Journal: Virologica Sinica

Article Title: Omicron variants breakthrough infection elicited higher specific memory immunity than third dose booster in healthy vaccinees.

doi: 10.1016/j.virs.2022.12.008

Figure Lengend Snippet: Fig. 1. Comparative analysis of anti-ancestral-spike-trimer-IgG. Serum were collected from vaccinated healthy volunteers (HVs) before third booster, at median of 33 days post inactivated vaccine (InV) booster and 30.5 days post Ad5-nCoV booster, and Omicron variants breakthrough infected patients (OBIPs) at median of 32 days post infection. A Anti-spike-trimer-IgG titer in InV boosted HVs, Ad5-nCoV boosted HVs, and OBIPs were determined by ELISA. B–G Comparative analysis of SARS- CoV-2 ancestral strain spike-trimer specific IgG subclass. Anti-spike-trimer-IgG1 (B), anti-Spike-trimer-IgG2 (C), anti-Spike-trimer-IgG3 (D), and anti-spike-trimer-IgG4 (E) titer in InV boosted HVs, Ad5-nCoV boosted HVs, and OBIPs were determined by ELISA. F The ratio of spike-trimer specific IgG1/IgG4 in InV boosted HVs, Ad5- nCoV boosted HVs, and OBIPs. G The ratio of spike-trimer specific IgG1þIgG3/IgG4þIgG2 in InV boosted HVs, Ad5-nCoV boosted HVs, and OBIPs. Data presented as geometric mean titers (GMT) and 95% confidence interval. Dotted line: the detection limits. Statistical analyses were performed by Mann-Whitney U test.

Article Snippet: After blocking, 100 μL 3-fold diluted plasma (initially dilute for IgG, IgG1, IgG2 and IgG3:1:20, IgG4:1:10) was added in each well, and incubated at 37 C for 1 h. After thorough washing, 100 μL HRP labeled secondary antibodies against human IgG (Cat#: 2010-05, 1:10,000, Southern Biotech, USA), IgG1 (Cat#: 9054-05, 1:4000, Southern Biotech, USA), IgG2 (Cat#: 9060- 05, 1:2000, Southern Biotech, USA), IgG3 (Cat#: 9210-05, 1:4000, Southern biotech, USA), and IgG4 (Cat#: 9200-05, 1:4000, Southern Biotech, USA) was added in each well.

Techniques: Infection, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY

Journal: Virologica Sinica

Article Title: Omicron variants breakthrough infection elicited higher specific memory immunity than third dose booster in healthy vaccinees.

doi: 10.1016/j.virs.2022.12.008

Figure Lengend Snippet: Fig. 2. Comparative analysis of anti-RBD antibody against SARS-CoV-2. Serum were collected vaccinated healthy volunteers (HVs) before third booster, at median of 33 days post InV booster and 30.5 days post Ad5-nCoV booster, and OBIPs at median of 32 days post infection. Anti-ancestral-receptor binding domain (RBD)-IgG (A), anti-Delta-RBD-IgG (B), and anti-Omicron-RBD-IgG (C) titer in pre-boosted HVs, InV boosted HVs, Ad5-nCoV boosted HVs, and OBIPs were determined by ELISA. D Paired analysis of anti-ancestral-RBD-, anti-Delta-RBD-, and anti-Omicron-RBD-IgG titer in pre-boosted HVs, InV boosted HVs, Ad5-nCoV boosted HVs, and OBIPs. Data presented as geometric mean titers (GMT) and 95% confidence interval. Dotted line: the detection limits. Statistical analyses were performed by Mann-Whitney U test.

