociation time. The real time binding sensorgrams (shown in black) represent duplicate injections for each Ag concentration while the fits generated by globally fitting the data using 1:1 binding model with mass transport limitation are shown as red lines. The highest Ag concentration used to fit the data is shown and Table 7 provides the results of the kinetic analyses. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: Different AHC pAbs were purchased from Jackson ImmunoResearch (Catalog# 109-005-098), MP Biomedicals (Catalog# 0855071) and Southern Biotech (Catalog# 2048–01).

Techniques: Binding Assay, Variant Assay, Injection, Concentration Assay, Generated

Journal: Analytical biochemistry

Article Title: High affinity human Fc specific monoclonal antibodies for capture kinetic analyses of antibody-antigen interactions.

doi: 10.1016/j.ab.2021.114455

Figure Lengend Snippet: Fig. 11. Capture kinetic analyses performed on four picomolar affinity hmAbs using three goat AHC pAb, REGN7942 or REGN7943 capture surfaces. Similar levels of hmAb binding to (A) 14.8kD, (B) 17.9kD, (C) 27.7kD, or (D) 57.8kD Ag were captured over three goat AHC pAb, REGN7942 or REGN7943 immobilized surfaces followed by the injection of different concentrations of their respective Ags (serially diluted by three-fold) for 3 min with an hour of dissociation phase. The real time binding sensorgrams (shown in black) represent at least duplicate injections for each Ag concentration while the fits generated by globally fitting the data using a 1:1 binding model with mass transport limitation is shown as red lines. The highest Ag concentration used to fit the data is provided and the results of the kinetic analyses are provided in Table 7. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: Different AHC pAbs were purchased from Jackson ImmunoResearch (Catalog# 109-005-098), MP Biomedicals (Catalog# 0855071) and Southern Biotech (Catalog# 2048–01).

Techniques: Binding Assay, Injection, Concentration Assay, Generated

Journal: Oncology Letters

Article Title: Generation of TIM3 inhibitory single-domain antibodies to boost the antitumor activity of chimeric antigen receptor T cells

doi: 10.3892/ol.2021.12803

Figure Lengend Snippet: Generation of TIM3 sdAbs. (A) Serum titer of rabbit R055 immunized with the recombinant TIM3-hFc. The control antigen, IAB-hFc, was a recombinant fragment of mesothelin (Q13421, amino acid 296–359). M0, pre-immunization serum; M2, post-immunization serum. (B) Serum titer of rabbit R056. The same experimental settings were followed. (C) Polyclonal phage ELISA. Eluted phage from the first, second and third round of panning was tested for its binding to TIM3-hFc. (D) Monoclonal phage ELISA. Monoclonal phage clones R23 and R53 were tested for their binding to TIM3-hFc. (E) Amino acid sequences of sdAbs R23 and R53. TIM3, T cell immunoglobulin and mucin domain 3; ELISA, enzyme-linked immunosorbent assay; sdAbs, single-domain antibodies.

Article Snippet: Cell binding of TIM3 sdAbs was detected with APC-conjugated goat anti-human polyclonal antibody (1:1,000; cat. no. 109-135-170, Jackson ImmunoResearch Laboratories, Inc.).

Techniques: Recombinant, Control, Enzyme-linked Immunosorbent Assay, Binding Assay, Clone Assay

Journal: Oncology Letters

Article Title: Generation of TIM3 inhibitory single-domain antibodies to boost the antitumor activity of chimeric antigen receptor T cells

doi: 10.3892/ol.2021.12803

Figure Lengend Snippet: Cell binding properties of TIM3 sdAbs. (A) Schematic illustration of the mesothelin-targeted CAR structure. Anti-mesothelin scFv YP158 was fused with the following domains: CD8α hinge, CD8α transmembrane region, 4-1BB intracellular domain, CD3ζ intracellular domain, internal ribosome entry site and a red fluorescent protein mScarlet-I. (B) Transduction efficiency analysis of CAR T cells. Following the transduction of PBMCs with a CAR-bearing lentiviral vector, CAR expression was indicated by the fluorescence of mScarletI. (C) Flow cytometric analysis of TIM3 sdAbs binding to the unstimulated PBMCs and CAR T cells. Briefly, 10 µg sdAbs were co-incubated with 1×10 6 cells. Antibody binding was detected by APC-conjugated goat-anti-human IgG. HN3, a human sdAb antibody that targeted the glypican-3, was used as the isotype control. Shaded area, secondary antibody staining; dashed lines, isotype control; red and blue solid line, sdAbs R53 and R23 staining, respectively. (D) Expression of TIM3 on CAR T cells cultured for different lengths of time. TIM3, T cell immunoglobulin and mucin domain 3; sdAbs, single-domain antibodies; CAR, chimeric antigen receptor; PBMCs, peripheral blood mononuclear cells.

Article Snippet: Cell binding of TIM3 sdAbs was detected with APC-conjugated goat anti-human polyclonal antibody (1:1,000; cat. no. 109-135-170, Jackson ImmunoResearch Laboratories, Inc.).

Techniques: Binding Assay, Transduction, Plasmid Preparation, Expressing, Fluorescence, Incubation, Control, Staining, Cell Culture