title: Generation of Recombinant Human IgG Monoclonal Antibodies from Immortalized Sorted B Cells.
abstract: Finding new methods for generating human monoclonal antibodies is an active research field that is important for both basic and applied sciences, including the development of immunotherapeutics. However, the techniques to identify and produce such antibodies tend to be arduous and sometimes the heavy and light chain pair of the antibodies are dissociated. Here, we describe a relatively simple, straightforward protocol to produce human recombinant monoclonal antibodies from human peripheral blood mononuclear cells using immortalization with Epstein-Barr Virus (EBV) and Toll-like receptor 9 activation. With an adequate staining, B cells producing antibodies can be isolated for subsequent immortalization and clonal expansion. The antibody transcripts produced by the immortalized B cell clones can be amplified by PCR, sequenced as corresponding heavy and light chain pairs and cloned into immunoglobulin expression vectors. The antibodies obtained with this technique can be powerful tools to study relevant human immune responses, including autoimmunity, and create the basis for new therapeutics.
authors.affiliation: School for Mental Health and Neuroscience, Maastricht University; Department of Neurosciences, Institut d'Investigació Germans Trias i Pujol.
authors.forename: Gisela
authors.initials: G
authors.lastname: Nogales-Gadea
authors.affiliation: School for Mental Health and Neuroscience, Maastricht University.
authors.forename: Abhishek
authors.initials: A
authors.lastname: Saxena
authors.affiliation: School for Mental Health and Neuroscience, Maastricht University.
authors.forename: Carolin
authors.initials: C
authors.lastname: Hoffmann
authors.affiliation: School for Mental Health and Neuroscience, Maastricht University.
authors.forename: Judith
authors.initials: J
authors.lastname: Hounjet
authors.affiliation: School for Mental Health and Neuroscience, Maastricht University.
authors.forename: Daniëlle
authors.initials: D
authors.lastname: Coenen
authors.affiliation: School for Mental Health and Neuroscience, Maastricht University.
authors.forename: Peter
authors.initials: P
authors.lastname: Molenaar
authors.affiliation: School for Mental Health and Neuroscience, Maastricht University.
authors.forename: Mario
authors.initials: M
authors.lastname: Losen
authors.affiliation: School for Mental Health and Neuroscience, Maastricht University; p.martinez@maastrichtuniversity.nl.
authors.forename: Pilar
authors.initials: P
authors.lastname: Martinez-Martinez
mesh_terms: D000911:Antibodies, Monoclonal / Q000096:biosynthesis* / Q000097:blood / Q000235:genetics; D001402:B-Lymphocytes / Q000166:cytology* / Q000276:immunology / Q000378:metabolism*; D018076:DNA, Complementary / Q000235:genetics; D005434:Flow Cytometry; D057809:HEK293 Cells; D004854:Herpesvirus 4, Human / Q000502:physiology; D006801:Humans; D007074:Immunoglobulin G / Q000096:biosynthesis* / Q000097:blood / Q000235:genetics; D007963:Leukocytes, Mononuclear / Q000166:cytology / Q000276:immunology / Q000378:metabolism; D016133:Polymerase Chain Reaction / Q000379:methods; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D051217:Toll-Like Receptor 9 / Q000378:metabolism; D014775:Virus Activation
chemical_list: D000911:Antibodies, Monoclonal; D018076:DNA, Complementary; D007074:Immunoglobulin G; D011994:Recombinant Proteins; C495371:TLR9 protein, human; D051217:Toll-Like Receptor 9