iga2 fitc  (Miltenyi Biotec)

Journal: Scientific Reports

Article Title: SARS-CoV-2-specific B cell responses in non-draining lymph nodes and antibody functionalities in immunized end-stage renal disease patients

doi: 10.1038/s41598-025-27815-y

Figure Lengend Snippet: The B cell isotypes in the LN and PB compartment, the horizontal black lines indicate the average percentage in both compartments ( a ) IgM in switched B cells, lower in LNs than in PB at 8% (IQR: 5% − 11%) or 13% (IQR: 11% − 26%) ( p = 0.0001), respectively; ( b ) IgM in S-binding B cells, no difference between the LNs and PB at 29% (IQR: 16% − 51%) or 15% (IQR 6% − 33%) ( p = 0.058), respectively; ( c ) IgA in switched B cells, no difference between LNs and PB at 36% (IQR 27% − 43%) or 37% (IQR 32% − 43%) ( p = 0.207), respectively; ( d ) IgA in S-binding B cells, in LNs higher than in PB at 22% (IQR 11% − 32%) or 8% (IQR 5% − 15%) ( p = 0.0002), respectively; ( e ) IgG in switched B cells, in LNs higher than in PB at 49% (IQR 42% − 53%) or 42% (IQR 32% − 48%) ( p = 0.0110), respectively; ( f ) IgG in S-binding B cells, in LNs lower than in PB at 53% (IQR: 44% − 67%) or 73% (IQR 61% − 80%) ( p = 0.0001), respectively. PRM (primary), two vaccinations; BST (booster three), three vaccinations; BSF (booster four), four vaccinations; INFX (infection), prior infection; HYB (hybrid), immunity in which the infection was prior to the last of two vaccinations.

Article Snippet: The following surface mAbs were used: CD19 BV785, CD20 APC-Fire750, CD27 BB700, IgD PE-CF594, CD24 BV650, CD38 BUV563, IgM BUV395, IgG BUV496 (all from BD Biosciences), CXCR3 R718 (eBioscience), IgG1 PE, IgG2 PE, IgG2 FITC, IgG3 FITC (all from SouthernBiotech), IgA PE-Vio770, IgA2 FITC (both from Miltenyi Biotec), and IgA1 PE (Abcam).

Techniques: Binding Assay, Infection

Journal: NPJ Vaccines

Article Title: BNT162b2 induces robust cross-variant SARS-CoV-2 immunity in children

doi: 10.1038/s41541-022-00575-w

Figure Lengend Snippet: Relative SARS-CoV-2 wild-type ( a ) spike (Wuhan) and ( b ) receptor binding domain (RBD) specific IgM, IgG1, and IgA1 binding levels were determined by Luminex in children receiving either 10 μg of BNT162b2 (ages 5-11 years old, yellow) or 30 μg BNT162b2 (ages 12-16 years old, blue and 16+ years old, red) before (V0 10μg (5-11y) : 32; V0 30μg (12-16y) : 29, V0 30μg (16+y) : 7), after the first dose (V1 10μg (5-11y) : 32; V1 30μg (12-16y) : 27, V1 30μg (16+y) : 8), or after the second dose (V2 10μg (5-11y) : 30; V2 30μg (12-16y) : 26, V2 30μg (16+y) : 11). A two-way ANOVA (two-sided) was used to calculate statistically significant differences between the groups at each timepoint. Exact p-values for statistically significant differences after Benjamini-Hochberg correction for multiple testing are shown above the graph. Horizontal lines indicate the median and error bars the 95% confidence interval.

Article Snippet: Following overnight incubation, non-specific antibodies were washed off and the immune complexes were incubated with Ig isotypes or subclasses with a 1:100 diluted PE-conjugated secondary antibody for IgG1 (clone: HP6001), IgG2 (clone: 31-7-4), IgG3 (clone: HP6050), IgG4 (clone: HP6025), IgM (clone: SA-DA4), IgA1 (clone: B3506B4), or IgA2 (clone: A9604D2) (all Southern Biotech).

