igg3 antibody  (SouthernBiotech)

Journal: Microbiology Spectrum

Article Title: A third COVID-19 vaccine dose in kidney transplant recipients induces antibody response to vaccine and Omicron variants but shows limited Ig subclass switching

doi: 10.1128/spectrum.02190-24

Figure Lengend Snippet: Serum IgG and nAb responses to SARS-CoV-2 ancestral and Omicron variants in KTRs in comparison to HCs. Total SARS-CoV-2 Spike (S)-specific IgG ( A ) and neutralizing antibody (nAb) ( B ) against the ancestral strain (red), Omicron BA.1 (green), and Omicron BA.5 (blue) variants measured in KTRs ( n = 81) or HCs ( n = 11) who were previously vaccinated with the initial two-dose mRNA vaccine series prior to dose 3 (pre-D3), 30 days post-dose 3 (30 days post-D3), and 90 days post-dose 3 (90 days post-D3). Total anti-S IgG ( C ) and nAb levels ( D ) at 30 days post-D3 and nAb levels at 90 days post-D3 ( E ) were compared between the ancestral strain, Omicron BA.1, and Omicron BA.5 variants. Total anti-S IgG ( F ) and nAb ( G ) levels against ancestral strain, Omicron BA.1, and Omicron BA.5 variants were compared between KTR and HCs at 30 days post-D3. Dotted lines indicate the limit of detection (LOD), which is −1.52 for the anti-S IgG ELISA assay ( A, C, F ) and 0.17 for the nAb assay ( B, D, E, G ). Open circles represent non-responders with negative serological responses that fall below the LOD value. Solid triangles represent patients with a confirmed SARS-CoV-2 infection during the course of the study. The mean ± 95% CI are shown in each panel. Significance is tested using one-way repeated measures ANOVA ( A–E ), and unpaired t-tests ( F, G ). * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001. Fold changes (X) are labeled below the significance lines. The number of positive samples out of the total number of samples tested is indicated in parentheses.

Article Snippet: For characterization of subclass-specific IgG, we used secondary IgG1 antibody (catalog 9054-05, SouthernBiotech) at a 1:4,000 dilution, IgG2 antibody (catalog 9060-05, SouthernBiotech) at a 1:4,000 dilution, IgG3 antibody (catalog 9210-05, SouthernBiotech) at a 1:4,000 dilution, and IgG4 antibody (catalog 9200-05, SouthernBiotech) at a 1:8,000 dilution.

Techniques: Comparison, Enzyme-linked Immunosorbent Assay, Infection, Labeling

Journal: Microbiology Spectrum

Article Title: A third COVID-19 vaccine dose in kidney transplant recipients induces antibody response to vaccine and Omicron variants but shows limited Ig subclass switching

doi: 10.1128/spectrum.02190-24

Figure Lengend Snippet: Serum IgG subclass profile to SARS-CoV-2 ancestral strain in KTRs. SARS-CoV-2 Spike (S)-specific IgG subclasses (IgG1-4): IgG1 (yellow), IgG2 (blue), IgG3 (green), and IgG4 (purple) against ancestral strain were measured in KTRs ( n = 81) prior to dose 3 (pre-D3), 30 days post-dose 3 (30 days post-D3), and 90 days post-dose 3 (90 days post-D3) ( A ). Anti-S IgG subclasses antibody levels against SARS-CoV-2 ancestral strain were compared at 30 days post-D3 ( B ) and 90 days post-D3 ( C ) in KTRs with anti-S total IgG (red) as a reference on the left of both panels. A summary panel of anti-S IgG subclass-specific antibody levels against ancestral strain is shown and connected by lines to show the changes of serological responses in IgG subclasses from pre-D3, 30 days post-D3, to 90 days post-D3 ( D ). Comparison of anti-S IgG subclass-specific antibody levels against ancestral strain was made between KTRs and healthy controls (HCs) ( n = 11) at 30 days post-D3 ( E ). Dotted lines indicate the limit of detection (LOD), which is −3.00 for the subclass-specific IgG ELISA assay. Open circles represent non-responders with negative serological responses that fall below the LOD value. olid triangles represent patients with a confirmed SARS-CoV-2 infection during the course of the study. The mean ± 95% CI are shown in each panel. Significance is tested using mixed-effects model ( A ), one-way repeated measures ANOVA ( B, C ), and unpaired t -test ( E ). * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001. Fold changes (X) are labeled below the significance lines. The number of positive samples out of the total number of samples tested is indicated in parentheses.

Article Snippet: For characterization of subclass-specific IgG, we used secondary IgG1 antibody (catalog 9054-05, SouthernBiotech) at a 1:4,000 dilution, IgG2 antibody (catalog 9060-05, SouthernBiotech) at a 1:4,000 dilution, IgG3 antibody (catalog 9210-05, SouthernBiotech) at a 1:4,000 dilution, and IgG4 antibody (catalog 9200-05, SouthernBiotech) at a 1:8,000 dilution.

Techniques: Comparison, Enzyme-linked Immunosorbent Assay, Infection, Labeling

Journal: bioRxiv

Article Title: Spike-specific IgG4 generated post BNT162b2 mRNA vaccination is inhibitory when directly competing with functional IgG subclasses

doi: 10.1101/2025.02.12.637095

Figure Lengend Snippet: Semiquantitative ELISA measurement of Wildtype (WT) SARS-CoV-2 Spike-specific Total IgG (black), IgG1 (blue), IgG2 (green), IgG3 (purple) and IgG4 (red) for (A) 3 week interval between 1 st and 2 nd dose group (short group, n=8), (B) 12-week interval between 1 st and 2 nd dose group (extended group, n=17), (C) subgroup of extended group with sample a 6 months post third dose (n=9), (D) prior infection group (n=8), (E) subgroup of extended group with a breakthrough infection after three doses (n=9), (F) subgroup of extended group with a bivalent WT/BA.1 booster 1 year after third dose (n=8). Samples paired from an individual in grey with coloured data showing geometric mean and standard deviation. Pre1 = before 1 st dose, Post1 = 2 weeks after 1 st dose, Pre2 = before 2 nd dose, Post2 = 2 weeks after 2 nd dose, Pre3 = before 3 rd dose, Post3 = 2 weeks after 3 rd dose, 6Month = 6 month after 3 rd dose, BTI = 2 weeks after breakthrough infection, Bivalent = 2 weeks after bivalent dose. See Supplementary Table 1 for demographics and Supplementary Fig. S1 for precise timings. Semiquantitative ELISA limit of detection 0.05 µg/ml.

