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Publication Detail
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
  • Publication Type:
    Journal article
  • Publication Sub Type:
  • Authors:
    Kumar S, Kalsi J, Ravirajan CT, Rahman A, Athwal D, Latchman DS, Isenberg DA, Pearl LH
  • Publication date:
  • Pagination:
    35129, 35136
  • Journal:
    Journal of Biological Chemistry
  • Volume:
  • Issue:
  • Print ISSN:
  • Keywords:
    Autoantibodies, cloning, Escherichia coli, expression, LIGHT, MB, Molecular, molecular cloning, analysis, Animal, antibodies, Antibody, ANTIGEN, ASSAY, Autoantibodies, BINDING, Biochemistry, biology, Blotting, Western, Cardiolipins, Cattle, Chromatography, Cloning, Molecular, culture, DNA, Electrophoresis, Polyacrylamide Gel, enzyme, enzyme linked immunosorbent assay, Enzyme-Linked Immunosorbent Assay, Form, functional, GEL ELECTROPHORESIS, genetics, Hiv, IgG, Immunoblotting, IMMUNOGLOBULIN, Immunoglobulins, Fab, Heavy-Chain, Light-Chain, immunology, LEVEL, medicine, metabolism, MG, Molecular Biology, optimum, plasmid, Plasmids, Polymerase Chain Reaction, PROTEIN, Protein Expression, Proteins, recombinant, Recombinant Proteins, Rheumatology, sensitive, size, STATE, STATES, Thymus Gland, UK, United States, western blot
  • Addresses:
    Centre for Rheumatology, Bloomsbury Rheumatology Unit, the Department of Medicine and the Department of Biochemistry and Molecular Biology, University College London, London W1P 9PG, UK. sanjeev@icr.ac.uk
  • Notes:
    UI - 20519604 LA - eng RN - 0 (Autoantibodies) RN - 0 (Cardiolipins) RN - 0 (IgG) RN - 0 (Immunoglobulins, Fab) RN - 0 (Immunoglobulins, Heavy-Chain) RN - 0 (Immunoglobulins, Light-Chain) RN - 0 (Plasmids) RN - 0 (Recombinant Proteins) RN - 9007-49-2 (DNA) PT - Journal Article DA - 20001127 IS - 0021-9258 SB - IM CY - UNITED STATES JC - HIV
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 Fab 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
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