cd16a nk 92 cells  (ATCC)


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    ATCC cd16a nk 92 cells
    Vaccine specific IgG induction of FcγRIIIa/CD16 effector functions correlate with neutralization of wildtype and SARS-CoV-2 clinical variants. Relationships between live SARS-CoV-2 WA1 neutralization (FRNT50) and receptor binding domain (RBD) specific relative binding to (A) <t>FcγRIIIa/CD16a,</t> (B) FcγRIIa/CD32a and (C) FcγRIIb/CD32b, RBD specific antibody dependent natural killer cell activation (ADNKA) determined by (D) CD107a expression, (E) IFNγ production and (F) TNFα secretion, and (G) RBD specific antibody dependent neutrophil phagocytosis (ADNP) are shown. (H) Heatmap summarizes Spearman correlations between neutralization of SARS-CoV-2 wildtype WA1 and variants with relative binding of RBD specific IgG to activating (FcγRIIIa/CD16a and FcγRIIa/CD32a), inhibitory (FcγRIIb/CD32b) and ratios of activating:inhibitory FcγR (FcγRIIIa/CD16a:FcγRIIb/CD32b and FcγRIIa/CD32a:FcγRIIb/CD32b) binding and Fc effector functions ADNKA, antibody dependent cellular phagocytosis (ADCP), ADNP and antibody dependent complement deposition (ADCD). * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001.
    Cd16a Nk 92 Cells, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "BNT162b2 induced neutralizing and non-neutralizing antibody functions against SARSCoV-2 diminish with age"

    Article Title: BNT162b2 induced neutralizing and non-neutralizing antibody functions against SARSCoV-2 diminish with age

    Journal: medRxiv

    doi: 10.1101/2022.08.12.22278726

    Vaccine specific IgG induction of FcγRIIIa/CD16 effector functions correlate with neutralization of wildtype and SARS-CoV-2 clinical variants. Relationships between live SARS-CoV-2 WA1 neutralization (FRNT50) and receptor binding domain (RBD) specific relative binding to (A) FcγRIIIa/CD16a, (B) FcγRIIa/CD32a and (C) FcγRIIb/CD32b, RBD specific antibody dependent natural killer cell activation (ADNKA) determined by (D) CD107a expression, (E) IFNγ production and (F) TNFα secretion, and (G) RBD specific antibody dependent neutrophil phagocytosis (ADNP) are shown. (H) Heatmap summarizes Spearman correlations between neutralization of SARS-CoV-2 wildtype WA1 and variants with relative binding of RBD specific IgG to activating (FcγRIIIa/CD16a and FcγRIIa/CD32a), inhibitory (FcγRIIb/CD32b) and ratios of activating:inhibitory FcγR (FcγRIIIa/CD16a:FcγRIIb/CD32b and FcγRIIa/CD32a:FcγRIIb/CD32b) binding and Fc effector functions ADNKA, antibody dependent cellular phagocytosis (ADCP), ADNP and antibody dependent complement deposition (ADCD). * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001.
    Figure Legend Snippet: Vaccine specific IgG induction of FcγRIIIa/CD16 effector functions correlate with neutralization of wildtype and SARS-CoV-2 clinical variants. Relationships between live SARS-CoV-2 WA1 neutralization (FRNT50) and receptor binding domain (RBD) specific relative binding to (A) FcγRIIIa/CD16a, (B) FcγRIIa/CD32a and (C) FcγRIIb/CD32b, RBD specific antibody dependent natural killer cell activation (ADNKA) determined by (D) CD107a expression, (E) IFNγ production and (F) TNFα secretion, and (G) RBD specific antibody dependent neutrophil phagocytosis (ADNP) are shown. (H) Heatmap summarizes Spearman correlations between neutralization of SARS-CoV-2 wildtype WA1 and variants with relative binding of RBD specific IgG to activating (FcγRIIIa/CD16a and FcγRIIa/CD32a), inhibitory (FcγRIIb/CD32b) and ratios of activating:inhibitory FcγR (FcγRIIIa/CD16a:FcγRIIb/CD32b and FcγRIIa/CD32a:FcγRIIb/CD32b) binding and Fc effector functions ADNKA, antibody dependent cellular phagocytosis (ADCP), ADNP and antibody dependent complement deposition (ADCD). * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001.

    Techniques Used: Neutralization, Binding Assay, Activation Assay, Expressing

    Differential fucose and sialic acid on vaccine specific IgG link to FcγRIIIa/CD16a effector functions. Stacked column graphs depict the relative abundance of individual glycoforms ( – ) with respect to (A) total bulk non-antigen specific and (B) receptor binding domain (RBD) specific IgG. Each column represents one individual study participant. Dot plots summarize differences between bulk non-antigen specific and RBD specific IgG in the collective relative abundance of all individual glycoforms ( and ) containing (C) fucose, (D) total sialic acid, (E) di-sialic acid, (F) mono-sialic acid and (G) di-galactose with statistical significance calculated by Wilcoxon matched-pairs signed rank test. Data for (H) asialylated fucosylated, (I) total sialic and (J) total di-sialic acid, the three RBD specific glycoforms that have a statistically significant relationship across all markers of ADNKA activation, are plotted with CD107a expression per RBD specific IgG, as well as IFNγ and TNFα . For comparison, data for (K) total mono-sialic acid is plotted.
    Figure Legend Snippet: Differential fucose and sialic acid on vaccine specific IgG link to FcγRIIIa/CD16a effector functions. Stacked column graphs depict the relative abundance of individual glycoforms ( – ) with respect to (A) total bulk non-antigen specific and (B) receptor binding domain (RBD) specific IgG. Each column represents one individual study participant. Dot plots summarize differences between bulk non-antigen specific and RBD specific IgG in the collective relative abundance of all individual glycoforms ( and ) containing (C) fucose, (D) total sialic acid, (E) di-sialic acid, (F) mono-sialic acid and (G) di-galactose with statistical significance calculated by Wilcoxon matched-pairs signed rank test. Data for (H) asialylated fucosylated, (I) total sialic and (J) total di-sialic acid, the three RBD specific glycoforms that have a statistically significant relationship across all markers of ADNKA activation, are plotted with CD107a expression per RBD specific IgG, as well as IFNγ and TNFα . For comparison, data for (K) total mono-sialic acid is plotted.

    Techniques Used: Binding Assay, Activation Assay, Expressing

    Age influences some but not all vaccine specific antibody FcγR functions. The relationships between relative binding of receptor binding domain (RBD) specific IgG to (A) FcγRIIIa/CD16a, (B) FcγRIIa/CD32a and (C) inhibitory FcγRIIb/CD32b and age are shown. (D) Heatmap of the coefficient of determination (r 2 ) summarizes the goodness of fit across RBD and control respiratory syncytial virus (RSV), influenza (Flu) and anthrax antigens in FcγR binding and age. * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; N/A not available given absence of significant detectable levels. The relationship between age and RBD antibody dependent natural killer cell activation (ADNKA) as measured by (E) CD107a and (F) TNFα, and (G) RBD antibody dependent cellular phagocytosis (ADCP) and (H) RBD antibody dependent neutrophil phagocytosis (ADNP) are shown. Linear regression with p values adjusted for sex are reported. (I) Radar plots depict vaccine specific IgG glycoforms calculated from the Z scored data for each individual RBD specific IgG glycoforms relative to bulk non-antigen specific IgG glycoforms with lines representing the median for each age group.
    Figure Legend Snippet: Age influences some but not all vaccine specific antibody FcγR functions. The relationships between relative binding of receptor binding domain (RBD) specific IgG to (A) FcγRIIIa/CD16a, (B) FcγRIIa/CD32a and (C) inhibitory FcγRIIb/CD32b and age are shown. (D) Heatmap of the coefficient of determination (r 2 ) summarizes the goodness of fit across RBD and control respiratory syncytial virus (RSV), influenza (Flu) and anthrax antigens in FcγR binding and age. * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; N/A not available given absence of significant detectable levels. The relationship between age and RBD antibody dependent natural killer cell activation (ADNKA) as measured by (E) CD107a and (F) TNFα, and (G) RBD antibody dependent cellular phagocytosis (ADCP) and (H) RBD antibody dependent neutrophil phagocytosis (ADNP) are shown. Linear regression with p values adjusted for sex are reported. (I) Radar plots depict vaccine specific IgG glycoforms calculated from the Z scored data for each individual RBD specific IgG glycoforms relative to bulk non-antigen specific IgG glycoforms with lines representing the median for each age group.