Techniques: Binding Assay, Luminex

Journal: NPJ Vaccines

Article Title: BNT162b2 induces robust cross-variant SARS-CoV-2 immunity in children

doi: 10.1038/s41541-022-00575-w

Figure Lengend Snippet: ( a ) Vaccine-induced IgM, IgA1, and IgG1 response to D614G (wild-type; blue), alpha (B.1.1.7, yellow), beta (B.1.1.7, purple), gamma (P.1, green), delta (B.1.617.2, orange), and omicron (B1.1.529/BA.1, red) to the full Spike in children receiving 10 μg of BNT162b2 (ages 5-11 years old, n = 30) or adolescent receiving 30 μg BNT162b2 (ages 12–15 years old, n26) or adults (16 + years old, n17) at V2. Geometric mean of each group is shown, error bars indicate the 95% confidence interval (CI). A two-sided Kruskal-Wallis test with Benjamini-Hochberg correction for multiple testing was performed to compare D614G and omicron-specific antibody titers. P-values for significant different comparisons are shown above the dataset. b ) Heatmaps show the relative fold change (red = increase, white = no change, blue= decrease) for the different VOCs compared to the original D614G variant for Spike and RBD-specific IgG1, Ig3, IgA1 and IgM titers or binding to FcγR2a, FcγR2b, FcγR3a, FcγR3b. C ) Flower plots summarize ADCD, ADCP, ADNP, ADNKA (CD107a, IFNγ, MIP-1β) and neutralization (VNT) at V1 and V2 against D614G (upper panel) or omicron (lower panel) Spike in 10 μg of BNT162b2 (ages 5-11 years old) or adolescent receiving 30 μg BNT162b2 (ages 12–15 years old) or adults (16 + years old) at V2. Each petal represents a specific function (compare color key) and the length of the petal corresponds of the intensity of Z-scored and normalized data. Asterisks in b ) indicate significant differences of the respective variant in a paired two-sided Wilcoxon rank test. P -values were corrected for multiple testing using Benjamini-Hochberg correction. *: p < 0.05, **: p < 0.01,***: p < 0.001, ****: p < 0.0001.

Article Snippet: Following overnight incubation, non-specific antibodies were washed off and the immune complexes were incubated with Ig isotypes or subclasses with a 1:100 diluted PE-conjugated secondary antibody for IgG1 (clone: HP6001), IgG2 (clone: 31-7-4), IgG3 (clone: HP6050), IgG4 (clone: HP6025), IgM (clone: SA-DA4), IgA1 (clone: B3506B4), or IgA2 (clone: A9604D2) (all Southern Biotech).

Techniques: Variant Assay, Binding Assay, Neutralization

Journal: Frontiers in Immunology

Article Title: Maternal IgA2 Recognizes Similar Fractions of Colostrum and Fecal Neonatal Microbiota

doi: 10.3389/fimmu.2021.712130

Figure Lengend Snippet: Microbiota from the human colostrum is differentially coated with IgA subclasses. (A) Staining profiles of bacteria in human colostrum characterized by flow cytometry. (B) Analysis of bacterial populations by their associations with IgA1 (APC Cy7) and IgA2 (FITC) in human colostrum. Plots are representative of thirty-six biological samples analyzed. (C) Quantification of free IgA subclasses in colostrum, titled “Colostrum,” and pasteurized milk, titled “Pasteurized”: by ELISA. Data are expressed in milligrams of IgA subclasses per milliliter of the sample (mg/mL). (D) Semi-quantification by Western blot of IgA subclasses’ levels bound to bacteria, data are expressed in nanograms of IgA subclasses per milliliter of the sample (ng/mL). IgA1 (black bars) and IgA2 levels (filled bars) from Colostrum and Pasteurized milk. (E) Numbers of bacteria determined by flow cytometry. Data are expressed in millions of bacteria per milliliter of the sample (10 6 bacteria/mL). IgA1+ (black bars), IgA2+ (filled bars) and total bacteria (gray bars). Bars indicate the mean ± SD of triplicates from thirty-six “Colostrum” and twenty-eight “Pasteurized” milk samples. Statistical analysis was performed using the Mann-Whitney U test, comparing each IgA subclasses level with the pasteurized milk sample as a control. *p < 0.05 and ***p < 0.001.