Article Snippet: Secondary antibodies were added (1:1000 dilution in 5% No Fat Milk in PBS-T) and incubated for 1 h at room temperature, including goat-anti-human Total IgG-alkaline phosphatase (AP) (Jackson Immunoresearch, 109-055-098), mouse-anti-human IgG1-horseradish peroxidase (HRP) (Invitrogen A10648), mouse-anti-human IgG2-HRP (Invitrogen MH1722), mouse-anti-human IgG3-HRP (Southern Biotech 9210-05), mouse-anti-human IgG4-HRP (Invitrogen MH174225)).

Techniques: Enzyme-linked Immunosorbent Assay, Infection, Standard Deviation

Journal: bioRxiv

Article Title: Spike-specific IgG4 generated post BNT162b2 mRNA vaccination is inhibitory when directly competing with functional IgG subclasses

doi: 10.1101/2025.02.12.637095

Figure Lengend Snippet: Concentration of Spike-specific Total IgG (black/grey) and IgG4 (red/pink) in sera for (A) a mild infection group and (B) severe, ITU admission group. (C) Concentration of SARS-CoV-2 N-specific Total IgG (filled black circles), Spike-specific Total IgG (clear black circles), N-specific IgG4 (filled red circles), Spike-specific IgG4 (clear red circles), 2 weeks after 3 rd dose (Post3), 3 weeks after 2 nd breakthrough infection (3W Post 2xBTI) and 6 months after 2 nd breakthrough infection (6M Post 2xBTI). (D) Concentration of Haemagglutinin-specific Total IgG (black/grey) or IgG4 (red/pink) with or without influenza booster given with 12-week interval 3 rd Dose. (E) ELISA measurement of Total IgG (black/grey) and IgG4 (red/pink) targeted against WT SARS-CoV-2 Full Spike, Receptor-Binding Domain (RBD) and N-Terminal Domain (NTD) in 12-week interval Post3 samples. (F) RBD and NTD binding Total IgG (black) and IgG4 (red) as a proportion of binding to Full Spike. (G) Total IgG (black/grey) and IgG4 (red/pink) binding to WT, Beta, Delta and BA.1 Spike in 12-week interval Post3 samples. (H) Serum antibody avidity of Total IgG (black) binding to WT Spike in Post2, Pre3, Post3 and Bivalent samples from the Bivalent group. (I) As in (H), but for IgG1 (blue) and IgG4 (red). (J) Serum antibody avidity of Total IgG (black/grey) and IgG4 (red/pink) against WT and BA.1 recombinant Spikes for Post3 and Bivalent samples from the Bivalent group. In all panels, samples paired from an individual are in lighter colour, with darker coloured data showing geometric mean and standard deviation. Data representative of 3 technical replicates. ns=not significant, * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001

Article Snippet: Secondary antibodies were added (1:1000 dilution in 5% No Fat Milk in PBS-T) and incubated for 1 h at room temperature, including goat-anti-human Total IgG-alkaline phosphatase (AP) (Jackson Immunoresearch, 109-055-098), mouse-anti-human IgG1-horseradish peroxidase (HRP) (Invitrogen A10648), mouse-anti-human IgG2-HRP (Invitrogen MH1722), mouse-anti-human IgG3-HRP (Southern Biotech 9210-05), mouse-anti-human IgG4-HRP (Invitrogen MH174225)).

Techniques: Concentration Assay, Infection, Enzyme-linked Immunosorbent Assay, Binding Assay, Recombinant, Standard Deviation

Journal: bioRxiv

Article Title: Spike-specific IgG4 generated post BNT162b2 mRNA vaccination is inhibitory when directly competing with functional IgG subclasses

doi: 10.1101/2025.02.12.637095

Figure Lengend Snippet: (A) Neutralising 50% Inhibitory Dose (ID 50 ) titre against WT SARS-CoV-2 Spike pseudotyped virus for 12-week interval Post2 and Post3 samples. (B) Correlation between Neutralising ID50 and IgG1 (blue) or IgG4 (red) concentration, with lighter colour for Post2 and darker colour for Post3 samples. (C) Neutralising ID50 titre for Bivalent group Post2, Post3 and Bivalent samples. (D, E & F) as (A, B & C) but for Antibody Dependent Cellular Cytotoxicity (ADCC) assay, measuring surface CD107a on activated NK cells. (G, H & I) as (A, B & C) but for Antibody Dependent Complement Deposition (ADCD) assay, measuring deposited C3. (J, K & L) as (A, B & C) but for Antibody Dependent Cellular Phagocytosis (ADCP) assay, measuring THP-1 macrophage uptake of WT Spike-coated fluorescent beads incubated with plasma. For all correlations, best fit line drawn for all IgG1 data, and include data points with non-zero values of IgG4. The vertical dotted line represents the limit of IgG4 detection. Data representative of 3 technical replicates. ns=not significant, *** p<0.001

Article Snippet: Secondary antibodies were added (1:1000 dilution in 5% No Fat Milk in PBS-T) and incubated for 1 h at room temperature, including goat-anti-human Total IgG-alkaline phosphatase (AP) (Jackson Immunoresearch, 109-055-098), mouse-anti-human IgG1-horseradish peroxidase (HRP) (Invitrogen A10648), mouse-anti-human IgG2-HRP (Invitrogen MH1722), mouse-anti-human IgG3-HRP (Southern Biotech 9210-05), mouse-anti-human IgG4-HRP (Invitrogen MH174225)).

