Superantigen-like interaction of IVIG with antibody Fab fragments cloned by phage display technology

2 Superantigen-like interaction of IVIG with antibody Fab fragments cloned by phage display technology Dissertation for the award of doctor rerum naturalium bp base pairs CD cluster of differentiation CDR complementarity-determining region cfu colony forming units C H 1 first constant region of antibody heavy chain C L light chain consant region cp coat protein D diversity gene DTT dithiothreitol EBV Epstein-Barr Virus ELISA enzyme linked immunosorbent assay Fc crystallizable fragment of antibody molecule Fd V H + C H 1 of the antibody molecule FR Hc framework region heavy chain HIV Human immunodeficiency virus HLA Human Leukocyte Antigen HRP horse radish peroxidase Ig immunoglobulin ITP idiopathic thrombocytopenic purpura IU international units IVIG intravenous immunoglobulins J H heavy chain joining gene J L Lc light chain joining gene light chain M13 VCSM13 helper phage PBL peripheral blood lymphocytes PEG polyethylene glycol RIBS ribosome binding site rpm rounds per minute SCID severe combined immune deficiency SLE systemic lupus erythematosus SpA staphylococcal protein A TNF Tumor necrosis factor tu transforming units V H heavy chain variable region V κ kappa chain variable region V L light chain variable region V λ lambda chain variable region w/v weight by volume 6 1. 0 INTRODUCTION 1. 1 GENERATION OF ANTIBODIES Mammalian B lymphocytes are formed first in the liver during fetal life but the bone marrow becomes the site of lymphopoeisis after birth and remains so throughout adult life (Rolink and Melchers, 1991; Kincade, 1987). Genes expressed in B lymphocytes encode the immunoglobulin (Ig) molecule. The monomeric immunoglobulin is composed of two identical heavy (H) and light (L) polypeptide chains. The amino-terminal domain of each polypeptide chain consists of a variable region whilst the carboxy-terminus contains the constant domain(s). Pairing together of a variable light and heavy chain region form the antigen-combining site responsible for antigen-antibody interactions. Each separate variable domain has three areas of high amino acid sequence variability (hypervariable regions) known as complementarity-determining regions (CDR) interspersed by regions of relatively constant amino acid sequence termed framework regions (FR) (Kabat et. al., 1991). CDR3 is the region of greatest variability. The immunoglobulin heavy chain variable region segment is encoded by a variable (V H), diversity (D), and a joining (J H) gene segments while the light chain variable region is encoded by a variable (V L), and joining (J L) segment. A site-specific recombination reaction known as V H (D)J H recombination brings …