Purification and immunochemical characterization of a natural human polyreactive monoclonal IgM antibody

Roggenbuck, Dirk; Marx, Uwe; Kießig, S.T.; Schoenherr, G; Jahn, S.; Porstmann, T

doi:10.1016/0022-1759(94)90089-2

Cited by 27 publications

(5 citation statements)

References 22 publications

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“…In contrast to the complete CB03 antibody, the kinetic constants were nearly the same for both Fab and scFv regardless whether they or their antigens, K casein and human myoglobin, were insolubilized. The high dissociation rates of complexes formed by Fab or scFv could explain difficulties in determining binding constants by ELISA [10,24].…”

Section: Discussionmentioning

confidence: 99%

“…The CB03 antibody was purified from cell culture supernatant obtained by fermentation of the CB03 hybridoma in a hollow-fibre bioreactor. The method described in detail [10] comprises hydrophobic interaction chromatography (HIC) and gel filtration. HIC was performed on Phenyl-Superose HR 10/10 using FPLC equipment (Pharmacia) and gel filtration on a Superose 12 column (Pharmacia).…”

Section: Methodsmentioning

confidence: 99%

“…This antibody is encoded by a V}i\.l gene, which only differs by two mutations in the CDRs (complementarity determining region) from its germline gene equivalent [9]. The purified complete antibody as well as its Fab reacted in solid-phase ELISAs with various endogenous and exogenous antigens [10]. Single-chain fragments (scFv) of this antibody were produced by genetic engineering and purified by affinity chromatography [Niemann et al, unpublished observations], in contrast to the complete antibody, no binding constants for the Fab and the scFv could be derived from ELISA experiments as described by Friguet et al [11] although the reaction pattern of the antibody fragments with different antigens was similar to that of the complete antibody.…”

Section: Introductionmentioning

confidence: 99%

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Real‐Time Biospecific Interaction Analysis of a Natural Human Polyreactive Monoclonal IgM Antibody and its Fab and scFv Fragments with Several Antigens

Roggenbuck

König²,

Niemann

et al. 1994

Scand J Immunol

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Surface plasmon resonance (SPR) was used to investigate the kinetics of interactions between the human monoclonal polyreactive IgM antibody CB03, its Fab as well as its single-chain variable fragment (scFv) and different antigens. From these experiments apparent binding constants were determined and compared with binding constants obtained by ELISA experiments. In SPR studies with the complete antibody, the polyreactivity of the CB03 antibody as derived from ELISA experiments was confirmed. Interaction of scFv with kappa casein and human myoglobin is strong evidence for the location of polyreactivity within the variable domains of the antibody. Apparent binding constants of the complete antibody to immobilized kappa casein (9.2 x 10(7) M-1) and to human myoglobin (1.6 x 10(7) M-1) are up to 83 times higher than those of Fab. The binding constants of the scFv to the above mentioned antigens are again about 10 times lower when compared with Fab, which is mainly due to the lower association rates of the complexes formed by the scFv.

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Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Real‐Time Biospecific Interaction Analysis of a Natural Human Polyreactive Monoclonal IgM Antibody and its Fab and scFv Fragments with Several Antigens

Roggenbuck

König²,

Niemann

et al. 1994

Scand J Immunol

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“…The hybridoma cells produce a polyspecific IgMA antibody binding to multiple antigens from both the external and the internal environments [15]. The CB03 cell line has been grown up in a hollow fibre fermentation system and the human monoclonal IgM antibody purified [21].…”

Section: Antibodies Ami Celt Linesmentioning

confidence: 99%

“…The binding of both the complete CB03 and the expressed antibody fragments to ssDNA (phage DNA), dsDNA (pUCl8 plasmid DNA) and to tetanus toxin was tested using established ELISA methods [21]. Complete antibodies and expressed c-myc fragments were incubated on antigen-coated plates for 4h at room temperature.…”

Section: Binding Specificity Of Complete Antibodies and Scfv Fragmentsmentioning

confidence: 99%

Tumour cell binding by a human monoclonal IgM antibody from the spleen of a non-tumour-associated patient is due to somatic mutations in the VH gene

Bohn

Niemann

Roggenbuck

et al. 1995

Clinical and Experimental Immunology

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SUMMARYRecently we described the occurrence of U cells producing polyspecific natural IgM wilh anlitumour specificity in lhe spleen of non-tumour-bearing individuals as well as in fetal organisms, immunoprecipitation and 2-D electrophoresis showed the binding of such antibodies to a 55-kD (pi 60) membrane surface glycoprotein. In vitro cultivation of human cancer cell lines in the presence of the purilicd IgM antibodies resulted in growth inhibition and complement-mediated cell lysis. Furthermore, the antibodies were shown to be able to induce MHC class I molecule expression on tumour cells. Because of this, a role for naturally occurring antibodies with antitumour specificity in preventing neoplasias had been suggested. We have constructed and expressed in Escherichia coli single-chain fragments (scFv: V|^-linker-Vi) derived from a polyspecific human monoclonal IgM autoantibody produced by a human x mouse heterohybridoma which was obtained from the spleen of an autoimmune patient. The mutated complementarity determining region (CDR) gene segments were replaced by the equivalent germ-line sequences and the CDR3 region was swapped for that from another potyspecific human natural antibody with no binding to tumours. Using these four scFv constructs for binding analyses and in vitro cultivation experiments we found: (i) scFv containing the mutated VH region of the original antibody were able to bind to tumour cells, to induce MHC class I molecule expression, and to inhibit tumour growth in a way similar to what had been described for the complete antibody; (ii) replacement of the mutated by the germ-line V,, gene independently of the CDR3 to which it had been recombined, resulted in failure to bind to tumour cells. Nevertheless, other antigens (ssDNA, tetanus toxin) were still recognized, although with lower affinity. We discuss the significance of the replacement mutations in the VH gene CDRs. selected probably by B cell contact to an (auto)antigen. for generating a tumour binding capacity, not encoded by the germ-line gene.

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Purification of Antibodies Other Than IgG: The Case of IgM and IgA

Cabanne¹,

Santarelli²

2008

Process Scale Purification of Antibodies

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Purification and immunochemical characterization of a natural human polyreactive monoclonal IgM antibody

Cited by 27 publications

References 22 publications

Real‐Time Biospecific Interaction Analysis of a Natural Human Polyreactive Monoclonal IgM Antibody and its Fab and scFv Fragments with Several Antigens

Real‐Time Biospecific Interaction Analysis of a Natural Human Polyreactive Monoclonal IgM Antibody and its Fab and scFv Fragments with Several Antigens

Tumour cell binding by a human monoclonal IgM antibody from the spleen of a non-tumour-associated patient is due to somatic mutations in the VH gene

Purification of Antibodies Other Than IgG: The Case of IgM and IgA

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