Article Snippet: The slide was washed five times with PBST and then incubated with 40 μL of secondary mouse antibody anti-human IgA2 coupled with FITC (Cat# 9140-02 SouthernBiotech ® , Birmingham, AL, USA) dilution 1:2500.

Techniques: Staining, Bacteria, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Western Blot, MANN-WHITNEY, Control

Journal: Frontiers in Immunology

Article Title: Maternal IgA2 Recognizes Similar Fractions of Colostrum and Fecal Neonatal Microbiota

doi: 10.3389/fimmu.2021.712130

Figure Lengend Snippet: Some bacteria have divergent recognition between IgA subclasses in the human colostrum. (A) Relative abundance of predominant bacterial taxa in colostrum samples. Abundance of each families/genera in IgA fractions (Total bacteria in colostrum = Total colostrum, bacteria coated with IgA1= IgA1+ colostrum, and bacteria coated with IgA2 = IgA2+ colostrum). IgA1+ fraction is plotted as red dots and IgA2+ fraction as green dots. Previous graphs represent data from thirty-six “Total bacteria,” thirty “IgA1+ colostrum,” and thirty-three “IgA2+ colostrum” samples. Both groups significantly differed according to the ANOSIM similarity test (R 2 = 0.289, p = 0.001) and Adonis statistical test (R 2 = 0.949, p = 0.001). (B) LEfSe analysis of the most representative bacterial genera in colostrum by IgA1 and IgA2. Linear discriminant analysis (LDA) effect size (LEfSe) compares differential abundant bacterial taxa between IgA fractions. Horizontal bars represent each taxon’s effect size: green indicates taxa enriched in the IgA2+ fraction group, and red indicates taxa enriched IgA1+ fraction. LDA score cutoff of 2.0 was used to discriminate bacterial taxon. (C) Beta diversity analysis. Two-dimensional scatterplots were generated using PCoA based on the unweighted UniFrac distance metric. PC3 vs PC2, PC1 vs PC2, and PC1 vs PC3.

Article Snippet: The slide was washed five times with PBST and then incubated with 40 μL of secondary mouse antibody anti-human IgA2 coupled with FITC (Cat# 9140-02 SouthernBiotech ® , Birmingham, AL, USA) dilution 1:2500.

Techniques: Bacteria, Generated

Journal: Frontiers in Immunology

Article Title: Maternal IgA2 Recognizes Similar Fractions of Colostrum and Fecal Neonatal Microbiota

doi: 10.3389/fimmu.2021.712130

Figure Lengend Snippet: IgA2 is predominantly found coating bacteria in newborn feces of breastfed children. (A) Dot blot of total bacteria in neonate’s fecal samples. (B) Bacteria found in meconium characterized by flow cytometry. (C) Bacterial populations coated with IgA subclasses in stool samples. Plots are representative of five pool meconium (B) and nineteen feces’ samples (C) , obtained after three days of colostrum-fed newborn children. (D) Quantification by ELISA of IgA subclasses in “Meconium,” a stool sample from children fed with pasteurized milk, titled “Feces Pasteurized,” or children fed with maternal colostrum, titled “Feces Colostrum”; data are expressed in milligrams of IgA per gram of fecal sample (mg/g). (E) Semi-quantification by Western blot of IgA subclasses’ levels bound to bacteria; data are expressed in nanograms of IgA per gram of fecal sample (ng/g). IgA1 (black bars) and IgA2 levels (filled bars). (F) Number of bacteria determined by flow cytometry; data are expressed in millions of bacteria per gram of fecal sample (10 6 bacteria/g). IgA2+ (filled bars) and total bacteria (gray bars). Bars indicate the mean ± SD of triplicates from five “Meconium,” twenty-seven “Feces Pasteurized,” and nineteen “Feces Colostrum” samples. Statistical analysis was performed using the Mann-Whitney U test between IgA subclasses in the same sample and comparing each level with feces pasteurized levels as control. *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001.

Article Snippet: The slide was washed five times with PBST and then incubated with 40 μL of secondary mouse antibody anti-human IgA2 coupled with FITC (Cat# 9140-02 SouthernBiotech ® , Birmingham, AL, USA) dilution 1:2500.

