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Molecular cloning and expression of the fabs of human autoantibodies in Escherichia coli - Determination of the heavy or light chain contribution to the anti-DNA/-cardiolipin activity of the Fab

Kumar, S; Kalsi, J; Ravirajan, CT; Rahman, A; Athwal, D; Latchman, DS; Isenberg, DA; (2000) Molecular cloning and expression of the fabs of human autoantibodies in Escherichia coli - Determination of the heavy or light chain contribution to the anti-DNA/-cardiolipin activity of the Fab. J BIOL CHEM , 275 (45) 35129 - 35136. Gold open access

Abstract

The Fabs of three human autoantibodies (B3/33H11, anti-DNA; UK4, anti-phospholipid) and six related hybrids have been cloned, expressed in Escherichia coli, and purified to homogeneity, SDS-polyacrylamide gel electrophoresis and Western blot analysis of the recombinant Feb demonstrated the purified Fab to be of correct size and in assembled form. Protein expression levels of up to 5-9 mg per liter of culture were achievable. A sensitive and reliable comparative anti-DNA enzyme-linked immunosorbent assay, involving a defined biotinylated 35-mer oligonucleotide in its single- or double-stranded form, is also described. Crithidia assay and anti-DNA or anti-cardiolipin antibody enzyme-linked immunosorbent assay analyses demonstrated convincing binding of the recombinant Fab proteins to DNA/cardiolipin, confirming the expression of functional molecule. The comparative DNA/cardiolipin binding analyses of the nine Fabs revealed that the anti-DNA (light, B3/33H11) or anti-cardiolipin (heavy, UK4) activity lies predominantly on one of the two chains. However, a compatible partner chain is necessary for optimum antigen binding activity of the antibody.

Type: Article
Title: Molecular cloning and expression of the fabs of human autoantibodies in Escherichia coli - Determination of the heavy or light chain contribution to the anti-DNA/-cardiolipin activity of the Fab
Open access status: An open access publication
Publisher version: http://www.jbc.org/content/early/recent/0
Keywords: SYSTEMIC LUPUS-ERYTHEMATOSUS, V-LAMBDA REPERTOIRE, MONOCLONAL-ANTIBODY, BINDING, SPECIFICITY, NEPHRITIS, CDR3, REACTIVITY, DIVERSITY, PROFILES
UCL classification: UCL > Provost and Vice Provost Offices
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Medical Sciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Medical Sciences > Div of Medicine
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Medical Sciences > Div of Medicine > Inflammation
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Pop Health Sciences > UCL GOS Institute of Child Health
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Pop Health Sciences > UCL GOS Institute of Child Health > ICH - Directors Office
URI: http://discovery.ucl.ac.uk/id/eprint/130952
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