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

E. Blanco, M. Perez-Andres, L. Sanoja-Flores, M. Wentink, O. Pelak, M. Martín-Ayuso, G. Grigore, J. Torres-Canizales, E. López-Granados, T. Kalina, M. van der Burg, S. Arriba-Méndez, S. Santa Cruz, N. Puig, JJM. van Dongen, A. Orfao,

. 2019 ; 475 (-) : 112372. [pub] 20170928

Language English Country Netherlands

Document type Journal Article, Research Support, Non-U.S. Gov't, Validation Study

Persistent link   https://www.medvik.cz/link/bmc20023415

The clinical value of assessing immunoglobulin (Ig)G and IgA subclasses in addition to the isotypes of soluble Igs in serum has been well established. >20years ago, the International Union of Immunological Societies and the World Health Organization performed collaborative studies in order to validate antibody (Ab) clones for the detection of IgG and IgA subclasses for a broad range of laboratory assays, except for flow cytometry. Here we analyzed the performance of commercially available Ab clones to detect IgG and IgA subclasses in memory B-cells and plasma cells (PCs) by flow cytometry. In a first step, 28 Ab clones were evaluated in peripheral blood from healthy donors. Only 17/28 clones showed reactivity against IgG and IgA subclasses expressed on the B-cell and PC surface membrane, including Ab clones for IgG1 (SAG1, HP6188, HP6001 and HP6186), IgG2 (SAG2, HP6014 and HP6002), IgG3 (SAG3, HP6095 and HP6050), IgG4 (SAG4), IgA1 (SAA1, H69-11.4 and B3506B4) and IgA2 (SAA2, 2E2, and A9604D2). In a second step, for each Ig subclass a single clone was 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 clones in human peripheral blood, bone marrow and tonsil samples, using different staining protocols (e.g. surface membrane and/or cytoplasmic staining). All selected Ab clones displayed strong positivity, high specificity and optimal resolution between negative and positive cells. Alternative Ab clones were also validated. Thus, our results show the feasibility of using the validated Ig subclass Ab clones in combination with other B cell-associated markers for detailed dissection of the memory B-cell and PC compartments that express distinct Ig subclasses in different human tissues.

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$a The clinical value of assessing immunoglobulin (Ig)G and IgA subclasses in addition to the isotypes of soluble Igs in serum has been well established. >20years ago, the International Union of Immunological Societies and the World Health Organization performed collaborative studies in order to validate antibody (Ab) clones for the detection of IgG and IgA subclasses for a broad range of laboratory assays, except for flow cytometry. Here we analyzed the performance of commercially available Ab clones to detect IgG and IgA subclasses in memory B-cells and plasma cells (PCs) by flow cytometry. In a first step, 28 Ab clones were evaluated in peripheral blood from healthy donors. Only 17/28 clones showed reactivity against IgG and IgA subclasses expressed on the B-cell and PC surface membrane, including Ab clones for IgG1 (SAG1, HP6188, HP6001 and HP6186), IgG2 (SAG2, HP6014 and HP6002), IgG3 (SAG3, HP6095 and HP6050), IgG4 (SAG4), IgA1 (SAA1, H69-11.4 and B3506B4) and IgA2 (SAA2, 2E2, and A9604D2). In a second step, for each Ig subclass a single clone was selected according to its specificity and fluorescence intensity (resolution power), for further more detailed validation (SAG1, SAG2, SAG3, SAG4, SAA1 and SAA2). This validation process was carried out in 4 different laboratories by testing the selected Ab clones in human peripheral blood, bone marrow and tonsil samples, using different staining protocols (e.g. surface membrane and/or cytoplasmic staining). All selected Ab clones displayed strong positivity, high specificity and optimal resolution between negative and positive cells. Alternative Ab clones were also validated. Thus, our results show the feasibility of using the validated Ig subclass Ab clones in combination with other B cell-associated markers for detailed dissection of the memory B-cell and PC compartments that express distinct Ig subclasses in different human tissues.
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$a Perez-Andres, Martin $u Cancer Research Centre (IBMCC, USAL-CSIC), Institute for Biomedical Research of Salamanca (IBSAL) and Department of Medicine and Cytometry Service (NUCLEUS Research Support Platform), University of Salamanca (USAL), Salamanca, Spain.
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$a Wentink, Marjolein $u Laboratory for Medical Immunology, Department of Immunology, Erasmus MC, University Medical Center Rotterdam, Rotterdam, The Netherlands.
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$a Pelak, Ondrej $u CLIP - Childhood Leukaemia Investigation Prague, Department of Paediatric Haematology and Oncology, Second Faculty of Medicine, Charles University and University Hospital Motol, Prague, Czech Republic.
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$a Kalina, Tomas $u CLIP - Childhood Leukaemia Investigation Prague, Department of Paediatric Haematology and Oncology, Second Faculty of Medicine, Charles University and University Hospital Motol, Prague, Czech Republic.
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$a van Dongen, Jacques J M $u Department of Immunohematology and Blood Transfusion, Leiden University Medical Center, Leiden, Netherlands.
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$a Orfao, Alberto $u Cancer Research Centre (IBMCC, USAL-CSIC), Institute for Biomedical Research of Salamanca (IBSAL) and Department of Medicine and Cytometry Service (NUCLEUS Research Support Platform), University of Salamanca (USAL), Salamanca, Spain. Electronic address: orfao@usal.es.
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