    Techniques Used: Binding Assay, Activation Assay

    Enhanced BNT162b2 induced polyfunctional antibody breadth and magnitude against SARS-CoV-2 in younger compared to older adults. For each individual, the neutralization breadth across variants and cumulative vaccine specific Fc functional magnitude from the sum of the Z scores for each of the individual effector functions were calculated. (A) Grouped by neutralization breadth (top), each column shows the cumulative Fc functional score for one individual. Median, minimum and maximum ages characterizing each neutralization breadth group are shown (bottom). Polyfunctional antibody breadth was calculated for each individual and used to categorize individuals into high (90–100%), medium (80–90%) or low (<80%) responders. The proportions of high, median and low responders are grouped by (B) age and (C) sex. (D) Radar plots depict vaccine specific polyfunctional antibody magnitude calculated from the Z scored data for each antibody function with lines representing the median for each age group. (E) Heatmap summarizes Spearman correlations between neutralization of SARS-CoV-2 wildtype WA1 and variants with RBD specific IgG/M/A, IgG and IgA levels, relative binding of RBD specific IgG to activating (FcγRIIIa/CD16a and FcγRIIa/CD32a), inhibitory (FcγRIIb/CD32b) and ratios of activating:inhibitory FcγR (FcγRIIIa/CD16a:FcγRIIb/CD32b and FcγRIIa/CD32a:FcγRIIb/CD32b) binding and Fc effector functions antibody dependent natural killer cell activation (ADNKA), antibody dependent cellular phagocytosis (ADCP), antibody dependent neutrophil phagocytosis (ADNP) and antibody dependent complement deposition (ADCD) for each age group. * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001.
    Figure Legend Snippet: Enhanced BNT162b2 induced polyfunctional antibody breadth and magnitude against SARS-CoV-2 in younger compared to older adults. For each individual, the neutralization breadth across variants and cumulative vaccine specific Fc functional magnitude from the sum of the Z scores for each of the individual effector functions were calculated. (A) Grouped by neutralization breadth (top), each column shows the cumulative Fc functional score for one individual. Median, minimum and maximum ages characterizing each neutralization breadth group are shown (bottom). Polyfunctional antibody breadth was calculated for each individual and used to categorize individuals into high (90–100%), medium (80–90%) or low (<80%) responders. The proportions of high, median and low responders are grouped by (B) age and (C) sex. (D) Radar plots depict vaccine specific polyfunctional antibody magnitude calculated from the Z scored data for each antibody function with lines representing the median for each age group. (E) Heatmap summarizes Spearman correlations between neutralization of SARS-CoV-2 wildtype WA1 and variants with RBD specific IgG/M/A, IgG and IgA levels, relative binding of RBD specific IgG to activating (FcγRIIIa/CD16a and FcγRIIa/CD32a), inhibitory (FcγRIIb/CD32b) and ratios of activating:inhibitory FcγR (FcγRIIIa/CD16a:FcγRIIb/CD32b and FcγRIIa/CD32a:FcγRIIb/CD32b) binding and Fc effector functions antibody dependent natural killer cell activation (ADNKA), antibody dependent cellular phagocytosis (ADCP), antibody dependent neutrophil phagocytosis (ADNP) and antibody dependent complement deposition (ADCD) for each age group. * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001.

    Techniques Used: Neutralization, Functional Assay, Binding Assay, Activation Assay

    cd16a nk 92 cells  (ATCC)


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    ATCC cd16a nk 92 cells
    Increasing maternal and cord blood neutralizing and Fc effector functional magnitude and breadth were observed after infection, vaccination, and vaccination and infection in pregnancy. The bars depict the median of matched maternal (white, left) and cord (blue, right) blood levels of ( A ) neutralization (FRNT 50 ) against SARS-CoV-2 WA1 (infection n = 14, vaccine n = 13, vaccine + infection n = 19), Delta, and Omicron viruses (infection n = 12, vaccine n = 8, vaccine + infection n = 14); ( B ) ADNKA by CD107a, IFN-γ, and TNF-α; ( C ) ADCD; ( D ) ADCP; and relative binding to ( E ) FcRN and ( F ) <t>FcγRIIIa/CD16a,</t> FcγRIIa/CD32a, and FcγRIIb/CD32b specific to receptor-binding domain. B – F , sample sizes are infection n = 20, vaccine n = 18, vaccine + infection n = 27. P values are adjusted for maternal age and body mass index using linear regression. ^ Marks significance after adjustment for multiple comparisons by Benjamini-Hochberg. The proportion of detectable functions was used to categorize individuals as a high, medium, or low responder . Polyfunctional breadth is depicted as the percentages of each type of responder within each group in ( G ) maternal and matched ( H ) cord samples. Abbreviations: ADCD, antibody-dependent complement deposition; ADCP, antibody-dependent cellular phagocytosis; ADNKA, antibody-dependent natural killer cell activation; FRNT 50 , 50% reduction neutralization test; IFN-γ, interferon-γ; MFI, mean fluorescence intensity; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2; TNF-α, tumor necrosis factor-α.
    Cd16a Nk 92 Cells, supplied by ATCC, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Diverging Maternal and Cord Antibody Functions From SARS-CoV-2 Infection and Vaccination in Pregnancy"

    Article Title: Diverging Maternal and Cord Antibody Functions From SARS-CoV-2 Infection and Vaccination in Pregnancy

    Journal: The Journal of Infectious Diseases

    doi: 10.1093/infdis/jiad421

    Increasing maternal and cord blood neutralizing and Fc effector functional magnitude and breadth were observed after infection, vaccination, and vaccination and infection in pregnancy. The bars depict the median of matched maternal (white, left) and cord (blue, right) blood levels of ( A ) neutralization (FRNT 50 ) against SARS-CoV-2 WA1 (infection n = 14, vaccine n = 13, vaccine + infection n = 19), Delta, and Omicron viruses (infection n = 12, vaccine n = 8, vaccine + infection n = 14); ( B ) ADNKA by CD107a, IFN-γ, and TNF-α; ( C ) ADCD; ( D ) ADCP; and relative binding to ( E ) FcRN and ( F ) FcγRIIIa/CD16a, FcγRIIa/CD32a, and FcγRIIb/CD32b specific to receptor-binding domain. B – F , sample sizes are infection n = 20, vaccine n = 18, vaccine + infection n = 27. P values are adjusted for maternal age and body mass index using linear regression. ^ Marks significance after adjustment for multiple comparisons by Benjamini-Hochberg. The proportion of detectable functions was used to categorize individuals as a high, medium, or low responder . Polyfunctional breadth is depicted as the percentages of each type of responder within each group in ( G ) maternal and matched ( H ) cord samples. Abbreviations: ADCD, antibody-dependent complement deposition; ADCP, antibody-dependent cellular phagocytosis; ADNKA, antibody-dependent natural killer cell activation; FRNT 50 , 50% reduction neutralization test; IFN-γ, interferon-γ; MFI, mean fluorescence intensity; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2; TNF-α, tumor necrosis factor-α.
    Figure Legend Snippet: Increasing maternal and cord blood neutralizing and Fc effector functional magnitude and breadth were observed after infection, vaccination, and vaccination and infection in pregnancy. The bars depict the median of matched maternal (white, left) and cord (blue, right) blood levels of ( A ) neutralization (FRNT 50 ) against SARS-CoV-2 WA1 (infection n = 14, vaccine n = 13, vaccine + infection n = 19), Delta, and Omicron viruses (infection n = 12, vaccine n = 8, vaccine + infection n = 14); ( B ) ADNKA by CD107a, IFN-γ, and TNF-α; ( C ) ADCD; ( D ) ADCP; and relative binding to ( E ) FcRN and ( F ) FcγRIIIa/CD16a, FcγRIIa/CD32a, and FcγRIIb/CD32b specific to receptor-binding domain. B – F , sample sizes are infection n = 20, vaccine n = 18, vaccine + infection n = 27. P values are adjusted for maternal age and body mass index using linear regression. ^ Marks significance after adjustment for multiple comparisons by Benjamini-Hochberg. The proportion of detectable functions was used to categorize individuals as a high, medium, or low responder . Polyfunctional breadth is depicted as the percentages of each type of responder within each group in ( G ) maternal and matched ( H ) cord samples. Abbreviations: ADCD, antibody-dependent complement deposition; ADCP, antibody-dependent cellular phagocytosis; ADNKA, antibody-dependent natural killer cell activation; FRNT 50 , 50% reduction neutralization test; IFN-γ, interferon-γ; MFI, mean fluorescence intensity; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2; TNF-α, tumor necrosis factor-α.

