Selection and validation of antibody clones against IgG and IgA subclasses in switched memory B-cells and plasma cells

Abstract The clinical value of assessing immunoglobulin (Ig)G and IgA subclassesin addition to the isotypes of soluble Igs in serum has been well established. > 20 years ago, the International Union of Immunological Societies and the World Health Organization performed collaborative studies in order to validate antibody (Ab) clonesfor the detection of IgG and IgA subclassesfor a broad range of laboratory assays, but except for flow cytometry. Here we analyzed the performance of commercially available Ab clonesto detect IgG and IgA subclassesin memory B-cellsand plasma cells (PCs) by flow cytometry. In a first step, 28 Ab cloneswere evaluated in peripheral blood from healthy donors. Only 17/28 clonesshowed reactivity against IgG and IgA subclassesexpressed on the B-cell and PC surface membrane, including Ab clonesfor IgG 1 (SAG1, HP6188, HP6001 and HP6186), IgG 2 (SAG2, HP6014 and HP6002), IgG 3 (SAG3, HP6095 and HP6050), IgG 4 (SAG4), IgA 1 (SAA1, H69-11.4 and B3506B4) and IgA 2 (SAA2, 2E2, and A9604D2). In a second step, for each Ig subclassa single clonewas selected according to its specificity and fluorescence intensity (resolution power), for further more detailed validation (SAG1, SAG2, SAG3, SAG4, SAA1 and SAA2). This validation process was carried out in 4 different laboratories by testing the selected Ab clonesin human peripheral blood, bone marrow and tonsil samples, using different staining protocols ( e.g. surface membrane and/or cytoplasmic staining). All selected Ab clonesdisplayed strong positivity, high specificity and optimal resolution between negative and positive cells. Alternative Ab cloneswere also validated. Thus, our results show the feasibility of using the validated Ig subclassAb clonesin combination with other B cell-associated markers for detailed dissection of the memory B-celland PC compartments that express distinct Ig subclassesin different human tissues.