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Expression of monovalent fragments derived from a human IgM autoantibody in E. Coli. The input of the somatically mutated CDR1/CDR2 and of the CDR3 into antigen binding specificity

Expression of monovalent fragments derived from a human IgM autoantibody in E. Coli. The input of the somatically mutated CDR1/CDR2 and of the CDR3 into antigen binding specificity
Expression of monovalent fragments derived from a human IgM autoantibody in E. Coli. The input of the somatically mutated CDR1/CDR2 and of the CDR3 into antigen binding specificity

A hybridoma producing a polyspecific human monoclonal IgM antibody (named CB03) has been derived from a fusion of mouse myeloma cells with human spleen lymphocytes obtained from an autoimmune patient suffering from chronic idiopathic thrombocytopenia. The antibody was found to be encoded by somatically mutated VHI and VlambdaIII genes. To study the input of mutated complementarity regions (CDRs) into antibody specificity, the antigen binding features of the purified complete IgM antibody were compared with (i) a Fab fragment obtained by hot tryptic digestion and (ii) recombinant monovalent fragments expressed in E. coli. In detail, vectors were constructed encoding for (i) rFab03 and single chain Fv03 fragments containing the VH and VL genes connected by a linker sequence, (ii) scFcl.l. fragments containing the VH germline equivalent and the CB03 wild-type CDR3 region, and (iii) scFv fragments containing the CDR1 and CDR2 in germline configuration and the CDR3 expressed in the CB253 human fetal B cell hybridoma producing a polyspecific IgM antibody. The expression vectors contained at the 3' end either a (His)6 motif allowing purification on Ni2+-agarose or a e-m yc tag for specifically detecting the expression products by a murine monoclonal antibody. Western blotting and ELISA analyses of the expression

antibody fragment, CDR, complementarity determining region, ELISA, enzyme-linked immunosorbent assay, Fab, Fd, IPTG, isoprophylthiocyanat-galactose, PAGE, polyacrylamid gel electrophoresis, recombinant antibody fragment, rFab, scfv, single chain antibody fragment, containing the heavy and light chain variable domains connected by a linker peptid, VHCH1 fragment
0171-2985
400-419
Jahn, Sigbert
1a4b464c-c8a2-41ba-948a-8a1d64241110
Roggenbuck, Dirk
4898f538-b01b-4b24-a97c-1bb3676520f6
Niemann, Birgit
bc530f44-e8e3-4931-9251-61e41e870f4c
Ward, E. Sally
b31c0877-8abe-485f-b800-244a9d3cd6cc
Jahn, Sigbert
1a4b464c-c8a2-41ba-948a-8a1d64241110
Roggenbuck, Dirk
4898f538-b01b-4b24-a97c-1bb3676520f6
Niemann, Birgit
bc530f44-e8e3-4931-9251-61e41e870f4c
Ward, E. Sally
b31c0877-8abe-485f-b800-244a9d3cd6cc

Jahn, Sigbert, Roggenbuck, Dirk, Niemann, Birgit and Ward, E. Sally (1995) Expression of monovalent fragments derived from a human IgM autoantibody in E. Coli. The input of the somatically mutated CDR1/CDR2 and of the CDR3 into antigen binding specificity. Immunobiology, 193 (5), 400-419. (doi:10.1016/S0171-2985(11)80427-4).

Record type: Article

Abstract

A hybridoma producing a polyspecific human monoclonal IgM antibody (named CB03) has been derived from a fusion of mouse myeloma cells with human spleen lymphocytes obtained from an autoimmune patient suffering from chronic idiopathic thrombocytopenia. The antibody was found to be encoded by somatically mutated VHI and VlambdaIII genes. To study the input of mutated complementarity regions (CDRs) into antibody specificity, the antigen binding features of the purified complete IgM antibody were compared with (i) a Fab fragment obtained by hot tryptic digestion and (ii) recombinant monovalent fragments expressed in E. coli. In detail, vectors were constructed encoding for (i) rFab03 and single chain Fv03 fragments containing the VH and VL genes connected by a linker sequence, (ii) scFcl.l. fragments containing the VH germline equivalent and the CB03 wild-type CDR3 region, and (iii) scFv fragments containing the CDR1 and CDR2 in germline configuration and the CDR3 expressed in the CB253 human fetal B cell hybridoma producing a polyspecific IgM antibody. The expression vectors contained at the 3' end either a (His)6 motif allowing purification on Ni2+-agarose or a e-m yc tag for specifically detecting the expression products by a murine monoclonal antibody. Western blotting and ELISA analyses of the expression

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More information

Published date: August 1995
Keywords: antibody fragment, CDR, complementarity determining region, ELISA, enzyme-linked immunosorbent assay, Fab, Fd, IPTG, isoprophylthiocyanat-galactose, PAGE, polyacrylamid gel electrophoresis, recombinant antibody fragment, rFab, scfv, single chain antibody fragment, containing the heavy and light chain variable domains connected by a linker peptid, VHCH1 fragment

Identifiers

Local EPrints ID: 425270
URI: http://eprints.soton.ac.uk/id/eprint/425270
ISSN: 0171-2985
PURE UUID: ae980f8a-52e3-4f81-892e-14d861bb20b7
ORCID for E. Sally Ward: ORCID iD orcid.org/0000-0003-3232-7238

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Date deposited: 12 Oct 2018 16:30
Last modified: 07 Oct 2020 02:22

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Contributors

Author: Sigbert Jahn
Author: Dirk Roggenbuck
Author: Birgit Niemann
Author: E. Sally Ward ORCID iD

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