SummaryWe have produced a panel of monoclonal antibodies (mAbs) against rabbit platelet factor 4 (PF4). Two of these mAbs have been characterized in this study. In particular the antibody called 10B2, which also recognizes the human molecule, is able to block PF4’s ability to neutralize heparin in a modified Heparin-Factor Xa chromogenic assay. The inhibition appears to be more than 95% at 1:1 mAb/PF4 molar ratio both for purified rabbit and human PF4. Similar results were obtained using supernatants from stimulated human platelets (90% of inhibition at 1:1 mAb/ PF4 molar ratio) or using Fab fragments from 10B2. Studies to determine the antigenic determinant against which 10B2 is directed, show that this is an assembled epitope which involves disulfide bonds of the PF4.

No abstract

Human lymphocyte CD4 becomes undetectable in the presence of exogenous gangliosides (CD4 masking), as originally described by Offner et al. (J. Immunol. 1987. 139: 3295). CD4 masking is apparently due to in situ rearrangement of the glycoprotein; since no direct binding of ganglioside to CD4 could be demonstrated, it was suggested that the effect could be mediated by interactions with other, as yet unidentified, surface structures. To gain insight into the structural requirements of the interaction(s) that leads to CD4 masking, we assayed the effects of a battery of gangliosides and of ganglioside derivatives on (a) CD4 masking; (b) cholera toxin binding (as a well known ganglioside-protein interaction) and (c) inhibition of lymphocyte mitogenic proliferation (as a second ganglioside interaction with a lymphocyte surface target). Our results indicate that the three interactions are distinctly different, since ganglioside chemical groups which are essential for one of the interactions are irrelevant for the others, and lead to the conclusion that gangliosides can interact with lymphocyte surface targets in a number of ways, causing a number of independent biological effects.

Ten mouse hybridoma lines producing monoclonal antibodies (Mabs) against recombinant human ciliary neurotrophic factor (rhCNTF) have been obtained. Two monoclonal antibodies belonging to the IgG1 class were selected and characterized. Their specificity was established by ELISA and Western blotting. Epitopes recognized by the two Mabs were investigated with ELISA and Western blotting by using rhCNTF mutants, rhCNTF fragments and synthetic peptides mimicking different portions of the CNTF molecule. The carboxy-terminal part of the CNTF and particularly the sequence between aa 150 and 159 appeared to constitute the immunodominant group. The fact that certain amino acid sequences of CNTF are conserved among species was utilized to examine the crossreactivity patterns of the two Mabs with rat sciatic nerve CNTF by Western blotting and immunohistochemistry. These antibodies will be useful for studying the distribution of CNTF in the nervous system and in developing an enzyme-linked immunosorbent assay for the quantitative determinations of CNTF in various neuropathologies.

Ten hybridomas producing monoclonal antibodies (Mabs) against rabbit platelet factor 4 (PF4) were obtained from the fusion of splenocytes from mice immunized with purified rabbit PF4 and NSO mouse myeloma cells. When the reactivities of these monoclonal antibodies were determined by enzyme-linked immunosorbent assay and immunoblotting with human and rabbit PF4, they showed a high degree of specificity. Only one Mab recognized an epitope common to the human and rabbit molecules, the other nine reacted only with the rabbit protein. All the antibodies recognized, in crude platelet lysates, a band that comigrates with the purified PF4 protein. None of these antibodies cross-reacted with major rabbit or human platelet-poor plasma proteins. The significance of the Mabs in immunological and physiological studies is discussed.