Article Snippet: After blocking, 100 μL 3-fold diluted plasma (initially dilute for IgG, IgG1, IgG2 and IgG3:1:20, IgG4:1:10) was added in each well, and incubated at 37 C for 1 h. After thorough washing, 100 μL HRP labeled secondary antibodies against human IgG (Cat#: 2010-05, 1:10,000, Southern Biotech, USA), IgG1 (Cat#: 9054-05, 1:4000, Southern Biotech, USA), IgG2 (Cat#: 9060- 05, 1:2000, Southern Biotech, USA), IgG3 (Cat#: 9210-05, 1:4000, Southern biotech, USA), and IgG4 (Cat#: 9200-05, 1:4000, Southern Biotech, USA) was added in each well.

Techniques: Infection, Binding Assay, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY

Journal: Journal of Clinical Laboratory Analysis

Article Title: Evaluation of autoantibodies and immunoglobulin G subclasses in women with suspected macroprolactinemia

doi: 10.1002/jcla.23456

Figure Lengend Snippet: IgG subclass distributions in Group 1 and Group 2 patients

Article Snippet: The membranes were then probed with the anti‐human IgG1 antibody (ThermoFisher, A10648, USA), anti‐human IgG2 antibody (SouthernBiotech, 9070‐01, USA), anti‐human IgG3 antibody (SouthernBiotech, 9210‐01), or anti‐human IgG4 antibody (SouthernBiotech, 9200‐01) separately, followed by incubation with the IRDyeTM secondary antibodies (1:20 000).

Techniques:

Journal: Journal of Clinical Laboratory Analysis

Article Title: Evaluation of autoantibodies and immunoglobulin G subclasses in women with suspected macroprolactinemia

doi: 10.1002/jcla.23456

Figure Lengend Snippet: Immunoprecipitation with the serum samples of the patients suspected for macroprolactinemia. A, Relative levels of PRL‐specific IgG subclasses associated with PRL‐IgG complexes in patients with anti‐PRL autoantibodies are shown in bar graph. Data are expressed as mean ± standard deviation. B, Representative Western blots with the patients’ serum samples from Group 1 (#117, 159, 196, 229) and Group 2 (#120, 262, 302, 322). Lane 1: positive control loaded with pure IgG1‐IgG4 proteins

Article Snippet: The membranes were then probed with the anti‐human IgG1 antibody (ThermoFisher, A10648, USA), anti‐human IgG2 antibody (SouthernBiotech, 9070‐01, USA), anti‐human IgG3 antibody (SouthernBiotech, 9210‐01), or anti‐human IgG4 antibody (SouthernBiotech, 9200‐01) separately, followed by incubation with the IRDyeTM secondary antibodies (1:20 000).

Techniques: Immunoprecipitation, Standard Deviation, Western Blot, Positive Control

Journal: Vaccines

Article Title: Cross-Reactive Fc-Mediated Antibody Responses to Influenza HA Stem Region in Human Sera Following Seasonal Vaccination

doi: 10.3390/vaccines13020140

Figure Lengend Snippet: Characteristics of serum antibodies to each HA region induced by seasonal influenza vaccination. Total IgG, IgG1, and IgG2 antibodies against vaccine strains HA head-specific, H1 stem-specific, and LAH epitopes specific to vaccine strains H1 and H3 were quantified by ELISA using pre- and post-vaccination sera from seasonal influenza vaccines. Quantification was performed using ELISA. ( A ) Total IgG antibody titers against HA head-specific, H1 stem-specific, and LAH epitopes specific to vaccine strains H1 and H3 before and after vaccination. ( B ) IgG1 and IgG2 antibody titers against H1 HA head-specific before and after vaccination. ( C ) IgG1 and IgG2 antibody titers against H3 HA head-specific antibodies before and after vaccination. Total IgG, IgG1, and IgG2 antibody titers against the H1 stem before and after vaccination. ( D ) IgG1 and IgG2 antibody titers against H1 stem cells before and after vaccination. Antibody titers were quantified using standard antibodies, and the amounts are shown. Each circle represents the results for an individual participant; lines represent median values. The fold change is shown above the graph as the median antibody titer in the post-vaccination group divided by that in the pre-vaccination group. The data were analyzed using the Wilcoxon matched-pair signed-rank test. Significance is indicated by the following symbols: *** p < 0.001, and **** p < 0.0001.