Techniques: Virus, Concentration Assay, ADCC Assay, Incubation

Journal: bioRxiv

Article Title: Spike-specific IgG4 generated post BNT162b2 mRNA vaccination is inhibitory when directly competing with functional IgG subclasses

doi: 10.1101/2025.02.12.637095

Figure Lengend Snippet: (A) Neutralisation curves for 5 monoclonal antibodies; P008_60, P008_87, P008_99, VA14_1, VA14_R39 class-switched to IgG1 (blue), IgG2 (green), IgG3 (purple) and IgG4 (red), against WT SARS-COV-2 Spike pseudotyped virus. (B) as in (A) but for ADCC measured as percentage of NK cells with surface-accessible CD107a. (C) ADCD assay for P008_87 as IgG1 (blue) or IgG4 (red) with 3 µg/ml WT Spike ELISA plate coating. (D) ADCD assay for P008_87 as IgG1 (blue) or IgG4 (red) against Spike Virus-like Particles (VLP) (filled circles) or Naked VLPs (clear circles). (E) ADCD assay for P008_87 as IgG1 (blue) or IgG4 (red) with 0.3 µg/ml WT Spike plate coating. (F) ADCD for P008_60, P008_87, P008_99, VA14_1, VA14_R39 class-switched to IgG1 (blue), IgG2 (green), IgG3 (purple) and IgG4 (red) with 0.3 µg/ml WT Spike plate coating. (G) as in (A) but for ADCP, measuring THP-1 macrophage uptake of WT Spike-coated fluorescent beads incubated with antibody. Data representative of 3 technical replicates.

Article Snippet: Secondary antibodies were added (1:1000 dilution in 5% No Fat Milk in PBS-T) and incubated for 1 h at room temperature, including goat-anti-human Total IgG-alkaline phosphatase (AP) (Jackson Immunoresearch, 109-055-098), mouse-anti-human IgG1-horseradish peroxidase (HRP) (Invitrogen A10648), mouse-anti-human IgG2-HRP (Invitrogen MH1722), mouse-anti-human IgG3-HRP (Southern Biotech 9210-05), mouse-anti-human IgG4-HRP (Invitrogen MH174225)).

Techniques: Virus, Enzyme-linked Immunosorbent Assay, Incubation

Journal: bioRxiv

Article Title: Spike-specific IgG4 generated post BNT162b2 mRNA vaccination is inhibitory when directly competing with functional IgG subclasses

doi: 10.1101/2025.02.12.637095

Figure Lengend Snippet: (A) ADCC competition assay, with constant level of either P008_87 IgG1 or VA14_R39 IgG1 to give 10% NK cells with detectable surface CD107a, incubated with increasing amounts of the same antibody as an IgG1 (blue) or IgG4 (red). (B) ADCC competition assay with constant level of either P008_87 IgG1 or VA14_R39 IgG1 to give 10% of NK cells surface CD107a, incubated with increasing amounts of the other non-competitively binding antibody. (C) ADCD competition assay with constant level of either P008_87 IgG1 or VA14_R39 IgG1, incubated with increasing amounts of the same antibody as IgG1 (blue) or IgG4 (red). (D) ADCD competition assay with constant level of P008_87 IgG1 or VA14_R39 IgG1, incubated with increasing amounts of the other non-competitively binding antibody. Data representative of 2 technical replicates.

Article Snippet: Secondary antibodies were added (1:1000 dilution in 5% No Fat Milk in PBS-T) and incubated for 1 h at room temperature, including goat-anti-human Total IgG-alkaline phosphatase (AP) (Jackson Immunoresearch, 109-055-098), mouse-anti-human IgG1-horseradish peroxidase (HRP) (Invitrogen A10648), mouse-anti-human IgG2-HRP (Invitrogen MH1722), mouse-anti-human IgG3-HRP (Southern Biotech 9210-05), mouse-anti-human IgG4-HRP (Invitrogen MH174225)).

Techniques: Competitive Binding Assay, Incubation, Binding Assay

Journal: Signal Transduction and Targeted Therapy

Article Title: Safety and efficacy of trifluridine/tipiracil +/− bevacizumab plus XB2001 (anti-IL-1α antibody): a single-center phase 1 trial

doi: 10.1038/s41392-024-02116-4

Figure Lengend Snippet: Pharmacokinetic analysis. a – e Dose-dependent pharmacokinetic measures of XB2001. Area under the curve ( a ), Cmax ( b ), half-life ( c ), clearance ( d ) and volume of distribution ( e ) are presented for the doses 250, 500 and 1000 mg. f Evaluation of XB2001 concentration in patient plasma over the first 5 treatment cycles according to the dose given (250, 500 or 1000 mg of XB2001). Measurement of IgG1 ( g ) and IgG3 ( h ) anti-drug antibody concentrations in patient plasma at C1D1, C2D1 and C5D1. n.s, not significant, comparison using Wilcoxon matched-pairs test

Article Snippet: Briefly, a dilution of the capture antibody (XB2001) or coating buffer alone were added on each well of the microplates and plates were incubated at 37 °C for 1 h. After blocking and washing of microplates, plasma samples were diluted 1/500 with 20% of FBS-TBS solution and then were added in each well (coated or non-coated with Natrunix) and incubated at RT for 2 h. During incubation, the dilution of each secondary antibody (mouse anti-human IgG2 Fc-HRP conjugated (9060-05, Southern Biotech, Clone: 31-7-4), mouse anti-human IgG3 (hinge region specific)-HRP conjugated (9230-05, Southern Biotech, Clone: SB81a), mouse anti-human IgG1 (Fc specific)-HRP conjugated (9054-05, Southern Biotech, Clone: HP6001), goat anti-human IgA (a-chain specific)-HRP conjugated (2050-05, Southern Biotech), mouse anti-human IgM (μ-chain specific)-HRP conjugated (9040-05, Southern Biotech, Clone: JDC-10)) was prepared in 20% FBS-TBS buffer.

Techniques: Concentration Assay, Clinical Proteomics, Comparison

Journal: Communications Medicine

Article Title: Application of machine learning algorithms to identify serological predictors of COVID-19 severity and outcomes

doi: 10.1038/s43856-024-00658-w

Figure Lengend Snippet: a – d Anti-nucleocapsid (N) and anti-spike (S) secretory IgA or ( e – h ) IgG responses were measured as median fluorescence intensity (MFI) in nasopharyngeal (NP) or oropharyngeal (OP) samples and compared among COVID-19 hospitalized patients classified as moderate (WHO score 3–4), severe (WHO score 5–7), or deceased (WHO score 8). Data are analyzed using Welch’s ANOVA and presented as means with standard deviations, indicated by error bars.

