Figure S2. IgA profiles of pt7 bNAbs and... | Rockefeller University Press

Figure S2.

$IgA profiles of pt7 bNAbs and purified serum antibodies. (A) ELISA binding analysis comparing the reactivity of pt7 bNAbs expressed as IgG and IgA against HIV-1 Env proteins. Means ± SD of duplicate OD values are shown. (B) Purification of monomeric and dimeric 7-269 IgA antibodies. FPLC chromatograms show the protein separation of IgA monomers, dimers, and multimers by SEC in two separate experiments. The x axis shows the elution volume (eV) required to obtain the values of absorption units at 280 nm (mAU) indicated on the y axis. Light blue bars indicate selected fractions. Silver-stained SDS-PAGE gels on the right show the protein bands in the purified pooled fractions. (C) Graphs comparing the neutralizing activity of monomeric and dimeric 7-269 IgA antibodies (mIgA and dIgA, respectively) against the selected pseudoviruses as measured in the in vitro TZM-bl assay. Means ± SD of duplicate values for two independent experiments are shown. mIgA and dIgA antibodies from purification 1 were tested in neutralization experiment 1, and mIgA and dIgA antibodies from purification 2 were used in experiment 2. (D) Heatmap showing the ELISA binding analysis of selected pt7 bNAbs against consensus subtype B overlapping Env peptides. Darker colors indicate higher reactivity (OD values); white, no binding. Non–HIV-1 mGO53 and anti-V3crown 10-188 antibody are negative and positive controls, respectively. (E) Silver-stained SDS-PAGE gel shows purified pt7 serum IgAs (ranging from 100 to 500 ng) in nonreducing and nonheated conditions. Monomeric 10-1074 IgA1 antibodies were used as a control. * and ** indicate monomeric and dimeric IgA antibodies, respectively. (F) ELISA binding analysis comparing the reactivity of purified IgG and IgA antibodies from pt7 serum against HIV-1 Env gp120 and selected mutant proteins. Means ± SD of triplicate OD values are shown. 10-1074 IgG1 and IgA1 antibodies were used as controls.$

IgA profiles of pt7 bNAbs and purified serum antibodies. (A) ELISA binding analysis comparing the reactivity of pt7 bNAbs expressed as IgG and IgA against HIV-1 Env proteins. Means ± SD of duplicate OD values are shown. (B) Purification of monomeric and dimeric 7-269 IgA antibodies. FPLC chromatograms show the protein separation of IgA monomers, dimers, and multimers by SEC in two separate experiments. The x axis shows the elution volume (eV) required to obtain the values of absorption units at 280 nm (mAU) indicated on the y axis. Light blue bars indicate selected fractions. Silver-stained SDS-PAGE gels on the right show the protein bands in the purified pooled fractions. (C) Graphs comparing the neutralizing activity of monomeric and dimeric 7-269 IgA antibodies (mIgA and dIgA, respectively) against the selected pseudoviruses as measured in the in vitro TZM-bl assay. Means ± SD of duplicate values for two independent experiments are shown. mIgA and dIgA antibodies from purification 1 were tested in neutralization experiment 1, and mIgA and dIgA antibodies from purification 2 were used in experiment 2. (D) Heatmap showing the ELISA binding analysis of selected pt7 bNAbs against consensus subtype B overlapping Env peptides. Darker colors indicate higher reactivity (OD values); white, no binding. Non–HIV-1 mGO53 and anti-V3^crown 10-188 antibody are negative and positive controls, respectively. (E) Silver-stained SDS-PAGE gel shows purified pt7 serum IgAs (ranging from 100 to 500 ng) in nonreducing and nonheated conditions. Monomeric 10-1074 IgA1 antibodies were used as a control. * and ** indicate monomeric and dimeric IgA antibodies, respectively. (F) ELISA binding analysis comparing the reactivity of purified IgG and IgA antibodies from pt7 serum against HIV-1 Env gp120 and selected mutant proteins. Means ± SD of triplicate OD values are shown. 10-1074 IgG1 and IgA1 antibodies were used as controls.