Article Snippet: Next, the plates were washed and incubated for 1 h with various secondary anti-human IgG antibodies, including mouse anti-Human IgG Fc-BIOT (SouthernBiotech, USA), mouse anti-Human IgG1 Fc secondary antibody-HRP (Thermo Fisher Scientific, Waltham, MA, USA), mouse Anti-Human IgG2 Fc-BIOT (SouthernBiotech, Birmingham, AL, USA), mouse Anti-Human IgG3 Hinge-BIOT (SouthernBiotech, Birmingham, AL, USA), or mouse Anti-Human IgG4 Fc-BIOT (SouthernBiotech, Birmingham, AL, USA).

Techniques: Enzyme-linked Immunosorbent Assay, Vaccines

Journal: Vaccines

Article Title: Cross-Reactive Fc-Mediated Antibody Responses to Influenza HA Stem Region in Human Sera Following Seasonal Vaccination

doi: 10.3390/vaccines13020140

Figure Lengend Snippet: Cross-reactive neutralizing antibody activity in serum induced by current seasonal influenza vaccination. Post-vaccination sera were subjected to a microneutralization assay using ten different influenza viruses and representative data from two independent experiments. The results obtained were scored as follows: 0 for 20 and below, 1 for 21 to 80, 2 for 81 to 640, 3 for 641 to 1280, and 4 for 1280 and above. Neutralization breath scores were calculated by summing the respective scores for groups 1, 2, and group 1 + 2. Neutralization breath scores were calculated by summing the scores of groups 1, 2, and group 1 + 2. ( A ) Correlation between neutralization group 1 breath score and total IgG of H1, H3 HA head-specific and H1 stem-specific. ( B ) Correlation of neutralization group 2 breath score with total IgG for H1, H3 HA head-specific, and H1 stem-specific. Data were statistically analyzed using Spearman’s rank correlation coefficients. Lines indicate correlations determined by linear regression analysis ( n = 50 for graphs of H1 and H3 HA head-specific antibodies and n = 36 for graphs of H1 stem-specific antibodies).

Article Snippet: Next, the plates were washed and incubated for 1 h with various secondary anti-human IgG antibodies, including mouse anti-Human IgG Fc-BIOT (SouthernBiotech, USA), mouse anti-Human IgG1 Fc secondary antibody-HRP (Thermo Fisher Scientific, Waltham, MA, USA), mouse Anti-Human IgG2 Fc-BIOT (SouthernBiotech, Birmingham, AL, USA), mouse Anti-Human IgG3 Hinge-BIOT (SouthernBiotech, Birmingham, AL, USA), or mouse Anti-Human IgG4 Fc-BIOT (SouthernBiotech, Birmingham, AL, USA).

Techniques: Activity Assay, Microneutralization Assay, Neutralization

Journal: Vaccines

Article Title: Cross-Reactive Fc-Mediated Antibody Responses to Influenza HA Stem Region in Human Sera Following Seasonal Vaccination

doi: 10.3390/vaccines13020140

Figure Lengend Snippet: Cross-reactive ADCC activity induced by current seasonal influenza vaccination in serum. ADCC assays were performed on post-vaccination sera using 10 different influenza viruses, representative data from two independent experiments. The results obtained were scored as follows: 0 for 10 or less, 1 for 11 to 100, 2 for 101 to 500, 3 for 501 to 1000, and 4 for 1000 or more. Breath scores were calculated by summing the scores of group 1, group 2, and group 1 + 2. ( A ) Correlation between the ADCC group 1 breath score and H1 HA head-specific total IgG, IgG1, and IgG2 levels. ( B ) Correlation between the ADCC group 2 breath score and H3 HA head-specific total IgG and IgG1 levels. ( C ) Correlation between ADCC group 1 breath score and H1 stem-specific total IgG and IgG1 levels. Data were statistically analyzed using Spearman’s rank correlation coefficients. Lines indicate correlations determined by linear regression analysis ( n = 50 for graphs of H1 and H3 HA head-specific antibodies and n = 36 for graphs of H1 stem-specific antibodies).