    Techniques Used: Functional Assay, Infection, Neutralization, Binding Assay, Activation Assay, Fluorescence

    Fc effector functions are preferentially transferred across the placenta compared to neutralizing activities. The bars depict the median of matched maternal (grey) and cord (blue) blood levels of ( A ) neutralization against live SARS-CoV-2 WA1, Delta, and Omicron; ( B ) ADNKA; ( C ) ADCD; ( D ) ADCP; and relative binding to ( E ) FcRN and ( F ) FcγRIIIa/CD16a, FcγRIIa/CD32a, and FcγRIIb/CD32b specific to receptor-binding domain. Statistical significance was calculated by Wilcoxon-matched pairs test. Abbreviations: ADCD, antibody-dependent complement deposition; ADCP, antibody-dependent cellular phagocytosis; ADNKA, antibody-dependent natural killer cell activation; FRNT, reduction neutralization test; IFN-γ, interferon-γ; MFI, mean fluorescence intensity; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2; TNF-α, tumor necrosis factor-α.
    Figure Legend Snippet: Fc effector functions are preferentially transferred across the placenta compared to neutralizing activities. The bars depict the median of matched maternal (grey) and cord (blue) blood levels of ( A ) neutralization against live SARS-CoV-2 WA1, Delta, and Omicron; ( B ) ADNKA; ( C ) ADCD; ( D ) ADCP; and relative binding to ( E ) FcRN and ( F ) FcγRIIIa/CD16a, FcγRIIa/CD32a, and FcγRIIb/CD32b specific to receptor-binding domain. Statistical significance was calculated by Wilcoxon-matched pairs test. Abbreviations: ADCD, antibody-dependent complement deposition; ADCP, antibody-dependent cellular phagocytosis; ADNKA, antibody-dependent natural killer cell activation; FRNT, reduction neutralization test; IFN-γ, interferon-γ; MFI, mean fluorescence intensity; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2; TNF-α, tumor necrosis factor-α.

    Techniques Used: Neutralization, Binding Assay, Activation Assay, Fluorescence

    cd16a nk 92 cells  (ATCC)


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    ATCC cd16a nk 92 cells
    Cd16a Nk 92 Cells, supplied by ATCC, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    nk 92 cd16a cells  (ATCC)


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    ATCC nk 92 cd16a cells
    Flow cytometric gating to detect CD107a externalization. ( A – E ) sequential gating for CD107a-positive <t>NK-92-CD16A</t> cells incubated with antibody and Raji cells. The assay conditions were an NK to Raji E:T of 1:2, with or without 10 ng/mL GA101-GE antibody, and 40 min of incubation at 37 °C. ( A ) Starting NK and Raji cells; ( B ) gating for single cells; ( C ) gating for GFP-bright NK-92-CD16A cells; ( D ) gating for CD56 bright GFP positive double-positive cells; ( E ) gating for the % CD107a-positive cells. ( F ) NK cells with Rajis without mAb. The data are from experiment JCA044, illustrated in .
    Nk 92 Cd16a Cells, supplied by ATCC, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Detection of Antibody-Dependent Cell-Mediated Cytotoxicity—Supporting Antibodies by NK-92-CD16A Cell Externalization of CD107a: Recognition of Antibody Afucosylation and Assay Optimization"

    Article Title: Detection of Antibody-Dependent Cell-Mediated Cytotoxicity—Supporting Antibodies by NK-92-CD16A Cell Externalization of CD107a: Recognition of Antibody Afucosylation and Assay Optimization

    Journal: Antibodies

    doi: 10.3390/antib12030044

    Flow cytometric gating to detect CD107a externalization. ( A – E ) sequential gating for CD107a-positive NK-92-CD16A cells incubated with antibody and Raji cells. The assay conditions were an NK to Raji E:T of 1:2, with or without 10 ng/mL GA101-GE antibody, and 40 min of incubation at 37 °C. ( A ) Starting NK and Raji cells; ( B ) gating for single cells; ( C ) gating for GFP-bright NK-92-CD16A cells; ( D ) gating for CD56 bright GFP positive double-positive cells; ( E ) gating for the % CD107a-positive cells. ( F ) NK cells with Rajis without mAb. The data are from experiment JCA044, illustrated in .
    Figure Legend Snippet: Flow cytometric gating to detect CD107a externalization. ( A – E ) sequential gating for CD107a-positive NK-92-CD16A cells incubated with antibody and Raji cells. The assay conditions were an NK to Raji E:T of 1:2, with or without 10 ng/mL GA101-GE antibody, and 40 min of incubation at 37 °C. ( A ) Starting NK and Raji cells; ( B ) gating for single cells; ( C ) gating for GFP-bright NK-92-CD16A cells; ( D ) gating for CD56 bright GFP positive double-positive cells; ( E ) gating for the % CD107a-positive cells. ( F ) NK cells with Rajis without mAb. The data are from experiment JCA044, illustrated in .

    Techniques Used: Incubation

    CD107a externalization after six assay conditions. The results are from JCA044 in which cells were cultured for 40 min, 1:2 NK to Raji target cells, without or with 0.04 or 10 ng/mL of GA-101 GE antibody or with phorbol myristic acid (PMA) and ionophore. ( A ) Unlabeled NK cells used to set gating for CD107a-positive cells. ( B – F ) Labeled cells. ( B ) NK-92-CD16A cells without Raji targets (repeat of F). ( C ) cells with PMA & calcium ionophore as a positive control. ( D ) NK activity towards Raji cells in the absence of anti-CD20 antibody. ( E ) NK plus Rajis with 0.04 ng/mL GA101 GE antibody. ( F ) NK plus Rajis with 10 ng/mL GA101-GE (repeat of E).
    Figure Legend Snippet: CD107a externalization after six assay conditions. The results are from JCA044 in which cells were cultured for 40 min, 1:2 NK to Raji target cells, without or with 0.04 or 10 ng/mL of GA-101 GE antibody or with phorbol myristic acid (PMA) and ionophore. ( A ) Unlabeled NK cells used to set gating for CD107a-positive cells. ( B – F ) Labeled cells. ( B ) NK-92-CD16A cells without Raji targets (repeat of F). ( C ) cells with PMA & calcium ionophore as a positive control. ( D ) NK activity towards Raji cells in the absence of anti-CD20 antibody. ( E ) NK plus Rajis with 0.04 ng/mL GA101 GE antibody. ( F ) NK plus Rajis with 10 ng/mL GA101-GE (repeat of E).

    Techniques Used: Cell Culture, Labeling, Positive Control, Activity Assay

    Assay by NK-92-CD16A cell externalization of CD107a for antibodies that can support ADCC. The Raji cells were pre-incubated with GA101 anti-CD20 antibodies, then NK-92-CD16A cells were added at a 1:2 effector NK to Raji target ratio (E:T), and the cells incubated for 40 min at 37 °C. NK and Raji cells were also incubated without antibodies to measure CD107a externalization associated with NK activity. ( A ) Antibody detection by NK CD107a vs. by fluorescent secondary anti-IgG to Raji-bound antibodies. For the anti-CD20 antibody bound to Raji cells, the Rajis were stained with AF647-labeled donkey anti-human IgG. Both PE-anti-CD107a and AF647 anti-human IgG were detected by flow cytometry. ( A1 ) Detection of target-cell bound antibody by NK CD107a or by fluorescent anti-human IgG. The EC 50 s for each method are indicated with arrows. EC 50 values are the effective concentrations of anti-CD20 that elicited 50% of maximum NK-92 antibody-specific (ADCC minus NK) CD107a externalization. The values for NK activity (without antibodies) are indicated by square symbols. ( A2 ) The median fluorescent intensities (MFIs) of the CD107a-positive NK cells or AF647-anti-human IgG labeled Raji cells. The NK MFIs are for only the CD107a-positive cells. ( B ) CD107a externalization in response to antibodies with different Fc-fucosylation. The antibodies are from one mAb clone, GA101. The WT antibody is ~10% afucosylated; the GE antibody 50% afucosylated. ( B1 ) EC 50 s for antibodies that differed in fucosylation. The EC 50 s associated with afucosylation were 20-fold apart in this experiment ( p < 0.05); similar differences were observed for three other experiments. ( B2 ) The MFIs of the CD107a positive cells. The MFIs are for the CD107a positive in ( B1 ). The CD107a per cell increased with afucosylation ( p < 0.001). ( C ) Antibody concentrations for CD107a externalization vs. death by ADCC. The CD107a values are the data of ( B1 ). ( C1 , C2 ) Antibody EC 50 s for CD107a vs. for cell death, with GA101-WT or GA101-GE antibody. NK CD107a was determined at an E:T of 1:2. ADCC-mediated death was determined with 51 Cr-Raji cells at an E:T of 20:1. Both assays were stopped at 40 min. The 95% confidence limits for each EC 50 are color-coded and indicated at the top of the graphs. ( D ) Antibody detection by NK-92CD16A cell CD107a or by ADCC by peripheral blood NK cells. The donors’ genotypes encoding CD16A AA158, either lower affinity for Fc-IgG phenylalanine (F) or higher affinity valine (V), are indicated. The CD16A-positive blood NK cell to target (E:T) ratios were 4:1, 3:1, and 1:1 for donors 030, 035, and 038, respectively.
    Figure Legend Snippet: Assay by NK-92-CD16A cell externalization of CD107a for antibodies that can support ADCC. The Raji cells were pre-incubated with GA101 anti-CD20 antibodies, then NK-92-CD16A cells were added at a 1:2 effector NK to Raji target ratio (E:T), and the cells incubated for 40 min at 37 °C. NK and Raji cells were also incubated without antibodies to measure CD107a externalization associated with NK activity. ( A ) Antibody detection by NK CD107a vs. by fluorescent secondary anti-IgG to Raji-bound antibodies. For the anti-CD20 antibody bound to Raji cells, the Rajis were stained with AF647-labeled donkey anti-human IgG. Both PE-anti-CD107a and AF647 anti-human IgG were detected by flow cytometry. ( A1 ) Detection of target-cell bound antibody by NK CD107a or by fluorescent anti-human IgG. The EC 50 s for each method are indicated with arrows. EC 50 values are the effective concentrations of anti-CD20 that elicited 50% of maximum NK-92 antibody-specific (ADCC minus NK) CD107a externalization. The values for NK activity (without antibodies) are indicated by square symbols. ( A2 ) The median fluorescent intensities (MFIs) of the CD107a-positive NK cells or AF647-anti-human IgG labeled Raji cells. The NK MFIs are for only the CD107a-positive cells. ( B ) CD107a externalization in response to antibodies with different Fc-fucosylation. The antibodies are from one mAb clone, GA101. The WT antibody is ~10% afucosylated; the GE antibody 50% afucosylated. ( B1 ) EC 50 s for antibodies that differed in fucosylation. The EC 50 s associated with afucosylation were 20-fold apart in this experiment ( p < 0.05); similar differences were observed for three other experiments. ( B2 ) The MFIs of the CD107a positive cells. The MFIs are for the CD107a positive in ( B1 ). The CD107a per cell increased with afucosylation ( p < 0.001). ( C ) Antibody concentrations for CD107a externalization vs. death by ADCC. The CD107a values are the data of ( B1 ). ( C1 , C2 ) Antibody EC 50 s for CD107a vs. for cell death, with GA101-WT or GA101-GE antibody. NK CD107a was determined at an E:T of 1:2. ADCC-mediated death was determined with 51 Cr-Raji cells at an E:T of 20:1. Both assays were stopped at 40 min. The 95% confidence limits for each EC 50 are color-coded and indicated at the top of the graphs. ( D ) Antibody detection by NK-92CD16A cell CD107a or by ADCC by peripheral blood NK cells. The donors’ genotypes encoding CD16A AA158, either lower affinity for Fc-IgG phenylalanine (F) or higher affinity valine (V), are indicated. The CD16A-positive blood NK cell to target (E:T) ratios were 4:1, 3:1, and 1:1 for donors 030, 035, and 038, respectively.

