goat anti-mouse igm, igg3, igg1, igg2b, igg2c, and iga antibodies  (SouthernBiotech)

Journal: Vaccines

Article Title: FLU-v, a Broad-Spectrum Peptide-Based Influenza Vaccine, Induces NK Cell Activating IgG1 and IgG3 Subclass Antibodies in Humans

doi: 10.3390/vaccines13111084

Figure Lengend Snippet: FLU-v IgG1 ( A ) and IgG3 ( B ) concentrations across all groups on days 0, 42, and 180. Data from the following groups are shown: adjuvanted FLU-v ( n = 52), non-adjuvanted FLU-v ( n = 58), adjuvanted placebo ( n = 26), and non-adjuvanted placebo ( n = 32). Box and whisker plot showing all individual values, 25th and 75th percentiles, and median. Whiskers represent ranges. The Mann–Whitney U test was used to compare different groups at each time point. The Wilcoxon signed-rank sum test was used to compare differences between time points within each group. Only significant differences are shown.

Article Snippet: Wells in the standard rows were coated with human IgG1 or IgG3 reference antibodies (16-16-090707-1/16-16-090707-3, Athens Research, Athens, GA, USA), performing double decreasing serial dilutions starting at 1000 ng/mL up to 1.95 ng/mL.

Techniques: Whisker Assay, MANN-WHITNEY

Journal: Vaccines

Article Title: FLU-v, a Broad-Spectrum Peptide-Based Influenza Vaccine, Induces NK Cell Activating IgG1 and IgG3 Subclass Antibodies in Humans

doi: 10.3390/vaccines13111084

Figure Lengend Snippet: Fold increase in FLU-v IgG1 ( A ) and IgG3 ( B ) concentrations across all groups from day 0 to days 42 and 180. Data from the following groups are shown: adjuvanted FLU-v ( n = 52), non-adjuvanted FLU-v ( n = 58), adjuvanted placebo (n = 26), and non-adjuvanted placebo ( n = 32). Box and whiskers plot showing all individual values, 25th and 75th percentiles, and median. Whiskers represent ranges. Fold change in IgG1 concentrations on day 42 and 180 is defined as the ratio of day 42 to day 0 and the ratio of day 180 to day 0, respectively. The Mann–Whitney U test was used to compare different groups at each time point. The Wilcoxon signed-rank sum test was used to compare differences between day 42 and 180 within each group. Significant differences are shown.

Article Snippet: Wells in the standard rows were coated with human IgG1 or IgG3 reference antibodies (16-16-090707-1/16-16-090707-3, Athens Research, Athens, GA, USA), performing double decreasing serial dilutions starting at 1000 ng/mL up to 1.95 ng/mL.

Techniques: MANN-WHITNEY

Journal: Vaccines

Article Title: FLU-v, a Broad-Spectrum Peptide-Based Influenza Vaccine, Induces NK Cell Activating IgG1 and IgG3 Subclass Antibodies in Humans

doi: 10.3390/vaccines13111084

Figure Lengend Snippet: Antibody-mediated NK cell activation after adjuvanted and non-adjuvanted FLU-v vaccination. For this analysis, a subset of paired samples ( n = 50) from day 0 and day 42 was selected based on high IgG1 and IgG3 fold increases. Data from the following groups are shown: adjuvanted FLU-v ( n = 32), non-adjuvanted FLU-v ( n = 16), and non-adjuvanted placebo ( n = 2). Antibody-dependent NK cell activation, or percentage of GFP + CD107a + CD16 (V176) NK-92 cells by flow cytometry, was measured in response to antibodies immobilized by plate-bound FLU-v ( A ) or non-related peptides used as negative controls ( B ). Medians ± 95% confidences intervals are shown. The Wilcoxon signed-rank sum test was used to compare differences between time points within each vaccine group. A Bonferroni test was used to correct for multiple comparisons, with a p value < 0.025 considered significant.

Article Snippet: Wells in the standard rows were coated with human IgG1 or IgG3 reference antibodies (16-16-090707-1/16-16-090707-3, Athens Research, Athens, GA, USA), performing double decreasing serial dilutions starting at 1000 ng/mL up to 1.95 ng/mL.

Techniques: Activation Assay, Flow Cytometry

Journal: Vaccines

Article Title: FLU-v, a Broad-Spectrum Peptide-Based Influenza Vaccine, Induces NK Cell Activating IgG1 and IgG3 Subclass Antibodies in Humans

doi: 10.3390/vaccines13111084

Figure Lengend Snippet: Correlation between antibody-mediated NK cell activation and FLU-v-specific IgG ( A ), IgG1 ( B ) and IgG3 ( C ) on days 0 and 42. Spearman rank order correlations were performed between the antibody-dependent NK cell activation assay, measuring the percentage of GFP + CD107a + CD16 (V176) NK-92 cells by flow cytometry, and total IgG, IgG1, and IgG3 with correlation coefficients (r) and p -values shown. IgG data for this analysis were obtained from a previous report .

Article Snippet: Wells in the standard rows were coated with human IgG1 or IgG3 reference antibodies (16-16-090707-1/16-16-090707-3, Athens Research, Athens, GA, USA), performing double decreasing serial dilutions starting at 1000 ng/mL up to 1.95 ng/mL.