Article Snippet: Plates were incubated for 2 h at RT, washed, and 50 μL of anti-human HRP IgG (1:5000, #A18823, Invitrogen, Thermo Fisher Scientific), IgA (1:5000, #A18787, Invitrogen, Thermo Fisher Scientific), IgG1 (1:4000, #9054-05, Southern Biotech), IgG2 (1:4000, #9060-05, Southern Biotech), IgG3 (1:4000, #9210-05, Southern Biotech) or IgG4 (1:8000, #9200-05, Southern Biotech) secondary antibody was added.

Techniques: Fluorescence

Journal: Communications Medicine

Article Title: Application of machine learning algorithms to identify serological predictors of COVID-19 severity and outcomes

doi: 10.1038/s43856-024-00658-w

Figure Lengend Snippet: a – d IgG binding antibody responses against ancestral spike (S), spike receptor binding domain (S-RBD), and nucleocapsid (N) were quantified by ELISA and calculated as the binding antibody units (BAU) per ml if international standards were available or as the area under the curve (AUC) if standards were not available and titration curves only could be generated; ( e ) ACE2 binding inhibition antibodies were measured using MSD V-PLEX SARS-CoV-2 ACE2 kits; and ( f – h ) Fc effector antibody responses were quantified using complement fixation and antibody-dependent cellular cytotoxicity (ADCC) assays. All assays were run using ancestral SARS-CoV-2. Data were compared using linear regression analysis, controlling for age and biological sex, to look at differences between unvaccinated non-hospitalized and hospitalized patients at 1 MPE. Data are presented as means with standard deviations, indicated by error bars. The limit of detection (LOD) is indicated by the dashed lines. p -values for statistically significant differences (p < 0.05) are shown in the figures.

Article Snippet: Plates were incubated for 2 h at RT, washed, and 50 μL of anti-human HRP IgG (1:5000, #A18823, Invitrogen, Thermo Fisher Scientific), IgA (1:5000, #A18787, Invitrogen, Thermo Fisher Scientific), IgG1 (1:4000, #9054-05, Southern Biotech), IgG2 (1:4000, #9060-05, Southern Biotech), IgG3 (1:4000, #9210-05, Southern Biotech) or IgG4 (1:8000, #9200-05, Southern Biotech) secondary antibody was added.

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, Titration, Generated, Inhibition

Journal: Communications Medicine

Article Title: Application of machine learning algorithms to identify serological predictors of COVID-19 severity and outcomes

doi: 10.1038/s43856-024-00658-w

Figure Lengend Snippet: The binding ( a – c ) IgG and ( d ) IgA antibodies recognizing ancestral SARS-CoV-2 spike (S), spike receptor binding domain (S-RBD), or nucleocapsid (N) were quantified by ELISA, and measured as the binding antibody units (BAU) per mL if international standards were available or as the area under the curve (AUC) if standards were not available and titration curves could only be generated. e The percentage of ACE2 inhibition for the ancestral SARS-CoV-2 variant was calculated and arcsine transformed for analyses. f – h The Fc effector antibody responses were measured based on C1q complement fixation in response to either the spike or S-RBD or antibody dependent cellular cytotoxicity and reported as arbitrary units (AU). Antibody responses were compared among COVID-19 hospitalized patients classified as moderate (WHO score 3–4), severe (WHO score 5–7), or deceased (WHO score 8) using samples collected at enrollment vs. 1 MPE. Data are presented as means with standard deviations, indicated by error bars. p -values for statistically significant differences (p < 0.05) by linear mixed-effects regression to compare change over time or Welch’s ANOVA to compare across groups within a time point are indicated. Limit of detection (LOD) are indicated by the dashed lines.

Article Snippet: Plates were incubated for 2 h at RT, washed, and 50 μL of anti-human HRP IgG (1:5000, #A18823, Invitrogen, Thermo Fisher Scientific), IgA (1:5000, #A18787, Invitrogen, Thermo Fisher Scientific), IgG1 (1:4000, #9054-05, Southern Biotech), IgG2 (1:4000, #9060-05, Southern Biotech), IgG3 (1:4000, #9210-05, Southern Biotech) or IgG4 (1:8000, #9200-05, Southern Biotech) secondary antibody was added.

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, Titration, Generated, Inhibition, Variant Assay, Transformation Assay

Journal: Communications Medicine

Article Title: Application of machine learning algorithms to identify serological predictors of COVID-19 severity and outcomes

doi: 10.1038/s43856-024-00658-w

Figure Lengend Snippet: The binding of IgG1 ( a ), IgG2 ( b ), IgG3 ( c ), and IgG4 ( d ) to ancestral SARS-CoV-2 S antigen was measured as the area under the curve (AUC). Spearman correlation of IgG1 ( e ), IgG2 ( f ), IgG3 ( g ), and IgG4 ( h ) with % ACE2 inhibition at enrollment. Hospitalized COVID-19 patients were classified as moderate (WHO score 3–4), severe (WHO score 5–7), or deceased (WHO score 8). Data are presented as means with standard deviations, indicated by error bars. The limit of detection (LOD) is indicated by the dashed lines. p -values for statistically significant differences (p < 0.05) by linear mixed-effects regression to compare change over time or Welch’s ANOVA to compare across groups within a time point are indicated.

Article Snippet: Plates were incubated for 2 h at RT, washed, and 50 μL of anti-human HRP IgG (1:5000, #A18823, Invitrogen, Thermo Fisher Scientific), IgA (1:5000, #A18787, Invitrogen, Thermo Fisher Scientific), IgG1 (1:4000, #9054-05, Southern Biotech), IgG2 (1:4000, #9060-05, Southern Biotech), IgG3 (1:4000, #9210-05, Southern Biotech) or IgG4 (1:8000, #9200-05, Southern Biotech) secondary antibody was added.