    Techniques Used: Incubation, Activity Assay, Staining, Labeling, Flow Cytometry

    Conditions that affect CD107a externalization. ( A ) Time courses. The E:T was 1:2, and the antibody concentration was 1 µg/mL GA101 WT. ( A1 ) Increases in the percentage of CD107a-positive NK-92-CD16A cells. The NK activity without antibodies is included to show its increase after antibody-dependent activity was complete. The inset illustrates that the antibody-dependent fraction (Total % − NK % positive) was unchanged after 40 min. ( A2 ) Increases in CD107a expression. The MFIs are for the CD107a-positive cells from ( A1 ). ( A3 ) Side-by-side comparison of the % CD107a positive cells vs. CD107a. MFIs. The antibody-dependent data are from ( A1 , A2 ). ( B ) Effects of E:T ratios on CD107a externalization and death of Raji cells. The E:Ts varied from excess effectors to excess targets, as illustrated for two separate assays, one for NK CD107a externalization and another for ADCC by 51 Cr release. Both assays were for 40 min with 1 µg/mL GA101 WT antibody. ( B1 ) % CD107a-pos cells vs. death by ADCC. The percentage of cells with external CD107a paradoxically decreased with increased E:T ratios. Note: each datum for the % CD107a positive cells represents the % of a varying number of effector cells that increased two-fold for each E:T. The color-matched asterisks indicate p < 0.01compared to the E:T 1:8 values. ( B2 ) Frequencies of CD107a-positive NK cell numbers vs. numbers of target cells killed by ADCC. The numbers (instead of percentages) of CD107a-positive cells at each E:T of ( B1 ) were calculated and then re-expressed as percentages of the initial Raji cells, indicated in orange. The % Raji cell death is also from ( B1 ). The CD107a-positive NK cells far exceeded the dead Raji cells (as indicated by the two ordinate scales). At the E:T of 8:1, the ratio of CD107a-pos NK cells to dead Raji cells was 13:1.
    Figure Legend Snippet: Conditions that affect CD107a externalization. ( A ) Time courses. The E:T was 1:2, and the antibody concentration was 1 µg/mL GA101 WT. ( A1 ) Increases in the percentage of CD107a-positive NK-92-CD16A cells. The NK activity without antibodies is included to show its increase after antibody-dependent activity was complete. The inset illustrates that the antibody-dependent fraction (Total % − NK % positive) was unchanged after 40 min. ( A2 ) Increases in CD107a expression. The MFIs are for the CD107a-positive cells from ( A1 ). ( A3 ) Side-by-side comparison of the % CD107a positive cells vs. CD107a. MFIs. The antibody-dependent data are from ( A1 , A2 ). ( B ) Effects of E:T ratios on CD107a externalization and death of Raji cells. The E:Ts varied from excess effectors to excess targets, as illustrated for two separate assays, one for NK CD107a externalization and another for ADCC by 51 Cr release. Both assays were for 40 min with 1 µg/mL GA101 WT antibody. ( B1 ) % CD107a-pos cells vs. death by ADCC. The percentage of cells with external CD107a paradoxically decreased with increased E:T ratios. Note: each datum for the % CD107a positive cells represents the % of a varying number of effector cells that increased two-fold for each E:T. The color-matched asterisks indicate p < 0.01compared to the E:T 1:8 values. ( B2 ) Frequencies of CD107a-positive NK cell numbers vs. numbers of target cells killed by ADCC. The numbers (instead of percentages) of CD107a-positive cells at each E:T of ( B1 ) were calculated and then re-expressed as percentages of the initial Raji cells, indicated in orange. The % Raji cell death is also from ( B1 ). The CD107a-positive NK cells far exceeded the dead Raji cells (as indicated by the two ordinate scales). At the E:T of 8:1, the ratio of CD107a-pos NK cells to dead Raji cells was 13:1.

    Techniques Used: Concentration Assay, Activity Assay, Expressing

    Variables that affect biosafety in future applications. Antibodies were heated to simulate inactivation of viruses. Formaldehyde treatment is routinely used to inactivate viruses. These assays were at an E:T of 1:2 for 40 min. ( A ) Heated antisera. GA101 WT antibody was heated with serum and then diluted with control heated serum to 1 μg/mL antibody in 10% heated human serum. The variables are indicated below each bar. ( B ) Formaldehyde fixation. Treatment was either (1) after addition of antibodies to Raji cells or (2) after the NK-92-CD16A-cells reacted with cell-bound antibodies. ( B ) Reactivity of the NK-92-CD16A cells to formaldehyde-treated GA101 IgG. Raji cells were treated with 1 or 0.01 µg/mL GA101 WT antibody, then 1% formaldehyde, washed, and used to stimulate NK-92-CD16A cells. The E:T was 1:2, and the assay was for 40 min. ( C ) Reactivity of PE-mAb anti-CD107a with formaldehyde-treated CD107a. Cells were treated with formaldehyde and washed prior to labeling.
    Figure Legend Snippet: Variables that affect biosafety in future applications. Antibodies were heated to simulate inactivation of viruses. Formaldehyde treatment is routinely used to inactivate viruses. These assays were at an E:T of 1:2 for 40 min. ( A ) Heated antisera. GA101 WT antibody was heated with serum and then diluted with control heated serum to 1 μg/mL antibody in 10% heated human serum. The variables are indicated below each bar. ( B ) Formaldehyde fixation. Treatment was either (1) after addition of antibodies to Raji cells or (2) after the NK-92-CD16A-cells reacted with cell-bound antibodies. ( B ) Reactivity of the NK-92-CD16A cells to formaldehyde-treated GA101 IgG. Raji cells were treated with 1 or 0.01 µg/mL GA101 WT antibody, then 1% formaldehyde, washed, and used to stimulate NK-92-CD16A cells. The E:T was 1:2, and the assay was for 40 min. ( C ) Reactivity of PE-mAb anti-CD107a with formaldehyde-treated CD107a. Cells were treated with formaldehyde and washed prior to labeling.