Article Snippet: Next, the plates were washed and incubated for 1 h with various secondary anti-human IgG antibodies, including mouse anti-Human IgG Fc-BIOT (SouthernBiotech, USA), mouse anti-Human IgG1 Fc secondary antibody-HRP (Thermo Fisher Scientific, Waltham, MA, USA), mouse Anti-Human IgG2 Fc-BIOT (SouthernBiotech, Birmingham, AL, USA), mouse Anti-Human IgG3 Hinge-BIOT (SouthernBiotech, Birmingham, AL, USA), or mouse Anti-Human IgG4 Fc-BIOT (SouthernBiotech, Birmingham, AL, USA).

Techniques: Activity Assay

Journal: Vaccines

Article Title: Cross-Reactive Fc-Mediated Antibody Responses to Influenza HA Stem Region in Human Sera Following Seasonal Vaccination

doi: 10.3390/vaccines13020140

Figure Lengend Snippet: Cross-reactive ADCP activity induced by current seasonal influenza vaccination in serum. ADCP assays were performed on post-vaccination plasma with 10 different influenza viruses, and representative data were obtained from two independent experiments. The results were scored as follows: 0, 10 and below; 1, 11–100; 2, 101–500; 3, 501–1000; and 4, 1000 and above. Breath scores were calculated by summing the respective scores for groups 1, 2, and 1 + 2. ( A ) Correlation between the ADCP group 1 breath score and H1 HA head-specific total IgG, IgG1, and IgG2 levels. ( B ) Correlation between ADCP group 2 breath score and H3 HA head-specific total IgG and IgG1 levels. ( C ) Correlation between the ADCP group 1 breath score and H1 stem-specific total IgG and IgG1. Data were statistically analyzed using Spearman’s rank correlation coefficients. Lines indicate correlations determined by linear regression analysis ( n = 50 for graphs of H1 and H3 HA head-specific antibodies and n = 36 for graphs of H1 stem-specific antibodies).

Article Snippet: Next, the plates were washed and incubated for 1 h with various secondary anti-human IgG antibodies, including mouse anti-Human IgG Fc-BIOT (SouthernBiotech, USA), mouse anti-Human IgG1 Fc secondary antibody-HRP (Thermo Fisher Scientific, Waltham, MA, USA), mouse Anti-Human IgG2 Fc-BIOT (SouthernBiotech, Birmingham, AL, USA), mouse Anti-Human IgG3 Hinge-BIOT (SouthernBiotech, Birmingham, AL, USA), or mouse Anti-Human IgG4 Fc-BIOT (SouthernBiotech, Birmingham, AL, USA).

Techniques: Activity Assay, Clinical Proteomics

Journal: Nature Communications

Article Title: Divergent trajectories of antiviral memory after SARS-CoV-2 infection

doi: 10.1038/s41467-022-28898-1

Figure Lengend Snippet: Humoral immune responses were assessed in acute and convalescent by binding antibody ELISA for total IgG specific to the a Spike glycoprotein and b Nucleocapsid, quantification of c IgG memory B cells specific to the spike glycoprotein, and d pseudoneutralisation antibody titres. A two-tailed Wilcoxon rank-sum test was used to compare between study time points. The boxplots all display the median values with the first and third quartile, and the whiskers represent the highest and lowest values no more than 1.5 times the interquartile range from the corresponding hinge. A generalised additive mixed model (GAMM) by restricted maximum likelihood—right-hand plots—was used to fit the immunological measures (log10 transformed) taken at multiple study time points, using Gaussian process smooth term. The GAMM plots the ribbon represents the 95% confidence interval around the fitted value. Disease severity group was included in the GAMM as a linear predictor and a participant identifier was included as a random effect. See Table for number of individuals evaluated per assay.