Techniques: Activation Assay, Flow Cytometry

Journal: ImmunoHorizons

Article Title: Aberrant humoral immune responses and intestinal homeostasis in Cd38 Bst1 double knockout mice

doi: 10.1093/immhor/vlaf029

Figure Lengend Snippet: Tissue distribution of BST-1 and CD38. (A) Expression levels of Cd38 and Bst1 in various organs from WT mice. RNA was prepared from various organs from 3-mo-old male C57BL/6J mice (n = 4). Amounts of transcripts of Cd38 and Bst1 relative to Actb in each tissue were determined using quantitative real-time reverse-transcription polymerase chain reaction assay. The amount in each tissue was normalized with that in the spleen as 1. (B-F) Immunohistochemical detection of BST-1 or CD38 localizations. Cryosections of 4-mo-old male C57BL/6J mice were stained with rabbit anti-BST-1 serum and rat anti-CD38 mAb, and the bound antibodies were visualized with Cy3-donkey anti-rabbit IgG (red) and Alexa 488 donkey anti-rat IgG antibodies (green), respectively. Reactivities of anti-BST-1 and anti-CD38 antibodies are indicated with red and green, respectively. Merged images of red (a) and green (b) are indicated in panel c. (B) The negative control (nc) was no primary antibody; (C, D) the nc was preimmune rabbit serum and rat IgG1 as primary antibodies. White rectangles in panels C, D, and E indicate the area to be magnified in the images below. (D) The bronchi are indicated by arrowheads. (E) The area of the renal tubules is indicated by dotted lines. A broken line indicates a capsule of the kidney. To take the photo of the kidney, a longer exposure time was applied to increase the visibility of faint signals by anti-CD38 reactivities at glomeruli. (F) For the analyses of intestines, tissues from Cd38 KO and Bst1 KO were used as ncs for staining with anti-CD38 mAb and anti-BST-1 serum, respectively. Representative data from 3 independent experiments are shown.

Article Snippet: Enzyme-linked immunosorbent assay (ELISA) plates were coated with goat anti-mouse IgM, IgG3, IgG1, IgG2b, IgG2c, and IgA antibodies (SouthernBiotech) at 10 μg/mL in coating buffer (carbonate-bicarbonate, pH 9.6) at room temperature for 1 h. After washing 3 times with phosphate-buffered saline (PBS) containing 0.1% Tween 20 (washing buffer), and blocking with 0.1% bovine serum albumin (BSA) in washing buffer for 1 h, a 50 μL aliquot of mouse serum appropriately diluted for each isotype in PBS was applied and incubated for 1 h at room temperature.

Techniques: Expressing, Reverse Transcription, Polymerase Chain Reaction, Immunohistochemical staining, Staining, Negative Control

Journal: ImmunoHorizons

Article Title: Aberrant humoral immune responses and intestinal homeostasis in Cd38 Bst1 double knockout mice

doi: 10.1093/immhor/vlaf029

Figure Lengend Snippet: Bst1 KO B cell–intrinsic hyperresponsiveness to LPS. (A) Total Ig productions by 4 strains at 4 to 5 mo old (female n = 6) after stimulation with TNP-LPS were determined using ELISA. Bar graphs indicate mean with SD, and all data points are included. Statistical significance was determined using 2-way analysis of variance test, and Tukey’s multiple comparison test was used for post hoc testing. (B) Histological analyses of polyclonal immunoglobulin production in the spleen in TI-1 antigen responses. Cryosections of the spleens from WT and Bst1 KO at day 14 were stained with ALP-labeled goat anti-mouse IgM, IgG2b, and IgG2c, antibodies and visualized with BCIP-NBT (dark blue). Nuclei were stained with methyl green. Representative data from 6 female mice of each genotype. (C) Immunohistochemical quantification of antibody-producing cells in the spleen. The ratio of the area occupied by polyclonal antibody-producing cells visualized with BCIP-NBT in panel B to the area of 1 field of each splenic section stained for IgM, IgG2b, or IgG2c was calculated with ImageJ software. The mean and SD of relative areas occupied by antibody-producing cells from 6 mice are indicated in the bar graph. Gray and pale coral pink bars indicate data of WT and Bst1 KO mice, respectively. (D) Increased polyclonal IgG2b and IgA productions by BM chimera lacking BST-1 in hematopoietic cells. Four combinations of BM chimera, WT (C57BL/6J)→WT, Bst1 KO→WT, WT→ Bst1 KO, and Bst1 KO→ Bst1 KO (6 BM chimeras for each combination), were stimulated with TNP-LPS, and concentrations of polyclonal serum immunoglobulins were determined using ELISA. Statistical significances were determined using 2-way analysis of variance test, and Tukey’s multiple comparison test was used for post hoc testing. Asterisks of IgG2b are Bst1 KO→WT vs. WT→WT and WT→ Bst1 KO. Asterisks of IgG2c are WT→ Bst1 KO vs. WT→WT and Bst1 KO→WT. Asterisks of IgA are Bst1 KO→ Bst1 KO vs. WT→WT. (E) Increased growth responses of Bst1 KO B cells stimulated with LPS. Splenic B cells purified with MACS B cell isolation kit were stimulated with 10 µg/mL LPS or F(ab’) 2 goat anti-µ antibody and cultured for 72 h. Growth of B cells was estimated by MTT assay, and relative absorbances at 590 nm are shown. Bar graphs indicate mean with SD, and all data points are included. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: Enzyme-linked immunosorbent assay (ELISA) plates were coated with goat anti-mouse IgM, IgG3, IgG1, IgG2b, IgG2c, and IgA antibodies (SouthernBiotech) at 10 μg/mL in coating buffer (carbonate-bicarbonate, pH 9.6) at room temperature for 1 h. After washing 3 times with phosphate-buffered saline (PBS) containing 0.1% Tween 20 (washing buffer), and blocking with 0.1% bovine serum albumin (BSA) in washing buffer for 1 h, a 50 μL aliquot of mouse serum appropriately diluted for each isotype in PBS was applied and incubated for 1 h at room temperature.

Techniques: Enzyme-linked Immunosorbent Assay, Comparison, Staining, Labeling, Immunohistochemical staining, Software, Purification, Cell Isolation, Cell Culture, MTT Assay