Techniques: Bacteria, Dot Blot, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Western Blot, MANN-WHITNEY, Control

Journal: Frontiers in Immunology

Article Title: Maternal IgA2 Recognizes Similar Fractions of Colostrum and Fecal Neonatal Microbiota

doi: 10.3389/fimmu.2021.712130

Figure Lengend Snippet: IgA2+ microbiota is present in fecal neonate’s samples. (A) Relative abundance of predominant bacterial taxa in stool samples (Meconium, neonates fed with pasteurized milk= “Pasteurized,” neonates fed with pasteurized milk= “Feces Pasteurized” and neonates fed with colostrum= “Feces Colostrum”; during first three days of life). The abundance of each family/genera in IgA fractions was compared between groups using a parametric test for paired samples followed by BH correction. (B) Alpha diversity based in observed number of species (p < 0.001), Chao1 (p < 0.001), Shannon (p < 0.001) and Simpson (p = 0.006) indexes. Mann-Whitney U test was used to find significant differences. Alpha diversity based on observed number species (p < 0.001), Chao1 (p < 0.001), Shannon (p < 0.001) and Simpson (p < 0.005) indexes. Mann–Whitney U test was used to find significant differences **p < 0.01, ***p < 0.001. (C) LEfSe analysis of the most representative bacterial genera in stool samples. Linear discriminant analysis (LDA) effect size (LEfSe) comparison of differentially abundant bacterial taxa among free fecal bacteria (“Total”) and IgA2+ fecal bacteria (“IgA2”); from neonates whose were fed with colostrum during the first three days of life. Horizontal bars represent each taxon’s effect size: red indicates taxa enriched in the IgA2+ fraction group, and green indicates free taxa enriched group. LDA score cutoff of 2.4 was used to discriminate bacterial taxon. Previous graphs represent data from five “Meconium”, twenty-eight “Pasteurized milk”, twenty-seven “Feces pasteurized”, thirty-six “Total colostrum”, thirty “IgA1+ colostrum”, thirty-three “IgA2+ colostrum”, nineteen “Feces Colostrum” and five “IgA2+ Feces” samples. (D) Two dimensional scatterplots were generated using PCoA based on the unweighted UniFrac distance metric. PC3 vs PC2, PC1 vs PC2, and PC1 vs PC3. Both groups significantly differed according to the ANOSIM similarity test (R 2 = 0.289, p = 0.001) and Adonis statistical test (R 2 = 0.949, p = 0.001). Total bacteria from colostrum samples are represented in red diamonds, IgA2+ fecal bacteria from neonates fed with colostrum in green squares, total bacteria from neonates fed colostrum in yellow circles, and total bacteria from pasteurized milk in purple triangles.

Article Snippet: The slide was washed five times with PBST and then incubated with 40 μL of secondary mouse antibody anti-human IgA2 coupled with FITC (Cat# 9140-02 SouthernBiotech ® , Birmingham, AL, USA) dilution 1:2500.

Techniques: MANN-WHITNEY, Comparison, Bacteria, Generated

Journal: Frontiers in Immunology

Article Title: Maternal IgA2 Recognizes Similar Fractions of Colostrum and Fecal Neonatal Microbiota

doi: 10.3389/fimmu.2021.712130

Figure Lengend Snippet: Microbial metabolic pathways among IgA2+ bacteria are enriched in intestine adherence and active carbohydrate metabolism. GO analysis of the most representative metabolic pathways in predicting functional microbial metabolic pathways using PICRUSt analysis (KEGG level three). The abundance of nine statistically significant metabolic pathways between five IgA2+ fecal bacteria samples from neonates fed with human colostrum for three days, in pink; and twenty-seven IgA- fecal bacteria samples from children fed with pasteurized milk, in gray. All values are represented as mean ± SD.

Article Snippet: The slide was washed five times with PBST and then incubated with 40 μL of secondary mouse antibody anti-human IgA2 coupled with FITC (Cat# 9140-02 SouthernBiotech ® , Birmingham, AL, USA) dilution 1:2500.

Techniques: Bacteria, Functional Assay