Techniques: Binding Assay, Inhibition

Journal: Communications Medicine

Article Title: Application of machine learning algorithms to identify serological predictors of COVID-19 severity and outcomes

doi: 10.1038/s43856-024-00658-w

Figure Lengend Snippet: Linear mixed-effects regression models for ( a – d ) anti-spike (S), anti-spike receptor binding domain (S-RBD), or anti-nucleocapsid (N) IgG or IgA, measured as the binding antibody units (BAU) per ml if international standards were available or as the area under the curve (AUC) if standards were not available and only titration curves could be generated; ( e ) the percentage ACE2 inhibition against ancestral SARS-CoV-2 as a surrogate of virus neutralization, and ( f – h ) Fc effector antibody responses as measured by complement fixation against spike or S-RBD or antibody-dependent cellular cytotoxicity (ADCC) up until 100 DPE or death among hospitalized patients classified as moderate (WHO score 3–4; n = 41), severe (WHO score 5–7; n = 40), or deceased (WHO score 8; n = 24). P -values for statistically significant differences (p < 0.05) by linear mixed-effects regression contrasts are shown within the figures.

Article Snippet: Plates were incubated for 2 h at RT, washed, and 50 μL of anti-human HRP IgG (1:5000, #A18823, Invitrogen, Thermo Fisher Scientific), IgA (1:5000, #A18787, Invitrogen, Thermo Fisher Scientific), IgG1 (1:4000, #9054-05, Southern Biotech), IgG2 (1:4000, #9060-05, Southern Biotech), IgG3 (1:4000, #9210-05, Southern Biotech) or IgG4 (1:8000, #9200-05, Southern Biotech) secondary antibody was added.

Techniques: Binding Assay, Titration, Generated, Inhibition, Virus, Neutralization

Journal: Vaccines

Article Title: Reproducibility Assessment of Enzyme-Linked Immunosorbent Assays to Detect Anti-HPV16 L1-Specific IgG1, IgG3, IgA, and IgM Antibodies

doi: 10.3390/vaccines12101108

Figure Lengend Snippet: Levey–Jennings plots for serum standards used in IgG1, IgG3, IgA, and IgM ELISAs. Calculated concentrations of serum standards in EU/mL from plates during reproducibility tests were plotted for ( A ) IgG1, ( B ) IgG3, ( C ) IgA, and ( D ) IgM. Mean (dotted line), 1 ± SD (thin solid lines), 2 ± SD (thin solid lines), and 3 ± SD (thick solid lines) are shown in each plot. Standard was tested a combined 72 times over several days by two technicians.

Article Snippet: The plates were washed and one of the following HRP-conjugated secondary antibodies was plated with the samples: goat anti-human total IgG antibody (KPL, Milford, CO, USA; cat# 214-1002); mouse anti-human IgG1 (Southern Biotech, Birmingham, AL, USA; cat# 9052-05), mouse anti-human IgG2 (Southern Biotech; cat# 9070-05), mouse anti-human IgG3 (Southern Biotech; cat# 9210-05), mouse anti-human IgG4 (Southern Biotech; cat# 9200-05), goat anti-human IgA (Jackson ImmunoResearch, West Grove, PA, USA; cat# 109-035-011), or goat anti-human IgM (Jackson ImmunoResearch, West Grove, USA; cat# 109-035-129).

Techniques:

Journal: Vaccines

Article Title: Reproducibility Assessment of Enzyme-Linked Immunosorbent Assays to Detect Anti-HPV16 L1-Specific IgG1, IgG3, IgA, and IgM Antibodies

doi: 10.3390/vaccines12101108

Figure Lengend Snippet: Levey–Jennings plots for positive controls used in IgG1, IgG3, IgA, and IgM ELISAs. On each plate, the same positive control was tested in different columns (PC1 and PC2). The concentrations of the controls were plotted for ( A ) IgG1, ( B ) IgG3, ( C ) IgA, and ( D ) IgM. Mean (dotted line), 1 ± SD (thin solid lines), 2 ± SD (thin solid lines), and 3 ± SD (thick solid lines) are shown in each plot. Each control (PC1 and PC2) was tested a combined 72 times over several days between two technicians.

Article Snippet: The plates were washed and one of the following HRP-conjugated secondary antibodies was plated with the samples: goat anti-human total IgG antibody (KPL, Milford, CO, USA; cat# 214-1002); mouse anti-human IgG1 (Southern Biotech, Birmingham, AL, USA; cat# 9052-05), mouse anti-human IgG2 (Southern Biotech; cat# 9070-05), mouse anti-human IgG3 (Southern Biotech; cat# 9210-05), mouse anti-human IgG4 (Southern Biotech; cat# 9200-05), goat anti-human IgA (Jackson ImmunoResearch, West Grove, PA, USA; cat# 109-035-011), or goat anti-human IgM (Jackson ImmunoResearch, West Grove, USA; cat# 109-035-129).

Techniques: Positive Control, Control

Journal: Vaccines

Article Title: Reproducibility Assessment of Enzyme-Linked Immunosorbent Assays to Detect Anti-HPV16 L1-Specific IgG1, IgG3, IgA, and IgM Antibodies

doi: 10.3390/vaccines12101108

Figure Lengend Snippet: The mean concentrations (EU/mL) of IgG1, IgG3, IgA, and IgM in the seventy-two CVT samples. Mean concentrations of different antibodies in the seventy-two CVT samples are listed. The means of eight replicates are shown. Twenty-four, twenty-five, and twenty-three samples from the low-, medium-, and high-concentration groups, respectively, were tested. The grouping was based on the concentration of anti-HPV16 L1-specific total IgG ( Table S2 ). The italicized numbers in parentheses are those samples with non-detectable levels of the antibodies. For these samples, half the cut-off values were assigned: IgG1 (6 EU/mL); IgG3 (0.625 EU/mL); IgA (0.24 EU/mL); IgM (2.40 EU/mL). IgG3 was detected in all samples.