    Techniques Used: Labeling

    nk 92 cd16a cells  (ATCC)


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    ATCC cd16a nk 92 cells
    A subset of infant cord SARS-CoV-2 neutralizing and RBD Fc effector antibody s is enhanced with vaccination compared to infection in pregnancy. The medians (bars) sample ( A ) neutralization (FRNT50) against SARS-CoV-2 WA1 (infection n=14, vaccine accine+infection n=19), Delta and Omicron viruses (infection n=12, vaccine n=8, infection n=14), ( B ) RBD antibody-dependent natural killer cell activation (ADNKA) by, IFNγ, and TNFα, ( C ) RBD antibody-dependent complement deposition (ADCD), ( D ) ibody-dependent cellular phagocytosis (ADCP), and ( E ) relative binding of RBD-specific es to <t>FcγRIIIa/CD16a,</t> FcγRIIa/CD32a, FcγRIIb/CD32b and FcRN are shown. For B-E, sizes are infection n=20, vaccine n=18, vaccine+infection n=27. P-values for A-E are for maternal age and body mass index using linear regression. ( F ) The magnitude of ctions are summarized in the radar plot. Each line represents the median Z-scored data clinical group. ( G ) The proportion of detectable functions was used to categorize ls as a high, medium or low responder. The percentages of each type of responder ch clinical group depict the polyfunctional antibody breadth.
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    1) Product Images from "Diverging maternal and infant cord antibody functions from SARS-CoV-2 infection and vaccination in pregnancy"

    Article Title: Diverging maternal and infant cord antibody functions from SARS-CoV-2 infection and vaccination in pregnancy

    Journal: bioRxiv

    doi: 10.1101/2023.05.01.538955

    A subset of infant cord SARS-CoV-2 neutralizing and RBD Fc effector antibody s is enhanced with vaccination compared to infection in pregnancy. The medians (bars) sample ( A ) neutralization (FRNT50) against SARS-CoV-2 WA1 (infection n=14, vaccine accine+infection n=19), Delta and Omicron viruses (infection n=12, vaccine n=8, infection n=14), ( B ) RBD antibody-dependent natural killer cell activation (ADNKA) by, IFNγ, and TNFα, ( C ) RBD antibody-dependent complement deposition (ADCD), ( D ) ibody-dependent cellular phagocytosis (ADCP), and ( E ) relative binding of RBD-specific es to FcγRIIIa/CD16a, FcγRIIa/CD32a, FcγRIIb/CD32b and FcRN are shown. For B-E, sizes are infection n=20, vaccine n=18, vaccine+infection n=27. P-values for A-E are for maternal age and body mass index using linear regression. ( F ) The magnitude of ctions are summarized in the radar plot. Each line represents the median Z-scored data clinical group. ( G ) The proportion of detectable functions was used to categorize ls as a high, medium or low responder. The percentages of each type of responder ch clinical group depict the polyfunctional antibody breadth.
    Figure Legend Snippet: A subset of infant cord SARS-CoV-2 neutralizing and RBD Fc effector antibody s is enhanced with vaccination compared to infection in pregnancy. The medians (bars) sample ( A ) neutralization (FRNT50) against SARS-CoV-2 WA1 (infection n=14, vaccine accine+infection n=19), Delta and Omicron viruses (infection n=12, vaccine n=8, infection n=14), ( B ) RBD antibody-dependent natural killer cell activation (ADNKA) by, IFNγ, and TNFα, ( C ) RBD antibody-dependent complement deposition (ADCD), ( D ) ibody-dependent cellular phagocytosis (ADCP), and ( E ) relative binding of RBD-specific es to FcγRIIIa/CD16a, FcγRIIa/CD32a, FcγRIIb/CD32b and FcRN are shown. For B-E, sizes are infection n=20, vaccine n=18, vaccine+infection n=27. P-values for A-E are for maternal age and body mass index using linear regression. ( F ) The magnitude of ctions are summarized in the radar plot. Each line represents the median Z-scored data clinical group. ( G ) The proportion of detectable functions was used to categorize ls as a high, medium or low responder. The percentages of each type of responder ch clinical group depict the polyfunctional antibody breadth.

    Techniques Used: Infection, Neutralization, Activation Assay, Binding Assay

    A subset of maternal SARS-CoV-2 neutralizing and RBD Fc effector antibody functions ced with vaccination compared to infection in pregnancy. The medians (bars) of the l pair of the cord samples in in ( A ) neutralization (FRNT50) against SARS-CoV-(infection n=14, vaccine n=13, vaccine+infection n=19), Delta and Omicron viruses n n=12, vaccine n=8, vaccine+infection n=14), ( B ) RBD antibody-dependent natural killer ation (ADNKA) as measured by CD107a, IFNγ, and TNFα, ( C ) RBD antibody-dependent ent deposition (ADCD), ( D ) RBD antibody-dependent cellular phagocytosis (ADCP), relative binding of RBD-specific antibodies to FcγRIIIa/CD16a, FcγRIIa/CD32a, CD32b and FcRN. For B-E, sample sizes are infection n=22, vaccine n=19, infection n=28. P-values for A-E are adjusted for maternal age and body mass index ear regression. ( F ) The magnitude of maternal functions are summarized in the radar h line represents the median Z-scored data for each clinical group. ( G ) The proportion of le functions was used to categorize individuals as a high, medium or low responder. The ges of each type of responder within each clinical group depict the polyfunctional breadth.
    Figure Legend Snippet: A subset of maternal SARS-CoV-2 neutralizing and RBD Fc effector antibody functions ced with vaccination compared to infection in pregnancy. The medians (bars) of the l pair of the cord samples in in ( A ) neutralization (FRNT50) against SARS-CoV-(infection n=14, vaccine n=13, vaccine+infection n=19), Delta and Omicron viruses n n=12, vaccine n=8, vaccine+infection n=14), ( B ) RBD antibody-dependent natural killer ation (ADNKA) as measured by CD107a, IFNγ, and TNFα, ( C ) RBD antibody-dependent ent deposition (ADCD), ( D ) RBD antibody-dependent cellular phagocytosis (ADCP), relative binding of RBD-specific antibodies to FcγRIIIa/CD16a, FcγRIIa/CD32a, CD32b and FcRN. For B-E, sample sizes are infection n=22, vaccine n=19, infection n=28. P-values for A-E are adjusted for maternal age and body mass index ear regression. ( F ) The magnitude of maternal functions are summarized in the radar h line represents the median Z-scored data for each clinical group. ( G ) The proportion of le functions was used to categorize individuals as a high, medium or low responder. The ges of each type of responder within each clinical group depict the polyfunctional breadth.

    Techniques Used: Infection, Neutralization, Binding Assay

    SARS-CoV-2 neutralizing and RBD Fc effector functions are differentially transferred the placenta. ( A ) Neutralization against live SARS-CoV-2 WA1, variant Delta and, ( B ) RBD ADNKA, ( C ) RBD ADCD, ( D ) RBD ADCP, and ( E ) relative binding of RBD-IgG to FcγRIIIa/CD16a, FcγRIIa/CD32a, FcγRIIb/CD32b and FcRN are compared with es of the medians for maternal (grey) and matched cord (blue) samples listed below. al significance was calculated by Wilcoxon-matched pairs test. ( F ) Antibody function ratios (the proportion of cord to maternal levels) are shown with medians (bars), rtile ranges (boxes), and ranges (whiskers).
    Figure Legend Snippet: SARS-CoV-2 neutralizing and RBD Fc effector functions are differentially transferred the placenta. ( A ) Neutralization against live SARS-CoV-2 WA1, variant Delta and, ( B ) RBD ADNKA, ( C ) RBD ADCD, ( D ) RBD ADCP, and ( E ) relative binding of RBD-IgG to FcγRIIIa/CD16a, FcγRIIa/CD32a, FcγRIIb/CD32b and FcRN are compared with es of the medians for maternal (grey) and matched cord (blue) samples listed below. al significance was calculated by Wilcoxon-matched pairs test. ( F ) Antibody function ratios (the proportion of cord to maternal levels) are shown with medians (bars), rtile ranges (boxes), and ranges (whiskers).

    Techniques Used: Neutralization, Variant Assay, Binding Assay

    Antibody functions highlight the effect of differential immune exposure in pregnancy cosylation marks diverging maternal and infant cord responses. Bubble plots ( A ) show lation between neutralizing activities against SARS-CoV-2 WA1, Delta and Omicron and ecific Fc effector functions of antibody-dependent natural killer cell activation (CD107a, Fα), antibody-dependent complement deposition (ADCD), antibody-dependent cellular tosis (ADCP) and relative binding to FcγR (FcγRIIIa/CD16a, FcγRIIa/CD32a, CD32b, FcRN). The Spearman’s rank correlation coefficient is shown by color and nce (-log p) by size with those p<0.05 depicted. Principle-component analysis (PCA) SARS-CoV-2 antibody functions and features show separations between infection and clinical groups and maternal and cord responses. Each symbol in the ( B ) score plot ts a single maternal or cord sample. Each antibody feature is represented in the ( C ) plot, where its location reflects the distribution of the individual samples in the ( B ) score plot
    Figure Legend Snippet: Antibody functions highlight the effect of differential immune exposure in pregnancy cosylation marks diverging maternal and infant cord responses. Bubble plots ( A ) show lation between neutralizing activities against SARS-CoV-2 WA1, Delta and Omicron and ecific Fc effector functions of antibody-dependent natural killer cell activation (CD107a, Fα), antibody-dependent complement deposition (ADCD), antibody-dependent cellular tosis (ADCP) and relative binding to FcγR (FcγRIIIa/CD16a, FcγRIIa/CD32a, CD32b, FcRN). The Spearman’s rank correlation coefficient is shown by color and nce (-log p) by size with those p<0.05 depicted. Principle-component analysis (PCA) SARS-CoV-2 antibody functions and features show separations between infection and clinical groups and maternal and cord responses. Each symbol in the ( B ) score plot ts a single maternal or cord sample. Each antibody feature is represented in the ( C ) plot, where its location reflects the distribution of the individual samples in the ( B ) score plot

