2S albumin-binding B-cells show specific binding to Ara h2 and/or 6
In order to verify the specificity of identified 2S albumin-binding B-cells, 42 different sets of heavy and corresponding light chain gene transcripts were heterologously expressed as IgE or IgG1 mAbs. The majority of these mAbs (81%, 34/42), mostly derived from allergic and tolerant patients, showed specific binding to Ara h2 and 6 at 10 µg/ml or lower in a concentration-dependent manner (Figure 2A). These heterologously expressed mAbs originated from 2S albumin-binding IgM+ (41%, 14/34), IgG1+ (3%, 1/34), IgG2+ (23%, 8/34), IgE+ (3%, 1/34), IgA1+ (18%, 6/34) and IgA2+ (9%, 3/34) B-cells. While the two mAbs with relatively high optical density (OD) values originated from IgG1+ and IgA1+ B-cells, mAbs with relatively low OD values originated predominately from IgM+ B-cells. About half of the specific mAbs (58%, 11/19) tested for separate binding to Ara h 2 and 62 or 6 recognized both allergens with comparable OD values. Eight mAbs exclusively recognized either Ara h2 or 6 (21%, 4/19 Ara h2 and 21%, 4/19 Ara h 6) (Figure 2B). The remaining antibodies without specific binding to Ara h2 or 6 at 10 µg/ml originated predominately from IgM+ B-cells of non-atopic controls (88%, 7/8). These data confirm the specificity of most selected 2S albumin-binding B-cells, supporting the relevance of the gene analyses described in the following paragraphs.