Article Snippet: The plates were washed and one of the following HRP-conjugated secondary antibodies was plated with the samples: goat anti-human total IgG antibody (KPL, Milford, CO, USA; cat# 214-1002); mouse anti-human IgG1 (Southern Biotech, Birmingham, AL, USA; cat# 9052-05), mouse anti-human IgG2 (Southern Biotech; cat# 9070-05), mouse anti-human IgG3 (Southern Biotech; cat# 9210-05), mouse anti-human IgG4 (Southern Biotech; cat# 9200-05), goat anti-human IgA (Jackson ImmunoResearch, West Grove, PA, USA; cat# 109-035-011), or goat anti-human IgM (Jackson ImmunoResearch, West Grove, USA; cat# 109-035-129).

Techniques: Concentration Assay

Journal: Vaccines

Article Title: Reproducibility Assessment of Enzyme-Linked Immunosorbent Assays to Detect Anti-HPV16 L1-Specific IgG1, IgG3, IgA, and IgM Antibodies

doi: 10.3390/vaccines12101108

Figure Lengend Snippet: Intraclass correlation coefficient (ICC) and coefficient of variation (CV) for each ELISA, overall and by the concentration groups. ICCs quantify the proportion of total assay variability due to real, inter-participant differences rather than assay noise. CVs approximated the assay distribution (i.e., mean-to-standard-deviation ratio) and were calculated to analyze the performance of each ELISA system. CVs were calculated overall as well as specific to variation between technicians and variation across testing days. ICCs and CVs were calculated specific to each ELISA using 8 replicates for each study participant (N = 72). Low, medium, and high groups were based on total IgG concentrations ( Table S2 ).

Article Snippet: The plates were washed and one of the following HRP-conjugated secondary antibodies was plated with the samples: goat anti-human total IgG antibody (KPL, Milford, CO, USA; cat# 214-1002); mouse anti-human IgG1 (Southern Biotech, Birmingham, AL, USA; cat# 9052-05), mouse anti-human IgG2 (Southern Biotech; cat# 9070-05), mouse anti-human IgG3 (Southern Biotech; cat# 9210-05), mouse anti-human IgG4 (Southern Biotech; cat# 9200-05), goat anti-human IgA (Jackson ImmunoResearch, West Grove, PA, USA; cat# 109-035-011), or goat anti-human IgM (Jackson ImmunoResearch, West Grove, USA; cat# 109-035-129).

Techniques: Enzyme-linked Immunosorbent Assay, Concentration Assay

Journal: Vaccines

Article Title: Reproducibility Assessment of Enzyme-Linked Immunosorbent Assays to Detect Anti-HPV16 L1-Specific IgG1, IgG3, IgA, and IgM Antibodies

doi: 10.3390/vaccines12101108

Figure Lengend Snippet: Summary of antibody measurements for IgG1, IgG3, IgA, and IgM in the ninety-five CVT samples. Samples with non-detectable levels of antibodies were excluded from the Range, Mean, and Median columns. For the % Detected column, all ninety-five samples were included in the calculations. The low, medium, and high groupings were based on the total IgG levels ( Table S2 ).

Article Snippet: The plates were washed and one of the following HRP-conjugated secondary antibodies was plated with the samples: goat anti-human total IgG antibody (KPL, Milford, CO, USA; cat# 214-1002); mouse anti-human IgG1 (Southern Biotech, Birmingham, AL, USA; cat# 9052-05), mouse anti-human IgG2 (Southern Biotech; cat# 9070-05), mouse anti-human IgG3 (Southern Biotech; cat# 9210-05), mouse anti-human IgG4 (Southern Biotech; cat# 9200-05), goat anti-human IgA (Jackson ImmunoResearch, West Grove, PA, USA; cat# 109-035-011), or goat anti-human IgM (Jackson ImmunoResearch, West Grove, USA; cat# 109-035-129).

Techniques:

Journal: Vaccines

Article Title: Reproducibility Assessment of Enzyme-Linked Immunosorbent Assays to Detect Anti-HPV16 L1-Specific IgG1, IgG3, IgA, and IgM Antibodies

doi: 10.3390/vaccines12101108

Figure Lengend Snippet: Correlation analyses of antibody concentrations between total IgG vs. IgG1, IgG3, IgA, and IgM. Measured antibody concentrations in EU/mL units were log transformed (log10) and plotted. Pearson r and p values are shown (N = 95). Measured antibody concentrations in EU/mL units were log transformed (log10) and plotted. Pearson r and p values are shown (N = 95). Pearson correlational analyses between total IgG and the respective isotype antibody level (IgG, IgG3, IgA, or IgM) were plotted ( A – D ). The correlations were statistically significant between total IgG and IgG1, IgG3, or IgA, but were not significant between total IgG and IgM.

Article Snippet: The plates were washed and one of the following HRP-conjugated secondary antibodies was plated with the samples: goat anti-human total IgG antibody (KPL, Milford, CO, USA; cat# 214-1002); mouse anti-human IgG1 (Southern Biotech, Birmingham, AL, USA; cat# 9052-05), mouse anti-human IgG2 (Southern Biotech; cat# 9070-05), mouse anti-human IgG3 (Southern Biotech; cat# 9210-05), mouse anti-human IgG4 (Southern Biotech; cat# 9200-05), goat anti-human IgA (Jackson ImmunoResearch, West Grove, PA, USA; cat# 109-035-011), or goat anti-human IgM (Jackson ImmunoResearch, West Grove, USA; cat# 109-035-129).

Techniques: Transformation Assay

Journal: Vaccines

Article Title: Reproducibility Assessment of Enzyme-Linked Immunosorbent Assays to Detect Anti-HPV16 L1-Specific IgG1, IgG3, IgA, and IgM Antibodies

doi: 10.3390/vaccines12101108

Figure Lengend Snippet: Correlation analyses of antibody concentrations between different antibody types. Measured antibody concentrations in EU/mL units were log transformed (log10) and plotted. Pearson r and p values are shown (N = 95). Pearson correlational analyses between IgG1 and IgG3, IgA, or IgM are plotted ( A – F ). Also, correlational analyses between IgG3 and IgA or IgM and between IgA and IgM are plotted. The correlations were statistically significant between IgG1 and IgG3 or IgA, and between IgG3 and IgA. Between IgG1 and IgM, IgG3 and IgM, and IgA and IgM were not significantly different.

