Establishment of the functional importance of thiamin carrier protein in pregnant rats by using monoclonal antibodies.

Monoclonal antibodies (mAbs) to chicken thiamin carrier protein (TCP) have been produced by hybridoma technology to identify the crucial epitopes involved in bioneutralization of the vitamin carrier. The monoclonality of these mAbs (A4C4, F3H6, H8H3, C8C1 and G7H10) was sought to be confirmed by sub-class isotyping; they all belong to IgG1, k type. The epitopes recognized by all the five mAbs are conserved in TCP from the chicken to the rat as assessed by liquid phase RIA and immunoprecipitation of 125I-labelled proteins from pregnant rat serum. Among these mAbs. passive immunization of pregnant rats with the mAb C8C1 only on three consecutive days (day 10, 11 and 12) resulted in embryonic resorption. These results demonstrate the importance of epitopic structure specified by the mAb C8C1 on TCP during pregnancy in rats.

Immunohistochemical localization of riboflavin carrier protein in testicular cells of mammals.

Using specific polyclonal antibodies against chicken riboflavin carrier protein (cRCP), immunocytochemical localization of riboflavin carrier protein was carried out in testicular sections and isolated cells of mammals. A positive reaction was observed in the developing germ cells of rat testis, especially in meiotic and post-meiotic germ cells such as pachytene spermatocytes, round spermatids and spermatozoa. In addition both the somatic cells of the testis, viz. Leydig and Sertoli cells with vital function in germ cell proliferation and differentiation, displayed a moderate to strong staining reaction. This was further confirmed using in utero X-irradiated rat testis devoid of germ cells. Different types of cells isolated from testis when subjected to immunostaining showed similar patterns of reaction as in the intact tissue. Mature spermatozoa from different mammals (rat, bull and monkey) exhibited strong staining reaction in their head regions localized mainly in acrosomal caps. It is suggested that the testicular riboflavin carrier protein has a role in cell to cell communication and may be crucial during development of germ cells especially at the meiotic and post-meiotic stages.

Characterization of poly- and monoclonal antibodies to physalis mottle (tymo) virus.

Polyclonal antibodies were raised against the Physalis mottle virus (PhMV) and its denatured coat protein (PhMV-P). Analysis of the reactivity of the polyclonal antibodies with tryptic peptides of PhMV-P in dot-blot assays revealed that many of the epitopes were common to intact virus and denatured coat protein. Five monoclonal antibodies to the intact virus were obtained using hybridoma technology. These monoclonal antibodies reacted well with the denatured coat protein. Epitope analysis suggested that probably these monoclonal antibodies recognize overlapping epitopes. This was substantiated by epitope mapping using the CNBr digest of PhMV-P in western blots. All the five monoclonals recognized the N-terminal 15 K fragment. Attempts to further delineate the specific region recognized by the monoclonals by various enzymatic cleavages resulted in the loss of reactivity in all the cases. The results indicate that these monoclonals probably recognize epitopes within the N-terminal 15 K fragment of the coat protein.

Early pregnancy termination in rats immunized with denatured chicken riboflavin carrier protein.

Immunoneutralization of the maternal riboflavin carrier protein in the pregnant rat with antibodies to chicken egg vitamin carrier has earlier been shown to terminate their pregnancies. In order to understand the nature of the epitopic conformations capable of eliciting antibodies bioneutralizing the endogenous riboflavin carrier protein in the pregnant rat, we compared pregnancy progression in the fertile rodents following active immunization with either the native, SDS-denatured, reduced-carboxymethylated or SDS-treated reduced carboxymethylated avian egg white riboflavin carrier protein. The data revealed that despite the total antibody titers being higher in the animals immunized with the native protein, the antibodies elicited against the denatured avian vitamin carrier exhibited relatively better potencies to bioneutralize the endogenous maternal protein as evidenced by higher rates of early fetal resorption.