Human monoclonal antibody derived from an autoimmune thrombocytopenic purpura patient, recognizing an intermediate filament's determinant common to vimentin and desmin.

Human monoclonal antibody (mAb) technology has been helpful in identifying autoantibodies that are involved in various autoimmune disorders. We report here the results of such an attempt to immortalize antibody-forming cells from spleen of an autoimmune thrombocytopenic purpura (ATP) patient and characterize the resulting mAb. The human mAb we derived, denoted (4G9), binds to the cytoskeletal network. Using immunofluorescence analyses of permeabilized and fixed cell lines and tissues, the 4G9 mAb was shown to be anti-vimentin specific by virtue of its intracellular staining pattern, decoration of cell lines of mesenchymal (but not epithelial) origin, and by the fact that polyclonal anti-vimentin (and not anti-actin, prekeratin, tubulin or vinculin) antibodies inhibited its binding to intermediate filaments of a fibroblastoid cell line. The presence of vimentin in platelets was also confirmed in the present study by immunoblotting of platelet extract using murine anti-vimentin mAb. Interestingly, in addition to vimentin the 4G9 mAb decorated intermediate filaments in desmin-expressing muscular cells, suggesting that the 4G9 epitope is most likely located within the homologous sequences that are known to be shared between vimentin and desmin.

Adjuvant effect of a peptidoglycan attached covalently to a synthetic antigen provoking anti-phage antibodies.

The synthetic antigen denoted P2-A--L, comprising the fragment P2 of the coat protein of MS-2 coliphage attached to multichain poly-DL-alanine, served for the immunization of guinea-pigs. Immunization was carried out either in phosphate buffered saline (PBS) or in Freund's incomplete adjuvant (FIA) in the presence or absence of a small molecular weight peptidoglycan prepared from Bacillus megaterium, which was checked for its adjuvant effect. The various antisera were assessed by their capacity to neutralize MS-2 bacteriophage viability. When injected in PBS or FIA, P2-A--L did not elicit any measurable anti-phage activity. Addition of the peptidoglycan by simple mixing did not bring about a significant increase in antibody production. However, when the peptidoglycan was chemically linked to the P2-A--L conjugate, it had a marked adjuvant effect when the material was administered in FIA, almost identical to the extent of the effect of Freund's complete adjuvant.

Antiviral effect on MS-2 coliphage obtained with a synthetic antigen.

The coat protein of bacteriophage MS-2 was cleaved with cyanogen bromide to yield three fragments, possessing the sequence 1-88 (P1), 89-108 (P2), and 109-129 (P3), respectively. The mixture of peptides P2 and P3, which could not be separated, was found capable of inhibiting the neutralization of the phage by antiserum to the whole MS-2. The peptides corresponding to P2 and P3 were therefore synthesized. The synthetic P3 had no capacity to interfere with neutralization of MS-2, not did its macromolecular conjugate with multichain poly(DL-alanine) elicit neutralizing antibodies. On the other hand, the synthetic P2 was very efficient in inhibiting the inactivation of the phage by the antiserum against phage. Furthermore, a synthetic antigen prepared by attachment of P2 to multichain poly(alanine) incuded antiserum in rabbits that was capable of neutralizing MS-2 activity almost as efficiently as the antiserum prepared against the intact coat protein. This inactivation is specific, because it can, in turn, be totally inhibited by P2 peptide.