Article Snippet: For detection of anti-spike IgG2 and IgG4 steps modified as follows: (1) Plates were additionally coated with commercially available human immunoglobulin control (recombinant human IgG2 lambda or recombinant human IgG4 lambda (Bio-Rad)) to serve as internal controls, (2) Mouse anti-human IgG2 Fd-AP or mouse anti-human IgG4 Fc-AP (Southern Biotech) were used, and (3) Optical density at 405 nm was measured using an ELx808 absorbance reader (BioTek) until the immunoglobulin control reached a specified OD405.

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, Two Tailed Test, Transformation Assay

Journal: Nature Communications

Article Title: Divergent trajectories of antiviral memory after SARS-CoV-2 infection

doi: 10.1038/s41467-022-28898-1

Figure Lengend Snippet: SARS-CoV-2 spike-specific antibody isotype and subclasses measured post-infection: a IgM, b IgA, c IgG1 and d IgG3. Antibody function measure post-SARS-CoV-2 infection: e antibody-dependent NK cell activation (ADNKA), f antibody-dependent neutrophil phagocytosis (ADNP), g antibody-dependent monocyte phagocytosis (ADMP) and h antibody-dependent complement deposition (ADCD). i Polar plot of various antibody isotype, subclass and function data, minimum-maximum normalised. The boxplots all display the median values with the first and third quartile, and the whiskers represent the highest and lowest values no more than 1.5 times the interquartile range from the corresponding hinge. A two-tailed Wilcoxon rank-sum test was used to compare between study time points. A generalised additive mixed model (GAMM) by restricted maximum likelihood—right-hand plots—was used to fit the immunological measures (log10 transformed) taken at multiple study time points, using Gaussian process smooth term. The GAMM plots the ribbon represents the 95% confidence interval around the fitted value. Disease severity group was included in the GAMM as a linear predictor and a participant identifier was included as a random effect. See Table for number of individuals evaluated per assay.

Article Snippet: For detection of anti-spike IgG2 and IgG4 steps modified as follows: (1) Plates were additionally coated with commercially available human immunoglobulin control (recombinant human IgG2 lambda or recombinant human IgG4 lambda (Bio-Rad)) to serve as internal controls, (2) Mouse anti-human IgG2 Fd-AP or mouse anti-human IgG4 Fc-AP (Southern Biotech) were used, and (3) Optical density at 405 nm was measured using an ELx808 absorbance reader (BioTek) until the immunoglobulin control reached a specified OD405.

Techniques: Infection, Activation Assay, Two Tailed Test, Transformation Assay

Journal: Journal of Translational Medicine

Article Title: New risk score for predicting progression of membranous nephropathy

doi: 10.1186/s12967-019-1792-8

Figure Lengend Snippet: Cox proportional hazards ratio model of primary outcomes

Article Snippet: IgG subclasses were tested by direct immunofluorescence methods with mouse anti-human IgG1 FITC, anti-human IgG2 FITC, anti-human IgG3 FITC and anti-human IgG4 FITC (Southern Biotech, CAT. No 9200-02; Southern Biotech, CAT. No9080-02; Southern Biotech, CAT. No 9052-02; Southern Biotech, CAT. No 9210-02).

Techniques: Staining

Journal: Journal of Translational Medicine

Article Title: New risk score for predicting progression of membranous nephropathy

doi: 10.1186/s12967-019-1792-8

Figure Lengend Snippet: Baseline characteristics of IMN patients

Article Snippet: IgG subclasses were tested by direct immunofluorescence methods with mouse anti-human IgG1 FITC, anti-human IgG2 FITC, anti-human IgG3 FITC and anti-human IgG4 FITC (Southern Biotech, CAT. No 9200-02; Southern Biotech, CAT. No9080-02; Southern Biotech, CAT. No 9052-02; Southern Biotech, CAT. No 9210-02).

Techniques: Staining