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.
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.
Kumar
S
S
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
Kalsi
J
J
Ravirajan
C T
CT
Rahman
A
A
Athwal
D
D
Latchman
D S
DS
Isenberg
D A
DA
Pearl
L H
LH
D000818:Animals; D001323:Autoantibodies / Q000235:genetics* / Q000378:metabolism*; D015153:Blotting, Western; D002308:Cardiolipins / Q000276:immunology / Q000378:metabolism; D002417:Cattle; D002845:Chromatography; D003001:Cloning, Molecular; D004247:DNA / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000276:immunology / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D007140:Immunoglobulin Fab Fragments / Q000235:genetics* / Q000276:immunology / Q000378:metabolism*; D007074:Immunoglobulin G / Q000276:immunology / Q000378:metabolism; D007143:Immunoglobulin Heavy Chains / Q000235:genetics; D007147:Immunoglobulin Light Chains / Q000235:genetics; D010957:Plasmids / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000276:immunology / Q000378:metabolism; D013950:Thymus Gland / Q000378:metabolism
D001323:Autoantibodies; D002308:Cardiolipins; D007140:Immunoglobulin Fab Fragments; D007074:Immunoglobulin G; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D011994:Recombinant Proteins; D004247:DNA