Enhanced sensitivity of E2-specific B cell detection using a sE2 cocktail.
(A) Hierarchical clustering of 89 genetically distinct sE2 proteins based upon relative binding of 12 E2-specific and 12 inferred reverted unmutated ancestor (rua) mAbs. Red lines indicate subtype 1a, and blue lines indicate subtype 1b proteins. Mean binding (OD450nm) of mature or rua mAbs to sE2s representative of each cluster is shown. (B) Representative staining of sE2-specific class switched B cells (csBC; CD3-, CD19+, IgM-, IgD-, CD10-, sE2+) in a healthy (HCV-) or an HCV+ subject. (C) Frequency (%) of csBC that are sE2+ in 15 HCV+ subjects, using either single sE2 proteins or a cocktail of three proteins. Dotted line indicates the true-positive threshold set using staining of cells from healthy, HCV- subjects. (D) Percentage of cultured csBC well supernatants that produced IgG or reacted in an ELISA with the sE2 antigens. (E) Percentage of E2-specific supernatants from 4 B cells/well cultures that reacted to each sE2 used in the cocktail. In C, non-normally distributed data by the Shapiro Wilk normality test was compared using the matched Friedman test with p values adjusted for multiple comparisons using the Two-stage linear step-up procedure of Benjamini, Krieger and Yekutieli. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001.