    Techniques Used: Activation Assay, Binding Assay, Infection

    cd16a nk 92 cells  (ATCC)


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    ATCC cd16a nk 92 cells
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    cd16a nk 92 cells  (ATCC)


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    ATCC cd16a nk 92 cells
    Vaccine specific IgG induction of FcγRIIIa/CD16 effector functions correlate with neutralization of wildtype and SARS-CoV-2 clinical variants. Relationships between live SARS-CoV-2 WA1 neutralization (FRNT50) and receptor binding domain (RBD) specific relative binding to (A) <t>FcγRIIIa/CD16a,</t> (B) FcγRIIa/CD32a and (C) FcγRIIb/CD32b, RBD specific antibody dependent natural killer cell activation (ADNKA) determined by (D) CD107a expression, (E) IFNγ production and (F) TNFα secretion, and (G) RBD specific antibody dependent neutrophil phagocytosis (ADNP) are shown. (H) Heatmap summarizes Spearman correlations between neutralization of SARS-CoV-2 wildtype WA1 and variants with relative binding of RBD specific IgG to activating (FcγRIIIa/CD16a and FcγRIIa/CD32a), inhibitory (FcγRIIb/CD32b) and ratios of activating:inhibitory FcγR (FcγRIIIa/CD16a:FcγRIIb/CD32b and FcγRIIa/CD32a:FcγRIIb/CD32b) binding and Fc effector functions ADNKA, antibody dependent cellular phagocytosis (ADCP), ADNP and antibody dependent complement deposition (ADCD). * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001.
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    1) Product Images from "BNT162b2 induced neutralizing and non-neutralizing antibody functions against SARSCoV-2 diminish with age"

    Article Title: BNT162b2 induced neutralizing and non-neutralizing antibody functions against SARSCoV-2 diminish with age

    Journal: medRxiv

    doi: 10.1101/2022.08.12.22278726

    Vaccine specific IgG induction of FcγRIIIa/CD16 effector functions correlate with neutralization of wildtype and SARS-CoV-2 clinical variants. Relationships between live SARS-CoV-2 WA1 neutralization (FRNT50) and receptor binding domain (RBD) specific relative binding to (A) FcγRIIIa/CD16a, (B) FcγRIIa/CD32a and (C) FcγRIIb/CD32b, RBD specific antibody dependent natural killer cell activation (ADNKA) determined by (D) CD107a expression, (E) IFNγ production and (F) TNFα secretion, and (G) RBD specific antibody dependent neutrophil phagocytosis (ADNP) are shown. (H) Heatmap summarizes Spearman correlations between neutralization of SARS-CoV-2 wildtype WA1 and variants with relative binding of RBD specific IgG to activating (FcγRIIIa/CD16a and FcγRIIa/CD32a), inhibitory (FcγRIIb/CD32b) and ratios of activating:inhibitory FcγR (FcγRIIIa/CD16a:FcγRIIb/CD32b and FcγRIIa/CD32a:FcγRIIb/CD32b) binding and Fc effector functions ADNKA, antibody dependent cellular phagocytosis (ADCP), ADNP and antibody dependent complement deposition (ADCD). * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001.
    Figure Legend Snippet: Vaccine specific IgG induction of FcγRIIIa/CD16 effector functions correlate with neutralization of wildtype and SARS-CoV-2 clinical variants. Relationships between live SARS-CoV-2 WA1 neutralization (FRNT50) and receptor binding domain (RBD) specific relative binding to (A) FcγRIIIa/CD16a, (B) FcγRIIa/CD32a and (C) FcγRIIb/CD32b, RBD specific antibody dependent natural killer cell activation (ADNKA) determined by (D) CD107a expression, (E) IFNγ production and (F) TNFα secretion, and (G) RBD specific antibody dependent neutrophil phagocytosis (ADNP) are shown. (H) Heatmap summarizes Spearman correlations between neutralization of SARS-CoV-2 wildtype WA1 and variants with relative binding of RBD specific IgG to activating (FcγRIIIa/CD16a and FcγRIIa/CD32a), inhibitory (FcγRIIb/CD32b) and ratios of activating:inhibitory FcγR (FcγRIIIa/CD16a:FcγRIIb/CD32b and FcγRIIa/CD32a:FcγRIIb/CD32b) binding and Fc effector functions ADNKA, antibody dependent cellular phagocytosis (ADCP), ADNP and antibody dependent complement deposition (ADCD). * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001.

    Techniques Used: Neutralization, Binding Assay, Activation Assay, Expressing

    Differential fucose and sialic acid on vaccine specific IgG link to FcγRIIIa/CD16a effector functions. Stacked column graphs depict the relative abundance of individual glycoforms ( – ) with respect to (A) total bulk non-antigen specific and (B) receptor binding domain (RBD) specific IgG. Each column represents one individual study participant. Dot plots summarize differences between bulk non-antigen specific and RBD specific IgG in the collective relative abundance of all individual glycoforms ( and ) containing (C) fucose, (D) total sialic acid, (E) di-sialic acid, (F) mono-sialic acid and (G) di-galactose with statistical significance calculated by Wilcoxon matched-pairs signed rank test. Data for (H) asialylated fucosylated, (I) total sialic and (J) total di-sialic acid, the three RBD specific glycoforms that have a statistically significant relationship across all markers of ADNKA activation, are plotted with CD107a expression per RBD specific IgG, as well as IFNγ and TNFα . For comparison, data for (K) total mono-sialic acid is plotted.
    Figure Legend Snippet: Differential fucose and sialic acid on vaccine specific IgG link to FcγRIIIa/CD16a effector functions. Stacked column graphs depict the relative abundance of individual glycoforms ( – ) with respect to (A) total bulk non-antigen specific and (B) receptor binding domain (RBD) specific IgG. Each column represents one individual study participant. Dot plots summarize differences between bulk non-antigen specific and RBD specific IgG in the collective relative abundance of all individual glycoforms ( and ) containing (C) fucose, (D) total sialic acid, (E) di-sialic acid, (F) mono-sialic acid and (G) di-galactose with statistical significance calculated by Wilcoxon matched-pairs signed rank test. Data for (H) asialylated fucosylated, (I) total sialic and (J) total di-sialic acid, the three RBD specific glycoforms that have a statistically significant relationship across all markers of ADNKA activation, are plotted with CD107a expression per RBD specific IgG, as well as IFNγ and TNFα . For comparison, data for (K) total mono-sialic acid is plotted.

    Techniques Used: Binding Assay, Activation Assay, Expressing

    Age influences some but not all vaccine specific antibody FcγR functions. The relationships between relative binding of receptor binding domain (RBD) specific IgG to (A) FcγRIIIa/CD16a, (B) FcγRIIa/CD32a and (C) inhibitory FcγRIIb/CD32b and age are shown. (D) Heatmap of the coefficient of determination (r 2 ) summarizes the goodness of fit across RBD and control respiratory syncytial virus (RSV), influenza (Flu) and anthrax antigens in FcγR binding and age. * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; N/A not available given absence of significant detectable levels. The relationship between age and RBD antibody dependent natural killer cell activation (ADNKA) as measured by (E) CD107a and (F) TNFα, and (G) RBD antibody dependent cellular phagocytosis (ADCP) and (H) RBD antibody dependent neutrophil phagocytosis (ADNP) are shown. Linear regression with p values adjusted for sex are reported. (I) Radar plots depict vaccine specific IgG glycoforms calculated from the Z scored data for each individual RBD specific IgG glycoforms relative to bulk non-antigen specific IgG glycoforms with lines representing the median for each age group.
    Figure Legend Snippet: Age influences some but not all vaccine specific antibody FcγR functions. The relationships between relative binding of receptor binding domain (RBD) specific IgG to (A) FcγRIIIa/CD16a, (B) FcγRIIa/CD32a and (C) inhibitory FcγRIIb/CD32b and age are shown. (D) Heatmap of the coefficient of determination (r 2 ) summarizes the goodness of fit across RBD and control respiratory syncytial virus (RSV), influenza (Flu) and anthrax antigens in FcγR binding and age. * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; N/A not available given absence of significant detectable levels. The relationship between age and RBD antibody dependent natural killer cell activation (ADNKA) as measured by (E) CD107a and (F) TNFα, and (G) RBD antibody dependent cellular phagocytosis (ADCP) and (H) RBD antibody dependent neutrophil phagocytosis (ADNP) are shown. Linear regression with p values adjusted for sex are reported. (I) Radar plots depict vaccine specific IgG glycoforms calculated from the Z scored data for each individual RBD specific IgG glycoforms relative to bulk non-antigen specific IgG glycoforms with lines representing the median for each age group.

