title: Efficient generation of human IgA monoclonal antibodies.
abstract: Immunoglobulin A (IgA) is the most abundant antibody isotype produced in humans. IgA antibodies primarily ensure immune protection of mucosal surfaces against invading pathogens, but also circulate and are present in large quantities in blood. IgAs are heterogeneous at a molecular level, with two IgA subtypes and the capacity to form multimers by interacting with the joining (J) chain. Here, we have developed an efficient strategy to rapidly generate human IgA1 and IgA2 monoclonal antibodies in their monomeric and dimeric forms. Recombinant monomeric and dimeric IgA1/IgA2 counterparts of a prototypical IgG1 monoclonal antibody, 10-1074, targeting the HIV-1 envelope protein, were produced in large amounts after expression cloning and transient transfection of 293-F cells. 10-1074 IgAs were FPLC-purified using a novel affinity-based resin engrafted with anti-IgA chimeric Fabs, followed by a monomers/multimers separation using size exclusion-based FPLC. ELISA binding experiments confirmed that the artificial IgA class switching of 10-1074 did not alter its antigen recognition. In summary, our technical approach allows the very efficient production of various forms of purified recombinant human IgA molecules, which are precious tools in dissecting IgA B-cell responses in physiological and pathophysiological conditions, and studying the biology, function and therapeutic potential of IgAs.
authors.affiliation: Laboratory of Humoral Response to Pathogens, Department of Immunology, Institut Pasteur, Paris, 75015, France; CNRS-URA 1961, Paris, 75015, France.
authors.forename: Valérie
authors.initials: V
authors.lastname: Lorin
authors.affiliation: Laboratory of Humoral Response to Pathogens, Department of Immunology, Institut Pasteur, Paris, 75015, France; CNRS-URA 1961, Paris, 75015, France. Electronic address: hugo.mouquet@pasteur.fr.
authors.forename: Hugo
authors.initials: H
authors.lastname: Mouquet
mesh_terms: D000911:Antibodies, Monoclonal / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D001402:B-Lymphocytes / Q000378:metabolism; D002460:Cell Line; D003001:Cloning, Molecular; D057809:HEK293 Cells; D015497:HIV-1 / Q000276:immunology*; D006801:Humans; D007070:Immunoglobulin A / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D017578:Immunoglobulin Class Switching / Q000276:immunology; D007132:Immunoglobulin Isotypes / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D054299:env Gene Products, Human Immunodeficiency Virus / Q000276:immunology*
chemical_list: D000911:Antibodies, Monoclonal; D007070:Immunoglobulin A; D007132:Immunoglobulin Isotypes; D011994:Recombinant Proteins; D054299:env Gene Products, Human Immunodeficiency Virus
keywords: Affinity/size-exclusion chromatography; Antibodies; IgA; Protein purification; Transient recombinant expression system