Article Snippet: The plates were washed and one of the following HRP-conjugated secondary antibodies was plated with the samples: goat anti-human total IgG antibody (KPL, Milford, CO, USA; cat# 214-1002); mouse anti-human IgG1 (Southern Biotech, Birmingham, AL, USA; cat# 9052-05), mouse anti-human IgG2 (Southern Biotech; cat# 9070-05), mouse anti-human IgG3 (Southern Biotech; cat# 9210-05), mouse anti-human IgG4 (Southern Biotech; cat# 9200-05), goat anti-human IgA (Jackson ImmunoResearch, West Grove, PA, USA; cat# 109-035-011), or goat anti-human IgM (Jackson ImmunoResearch, West Grove, USA; cat# 109-035-129).

Techniques: Transformation Assay

Journal: Vaccines

Article Title: Reproducibility Assessment of Enzyme-Linked Immunosorbent Assays to Detect Anti-HPV16 L1-Specific IgG1, IgG3, IgA, and IgM Antibodies

doi: 10.3390/vaccines12101108

Figure Lengend Snippet: Levey–Jennings plots for serum standards used in IgG1, IgG3, IgA, and IgM ELISAs. Calculated concentrations of serum standards in EU/mL from plates during reproducibility tests were plotted for ( A ) IgG1, ( B ) IgG3, ( C ) IgA, and ( D ) IgM. Mean (dotted line), 1 ± SD (thin solid lines), 2 ± SD (thin solid lines), and 3 ± SD (thick solid lines) are shown in each plot. Standard was tested a combined 72 times over several days by two technicians.

Article Snippet: The plates were washed and one of the following HRP-conjugated secondary antibodies was plated with the samples: goat anti-human total IgG antibody (KPL, Milford, CO, USA; cat# 214-1002); mouse anti-human IgG1 (Southern Biotech, Birmingham, AL, USA; cat# 9052-05), mouse anti-human IgG2 (Southern Biotech; cat# 9070-05), mouse anti-human IgG3 (Southern Biotech; cat# 9210-05), mouse anti-human IgG4 (Southern Biotech; cat# 9200-05), goat anti-human IgA (Jackson ImmunoResearch, West Grove, PA, USA; cat# 109-035-011), or goat anti-human IgM (Jackson ImmunoResearch, West Grove, USA; cat# 109-035-129).

Techniques:

Journal: Vaccines

Article Title: Reproducibility Assessment of Enzyme-Linked Immunosorbent Assays to Detect Anti-HPV16 L1-Specific IgG1, IgG3, IgA, and IgM Antibodies

doi: 10.3390/vaccines12101108

Figure Lengend Snippet: Levey–Jennings plots for positive controls used in IgG1, IgG3, IgA, and IgM ELISAs. On each plate, the same positive control was tested in different columns (PC1 and PC2). The concentrations of the controls were plotted for ( A ) IgG1, ( B ) IgG3, ( C ) IgA, and ( D ) IgM. Mean (dotted line), 1 ± SD (thin solid lines), 2 ± SD (thin solid lines), and 3 ± SD (thick solid lines) are shown in each plot. Each control (PC1 and PC2) was tested a combined 72 times over several days between two technicians.

Article Snippet: The plates were washed and one of the following HRP-conjugated secondary antibodies was plated with the samples: goat anti-human total IgG antibody (KPL, Milford, CO, USA; cat# 214-1002); mouse anti-human IgG1 (Southern Biotech, Birmingham, AL, USA; cat# 9052-05), mouse anti-human IgG2 (Southern Biotech; cat# 9070-05), mouse anti-human IgG3 (Southern Biotech; cat# 9210-05), mouse anti-human IgG4 (Southern Biotech; cat# 9200-05), goat anti-human IgA (Jackson ImmunoResearch, West Grove, PA, USA; cat# 109-035-011), or goat anti-human IgM (Jackson ImmunoResearch, West Grove, USA; cat# 109-035-129).

Techniques: Positive Control, Control

Journal: Vaccines

Article Title: Reproducibility Assessment of Enzyme-Linked Immunosorbent Assays to Detect Anti-HPV16 L1-Specific IgG1, IgG3, IgA, and IgM Antibodies

doi: 10.3390/vaccines12101108

Figure Lengend Snippet: The mean concentrations (EU/mL) of IgG1, IgG3, IgA, and IgM in the seventy-two CVT samples. Mean concentrations of different antibodies in the seventy-two CVT samples are listed. The means of eight replicates are shown. Twenty-four, twenty-five, and twenty-three samples from the low-, medium-, and high-concentration groups, respectively, were tested. The grouping was based on the concentration of anti-HPV16 L1-specific total IgG ( Table S2 ). The italicized numbers in parentheses are those samples with non-detectable levels of the antibodies. For these samples, half the cut-off values were assigned: IgG1 (6 EU/mL); IgG3 (0.625 EU/mL); IgA (0.24 EU/mL); IgM (2.40 EU/mL). IgG3 was detected in all samples.

Article Snippet: The plates were washed and one of the following HRP-conjugated secondary antibodies was plated with the samples: goat anti-human total IgG antibody (KPL, Milford, CO, USA; cat# 214-1002); mouse anti-human IgG1 (Southern Biotech, Birmingham, AL, USA; cat# 9052-05), mouse anti-human IgG2 (Southern Biotech; cat# 9070-05), mouse anti-human IgG3 (Southern Biotech; cat# 9210-05), mouse anti-human IgG4 (Southern Biotech; cat# 9200-05), goat anti-human IgA (Jackson ImmunoResearch, West Grove, PA, USA; cat# 109-035-011), or goat anti-human IgM (Jackson ImmunoResearch, West Grove, USA; cat# 109-035-129).