    Techniques Used: Binding Assay, Activation Assay

    Enhanced BNT162b2 induced polyfunctional antibody breadth and magnitude against SARS-CoV-2 in younger compared to older adults. For each individual, the neutralization breadth across variants and cumulative vaccine specific Fc functional magnitude from the sum of the Z scores for each of the individual effector functions were calculated. (A) Grouped by neutralization breadth (top), each column shows the cumulative Fc functional score for one individual. Median, minimum and maximum ages characterizing each neutralization breadth group are shown (bottom). Polyfunctional antibody breadth was calculated for each individual and used to categorize individuals into high (90–100%), medium (80–90%) or low (<80%) responders. The proportions of high, median and low responders are grouped by (B) age and (C) sex. (D) Radar plots depict vaccine specific polyfunctional antibody magnitude calculated from the Z scored data for each antibody function with lines representing the median for each age group. (E) Heatmap summarizes Spearman correlations between neutralization of SARS-CoV-2 wildtype WA1 and variants with RBD specific IgG/M/A, IgG and IgA levels, relative binding of RBD specific IgG to activating (FcγRIIIa/CD16a and FcγRIIa/CD32a), inhibitory (FcγRIIb/CD32b) and ratios of activating:inhibitory FcγR (FcγRIIIa/CD16a:FcγRIIb/CD32b and FcγRIIa/CD32a:FcγRIIb/CD32b) binding and Fc effector functions antibody dependent natural killer cell activation (ADNKA), antibody dependent cellular phagocytosis (ADCP), antibody dependent neutrophil phagocytosis (ADNP) and antibody dependent complement deposition (ADCD) for each age group. * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001.
    Figure Legend Snippet: Enhanced BNT162b2 induced polyfunctional antibody breadth and magnitude against SARS-CoV-2 in younger compared to older adults. For each individual, the neutralization breadth across variants and cumulative vaccine specific Fc functional magnitude from the sum of the Z scores for each of the individual effector functions were calculated. (A) Grouped by neutralization breadth (top), each column shows the cumulative Fc functional score for one individual. Median, minimum and maximum ages characterizing each neutralization breadth group are shown (bottom). Polyfunctional antibody breadth was calculated for each individual and used to categorize individuals into high (90–100%), medium (80–90%) or low (<80%) responders. The proportions of high, median and low responders are grouped by (B) age and (C) sex. (D) Radar plots depict vaccine specific polyfunctional antibody magnitude calculated from the Z scored data for each antibody function with lines representing the median for each age group. (E) Heatmap summarizes Spearman correlations between neutralization of SARS-CoV-2 wildtype WA1 and variants with RBD specific IgG/M/A, IgG and IgA levels, relative binding of RBD specific IgG to activating (FcγRIIIa/CD16a and FcγRIIa/CD32a), inhibitory (FcγRIIb/CD32b) and ratios of activating:inhibitory FcγR (FcγRIIIa/CD16a:FcγRIIb/CD32b and FcγRIIa/CD32a:FcγRIIb/CD32b) binding and Fc effector functions antibody dependent natural killer cell activation (ADNKA), antibody dependent cellular phagocytosis (ADCP), antibody dependent neutrophil phagocytosis (ADNP) and antibody dependent complement deposition (ADCD) for each age group. * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001.

    Techniques Used: Neutralization, Functional Assay, Binding Assay, Activation Assay

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    ATCC cd16a nk 92 cells
    Vaccine specific IgG induction of FcγRIIIa/CD16 effector functions correlate with neutralization of wildtype and SARS-CoV-2 clinical variants. Relationships between live SARS-CoV-2 WA1 neutralization (FRNT50) and receptor binding domain (RBD) specific relative binding to (A) <t>FcγRIIIa/CD16a,</t> (B) FcγRIIa/CD32a and (C) FcγRIIb/CD32b, RBD specific antibody dependent natural killer cell activation (ADNKA) determined by (D) CD107a expression, (E) IFNγ production and (F) TNFα secretion, and (G) RBD specific antibody dependent neutrophil phagocytosis (ADNP) are shown. (H) Heatmap summarizes Spearman correlations between neutralization of SARS-CoV-2 wildtype WA1 and variants with relative binding of RBD specific IgG to activating (FcγRIIIa/CD16a and FcγRIIa/CD32a), inhibitory (FcγRIIb/CD32b) and ratios of activating:inhibitory FcγR (FcγRIIIa/CD16a:FcγRIIb/CD32b and FcγRIIa/CD32a:FcγRIIb/CD32b) binding and Fc effector functions ADNKA, antibody dependent cellular phagocytosis (ADCP), ADNP and antibody dependent complement deposition (ADCD). * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001.
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    1) Product Images from "BNT162b2 induced neutralizing and non-neutralizing antibody functions against SARSCoV-2 diminish with age"

    Article Title: BNT162b2 induced neutralizing and non-neutralizing antibody functions against SARSCoV-2 diminish with age

    Journal: medRxiv

    doi: 10.1101/2022.08.12.22278726

    Vaccine specific IgG induction of FcγRIIIa/CD16 effector functions correlate with neutralization of wildtype and SARS-CoV-2 clinical variants. Relationships between live SARS-CoV-2 WA1 neutralization (FRNT50) and receptor binding domain (RBD) specific relative binding to (A) FcγRIIIa/CD16a, (B) FcγRIIa/CD32a and (C) FcγRIIb/CD32b, RBD specific antibody dependent natural killer cell activation (ADNKA) determined by (D) CD107a expression, (E) IFNγ production and (F) TNFα secretion, and (G) RBD specific antibody dependent neutrophil phagocytosis (ADNP) are shown. (H) Heatmap summarizes Spearman correlations between neutralization of SARS-CoV-2 wildtype WA1 and variants with relative binding of RBD specific IgG to activating (FcγRIIIa/CD16a and FcγRIIa/CD32a), inhibitory (FcγRIIb/CD32b) and ratios of activating:inhibitory FcγR (FcγRIIIa/CD16a:FcγRIIb/CD32b and FcγRIIa/CD32a:FcγRIIb/CD32b) binding and Fc effector functions ADNKA, antibody dependent cellular phagocytosis (ADCP), ADNP and antibody dependent complement deposition (ADCD). * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001.
    Figure Legend Snippet: Vaccine specific IgG induction of FcγRIIIa/CD16 effector functions correlate with neutralization of wildtype and SARS-CoV-2 clinical variants. Relationships between live SARS-CoV-2 WA1 neutralization (FRNT50) and receptor binding domain (RBD) specific relative binding to (A) FcγRIIIa/CD16a, (B) FcγRIIa/CD32a and (C) FcγRIIb/CD32b, RBD specific antibody dependent natural killer cell activation (ADNKA) determined by (D) CD107a expression, (E) IFNγ production and (F) TNFα secretion, and (G) RBD specific antibody dependent neutrophil phagocytosis (ADNP) are shown. (H) Heatmap summarizes Spearman correlations between neutralization of SARS-CoV-2 wildtype WA1 and variants with relative binding of RBD specific IgG to activating (FcγRIIIa/CD16a and FcγRIIa/CD32a), inhibitory (FcγRIIb/CD32b) and ratios of activating:inhibitory FcγR (FcγRIIIa/CD16a:FcγRIIb/CD32b and FcγRIIa/CD32a:FcγRIIb/CD32b) binding and Fc effector functions ADNKA, antibody dependent cellular phagocytosis (ADCP), ADNP and antibody dependent complement deposition (ADCD). * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001.

    Techniques Used: Neutralization, Binding Assay, Activation Assay, Expressing

    Vaccine specific IgG induction of FcγRIIIa/CD16 effector functions correlate with neutralization of SARS-CoV-2 variants. (A) The relationships between live SARS-CoV-2 WA1 wildtype neutralization and RBD specific antibody dependent cellular phagocytosis (ADCP), and RBD specific antibody dependent complement deposition (ADCD), and receptor binding domain (RBD) specific relative binding ratios of activating:inhibitory FcγR FcγRIIIa/CD16a:FcγRIIb/CD32b, FcγRIIa/CD32a:FcγRIIb/CD32b are shown. (B) The relationships between live SARS-CoV-2 variants neutralization and RBD specific FcγRIIIa/CD16a binding and effector function antibody dependent natural killer cell activation (ADNKA) are depicted. Statistical significances were determined by Spearman correlation.
    Figure Legend Snippet: Vaccine specific IgG induction of FcγRIIIa/CD16 effector functions correlate with neutralization of SARS-CoV-2 variants. (A) The relationships between live SARS-CoV-2 WA1 wildtype neutralization and RBD specific antibody dependent cellular phagocytosis (ADCP), and RBD specific antibody dependent complement deposition (ADCD), and receptor binding domain (RBD) specific relative binding ratios of activating:inhibitory FcγR FcγRIIIa/CD16a:FcγRIIb/CD32b, FcγRIIa/CD32a:FcγRIIb/CD32b are shown. (B) The relationships between live SARS-CoV-2 variants neutralization and RBD specific FcγRIIIa/CD16a binding and effector function antibody dependent natural killer cell activation (ADNKA) are depicted. Statistical significances were determined by Spearman correlation.

