A New method for the analysis and production of monoclonal antibody fragments originating from single human B cells

Autor: Peter G. A. Steenbakkers, W. J. Van Venrooij, F.H.J. van den Hoogen, A.H.M. Pennings, Rene Hoet, R.M.T. de Wildt
Jazyk: angličtina
Rok vydání: 1997
Předmět:
Phage display
medicine.drug_class
Cells
Immunology
Cell Culture Techniques
Immunoglobulin light chain
Monoclonal antibody
Polymerase Chain Reaction
Connective Tissue Cells (Non MeSH)
Ribonucleoprotein
U1 Small Nuclear
Fetus
Antigen
medicine
Humans
Immunology and Allergy
Single-chain variable fragment
Cloning
Molecular
Cells
Cultured
B cell
Autoantibodies
B-Lymphocytes
Cultured
Blood Cells
Leukemia
biology
Histocompatibility Testing
Stem Cells
Antibodies
Monoclonal
RNA-Binding Proteins
Dubbelblind vergelijkend onderzoek van methotrexaat versus placebo bij patiënten met systemischesclerose
Cell sorting
Flow Cytometry
Systemic sclerosis
methotrexate versus placebo. Double-blind controlled study
Molecular biology
Hematopoiesis and stem cell transplantation
Clone Cells
Bone Marrow Cells (Non MeSH)
medicine.anatomical_structure
biology.protein
Immunoglobulin Light Chains
Antibody
Immunoglobulin Heavy Chains
Zdroj: Journal of Immunological Methods, 207, 61-67
Journal of Immunological Methods, 207, pp. 61-67
ISSN: 0022-1759
Popis: The phage display approach has proven to be a major step forward in studies on the human autoimmune repertoire. However, it remains doubtful whether the heavy and light chains of the antibodies obtained from these libraries resemble original in vivo pairings. Here we describe a novel, simple method for the immortalization of the variable heavy and light chain regions originating from individual, nonboosted, autoantigen-specific human B cells. Our method is based on the clonal expansion of B cells in which cell-cell interactions (CD40-CD40L) as well as soluble factors were shown to be essential. This B cell culture system combined with a selection on antigen (the U1A protein, a frequent autoantigenic target in patients with systemic lupus erythematosus) and single cell sorting resulted in the isolation of U1A-specific human B cells and the subsequent expression of an U1A-specific single chain variable fragment (scFv). Our method circumvents laborious plating and screening and has the advantage that original heavy/light chain pairings can be isolated. Due to the high growth efficiency of single cultured B cells (50-70% outgrowth) even rare B cell activities can be studied using this system.
Databáze: OpenAIRE
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