Techniques: Concentration Assay

Journal: Vaccines

Article Title: Reproducibility Assessment of Enzyme-Linked Immunosorbent Assays to Detect Anti-HPV16 L1-Specific IgG1, IgG3, IgA, and IgM Antibodies

doi: 10.3390/vaccines12101108

Figure Lengend Snippet: Intraclass correlation coefficient (ICC) and coefficient of variation (CV) for each ELISA, overall and by the concentration groups. ICCs quantify the proportion of total assay variability due to real, inter-participant differences rather than assay noise. CVs approximated the assay distribution (i.e., mean-to-standard-deviation ratio) and were calculated to analyze the performance of each ELISA system. CVs were calculated overall as well as specific to variation between technicians and variation across testing days. ICCs and CVs were calculated specific to each ELISA using 8 replicates for each study participant (N = 72). Low, medium, and high groups were based on total IgG concentrations ( Table S2 ).

Article Snippet: The plates were washed and one of the following HRP-conjugated secondary antibodies was plated with the samples: goat anti-human total IgG antibody (KPL, Milford, CO, USA; cat# 214-1002); mouse anti-human IgG1 (Southern Biotech, Birmingham, AL, USA; cat# 9052-05), mouse anti-human IgG2 (Southern Biotech; cat# 9070-05), mouse anti-human IgG3 (Southern Biotech; cat# 9210-05), mouse anti-human IgG4 (Southern Biotech; cat# 9200-05), goat anti-human IgA (Jackson ImmunoResearch, West Grove, PA, USA; cat# 109-035-011), or goat anti-human IgM (Jackson ImmunoResearch, West Grove, USA; cat# 109-035-129).

Techniques: Enzyme-linked Immunosorbent Assay, Concentration Assay

Journal: Vaccines

Article Title: Reproducibility Assessment of Enzyme-Linked Immunosorbent Assays to Detect Anti-HPV16 L1-Specific IgG1, IgG3, IgA, and IgM Antibodies

doi: 10.3390/vaccines12101108

Figure Lengend Snippet: Summary of antibody measurements for IgG1, IgG3, IgA, and IgM in the ninety-five CVT samples. Samples with non-detectable levels of antibodies were excluded from the Range, Mean, and Median columns. For the % Detected column, all ninety-five samples were included in the calculations. The low, medium, and high groupings were based on the total IgG levels ( Table S2 ).

Article Snippet: The plates were washed and one of the following HRP-conjugated secondary antibodies was plated with the samples: goat anti-human total IgG antibody (KPL, Milford, CO, USA; cat# 214-1002); mouse anti-human IgG1 (Southern Biotech, Birmingham, AL, USA; cat# 9052-05), mouse anti-human IgG2 (Southern Biotech; cat# 9070-05), mouse anti-human IgG3 (Southern Biotech; cat# 9210-05), mouse anti-human IgG4 (Southern Biotech; cat# 9200-05), goat anti-human IgA (Jackson ImmunoResearch, West Grove, PA, USA; cat# 109-035-011), or goat anti-human IgM (Jackson ImmunoResearch, West Grove, USA; cat# 109-035-129).

Techniques:

Journal: Vaccines

Article Title: Reproducibility Assessment of Enzyme-Linked Immunosorbent Assays to Detect Anti-HPV16 L1-Specific IgG1, IgG3, IgA, and IgM Antibodies

doi: 10.3390/vaccines12101108

Figure Lengend Snippet: Correlation analyses of antibody concentrations between total IgG vs. IgG1, IgG3, IgA, and IgM. Measured antibody concentrations in EU/mL units were log transformed (log10) and plotted. Pearson r and p values are shown (N = 95). Measured antibody concentrations in EU/mL units were log transformed (log10) and plotted. Pearson r and p values are shown (N = 95). Pearson correlational analyses between total IgG and the respective isotype antibody level (IgG, IgG3, IgA, or IgM) were plotted ( A – D ). The correlations were statistically significant between total IgG and IgG1, IgG3, or IgA, but were not significant between total IgG and IgM.

Article Snippet: The plates were washed and one of the following HRP-conjugated secondary antibodies was plated with the samples: goat anti-human total IgG antibody (KPL, Milford, CO, USA; cat# 214-1002); mouse anti-human IgG1 (Southern Biotech, Birmingham, AL, USA; cat# 9052-05), mouse anti-human IgG2 (Southern Biotech; cat# 9070-05), mouse anti-human IgG3 (Southern Biotech; cat# 9210-05), mouse anti-human IgG4 (Southern Biotech; cat# 9200-05), goat anti-human IgA (Jackson ImmunoResearch, West Grove, PA, USA; cat# 109-035-011), or goat anti-human IgM (Jackson ImmunoResearch, West Grove, USA; cat# 109-035-129).

Techniques: Transformation Assay

Journal: Vaccines

Article Title: Reproducibility Assessment of Enzyme-Linked Immunosorbent Assays to Detect Anti-HPV16 L1-Specific IgG1, IgG3, IgA, and IgM Antibodies

doi: 10.3390/vaccines12101108

Figure Lengend Snippet: Correlation analyses of antibody concentrations between different antibody types. Measured antibody concentrations in EU/mL units were log transformed (log10) and plotted. Pearson r and p values are shown (N = 95). Pearson correlational analyses between IgG1 and IgG3, IgA, or IgM are plotted ( A – F ). Also, correlational analyses between IgG3 and IgA or IgM and between IgA and IgM are plotted. The correlations were statistically significant between IgG1 and IgG3 or IgA, and between IgG3 and IgA. Between IgG1 and IgM, IgG3 and IgM, and IgA and IgM were not significantly different.

Article Snippet: The plates were washed and one of the following HRP-conjugated secondary antibodies was plated with the samples: goat anti-human total IgG antibody (KPL, Milford, CO, USA; cat# 214-1002); mouse anti-human IgG1 (Southern Biotech, Birmingham, AL, USA; cat# 9052-05), mouse anti-human IgG2 (Southern Biotech; cat# 9070-05), mouse anti-human IgG3 (Southern Biotech; cat# 9210-05), mouse anti-human IgG4 (Southern Biotech; cat# 9200-05), goat anti-human IgA (Jackson ImmunoResearch, West Grove, PA, USA; cat# 109-035-011), or goat anti-human IgM (Jackson ImmunoResearch, West Grove, USA; cat# 109-035-129).

Techniques: Transformation Assay