    Techniques Used: Neutralization, Binding Assay, Activation Assay

    Differential fucose and sialic acid on vaccine specific IgG link to FcγRIIIa/CD16a effector functions. Stacked column graphs depict the relative abundance of individual glycoforms with respect to (A) total bulk non-antigen specific and (B) receptor binding domain (RBD) specific IgG. Each column represents one individual study participant. Dot plots summarize differences between bulk non-antigen specific and RBD specific IgG in the collective relative abundance of all individual glycoforms ( and ) containing (C) fucose, (D) total sialic acid, (E) di-sialic acid, (F) mono-sialic acid and (G) di-galactose with statistical significance calculated by Wilcoxon matched-pairs signed rank test. Data for (H) asialylated fucosylated, (I) total sialic and (J) total di-sialic acid, the three RBD specific glycoforms that have a statistically significant relationship across all markers of ADNKA activation, are plotted with CD107a expression per RBD specific IgG, as well as IFNγ and TNFα . For comparison, data for (K) total mono-sialic acid is plotted.
    Figure Legend Snippet: Differential fucose and sialic acid on vaccine specific IgG link to FcγRIIIa/CD16a effector functions. Stacked column graphs depict the relative abundance of individual glycoforms with respect to (A) total bulk non-antigen specific and (B) receptor binding domain (RBD) specific IgG. Each column represents one individual study participant. Dot plots summarize differences between bulk non-antigen specific and RBD specific IgG in the collective relative abundance of all individual glycoforms ( and ) containing (C) fucose, (D) total sialic acid, (E) di-sialic acid, (F) mono-sialic acid and (G) di-galactose with statistical significance calculated by Wilcoxon matched-pairs signed rank test. Data for (H) asialylated fucosylated, (I) total sialic and (J) total di-sialic acid, the three RBD specific glycoforms that have a statistically significant relationship across all markers of ADNKA activation, are plotted with CD107a expression per RBD specific IgG, as well as IFNγ and TNFα . For comparison, data for (K) total mono-sialic acid is plotted.

    Techniques Used: Binding Assay, Activation Assay, Expressing

    Differential fucose and sialic acid on vaccine specific IgG link FcγRIIIa/CD16a mediated IFNγ and TNFα production. Volcano plots depict slope and statistical significance (-log p) from linear regression assessing the dependency of receptor binding domain (RBD) ADNKA by (A) % CD56 CD107a, (B) IFNγ and (C) TNFα on different RBD specific IgG glycans. (D) Relationships where p<0.05 are enumerated and identified. Data for antibody dependent natural killer cell activation (ADNKA) markers of IFNγ (middle row) and TNFα (bottom row) per RBD IgG and relative abundance of RBD specific (E and H) asialylated fucosylated, (F and I) total sialic and (G and J) total di-sialic acid are plotted. Statistical significances were evaluated by linear regression.
    Figure Legend Snippet: Differential fucose and sialic acid on vaccine specific IgG link FcγRIIIa/CD16a mediated IFNγ and TNFα production. Volcano plots depict slope and statistical significance (-log p) from linear regression assessing the dependency of receptor binding domain (RBD) ADNKA by (A) % CD56 CD107a, (B) IFNγ and (C) TNFα on different RBD specific IgG glycans. (D) Relationships where p<0.05 are enumerated and identified. Data for antibody dependent natural killer cell activation (ADNKA) markers of IFNγ (middle row) and TNFα (bottom row) per RBD IgG and relative abundance of RBD specific (E and H) asialylated fucosylated, (F and I) total sialic and (G and J) total di-sialic acid are plotted. Statistical significances were evaluated by linear regression.

    Techniques Used: Binding Assay, Activation Assay

    Age influences some but not all vaccine specific antibody FcγR functions. The relationships between relative binding of receptor binding domain (RBD) specific IgG to (A) FcγRIIIa/CD16a, (B) FcγRIIa/CD32a and (C) inhibitory FcγRIIb/CD32b and age are shown. (D) Heatmap of the coefficient of determination (r2) summarizes the goodness of fit across RBD and control respiratory syncytial virus (RSV), influenza (Flu) and anthrax antigens in FcγR binding and age. * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; N/A not available given absence of significant detectable levels. The relationship between age and RBD antibody dependent natural killer cell activation (ADNKA) as measured by (E) CD107a and (F) TNFα, and (G) RBD antibody dependent cellular phagocytosis (ADCP) and (H) RBD antibody dependent neutrophil phagocytosis (ADNP) are shown. Linear regression with p values adjusted for sex are reported. (I) Radar plots depict vaccine specific IgG glycoforms calculated from the Z scored data for each individual RBD specific IgG glycoforms relative to bulk non-antigen specific IgG glycoforms with lines representing the median for each age group.
    Figure Legend Snippet: Age influences some but not all vaccine specific antibody FcγR functions. The relationships between relative binding of receptor binding domain (RBD) specific IgG to (A) FcγRIIIa/CD16a, (B) FcγRIIa/CD32a and (C) inhibitory FcγRIIb/CD32b and age are shown. (D) Heatmap of the coefficient of determination (r2) summarizes the goodness of fit across RBD and control respiratory syncytial virus (RSV), influenza (Flu) and anthrax antigens in FcγR binding and age. * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; N/A not available given absence of significant detectable levels. The relationship between age and RBD antibody dependent natural killer cell activation (ADNKA) as measured by (E) CD107a and (F) TNFα, and (G) RBD antibody dependent cellular phagocytosis (ADCP) and (H) RBD antibody dependent neutrophil phagocytosis (ADNP) are shown. Linear regression with p values adjusted for sex are reported. (I) Radar plots depict vaccine specific IgG glycoforms calculated from the Z scored data for each individual RBD specific IgG glycoforms relative to bulk non-antigen specific IgG glycoforms with lines representing the median for each age group.

    Techniques Used: Binding Assay, Activation Assay

    Enhanced BNT162b2 induced polyfunctional antibody breadth and magnitude against SARS-CoV-2 in younger compared to older adults. For each individual, the neutralization breadth across variants and cumulative vaccine specific Fc functional magnitude from the sum of the Z scores for each of the individual effector functions were calculated. (A) Grouped by neutralization breadth (top), each column shows the cumulative Fc functional score for one individual. Median, minimum and maximum ages characterizing each neutralization breadth group are shown (bottom). Polyfunctional antibody breadth was calculated for each individual and used to categorize individuals into high (90-100%), medium (80-90%) or low (<80%) responders. The proportions of high, median and low responders are grouped by (B) age and (C) sex. (D) Radar plots depict vaccine specific polyfunctional antibody magnitude calculated from the Z scored data for each antibody function with lines representing the median for each age group. (E) Heatmap summarizes Spearman correlations between neutralization of SARS-CoV-2 wildtype WA1 and variants with RBD specific IgG/M/A, IgG and IgA levels, relative binding of RBD specific IgG to activating (FcγRIIIa/CD16a and FcγRIIa/CD32a), inhibitory (FcγRIIb/CD32b) and ratios of activating:inhibitory FcγR (FcγRIIIa/CD16a:FcγRIIb/CD32b and FcγRIIa/CD32a:FcγRIIb/CD32b) binding and Fc effector functions antibody dependent natural killer cell activation (ADNKA), antibody dependent cellular phagocytosis (ADCP), antibody dependent neutrophil phagocytosis (ADNP) and antibody dependent complement deposition (ADCD) for each age group. * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001.
    Figure Legend Snippet: Enhanced BNT162b2 induced polyfunctional antibody breadth and magnitude against SARS-CoV-2 in younger compared to older adults. For each individual, the neutralization breadth across variants and cumulative vaccine specific Fc functional magnitude from the sum of the Z scores for each of the individual effector functions were calculated. (A) Grouped by neutralization breadth (top), each column shows the cumulative Fc functional score for one individual. Median, minimum and maximum ages characterizing each neutralization breadth group are shown (bottom). Polyfunctional antibody breadth was calculated for each individual and used to categorize individuals into high (90-100%), medium (80-90%) or low (<80%) responders. The proportions of high, median and low responders are grouped by (B) age and (C) sex. (D) Radar plots depict vaccine specific polyfunctional antibody magnitude calculated from the Z scored data for each antibody function with lines representing the median for each age group. (E) Heatmap summarizes Spearman correlations between neutralization of SARS-CoV-2 wildtype WA1 and variants with RBD specific IgG/M/A, IgG and IgA levels, relative binding of RBD specific IgG to activating (FcγRIIIa/CD16a and FcγRIIa/CD32a), inhibitory (FcγRIIb/CD32b) and ratios of activating:inhibitory FcγR (FcγRIIIa/CD16a:FcγRIIb/CD32b and FcγRIIa/CD32a:FcγRIIb/CD32b) binding and Fc effector functions antibody dependent natural killer cell activation (ADNKA), antibody dependent cellular phagocytosis (ADCP), antibody dependent neutrophil phagocytosis (ADNP) and antibody dependent complement deposition (ADCD) for each age group. * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001.

    Techniques Used: Neutralization, Functional Assay, Binding Assay, Activation Assay

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