Elution And Partial Characterisation Of Immunoglobulins Bound To Goat Placenta

Abstract

The success of viviparous pregnancy in the vertebrate species remains a major puzzle to immunologists. Thus, the fetus is antigenic ally foreign by virtue of its paternally derived genes yet if is not rejected. The uncharacteristic failure of the mother to immunologically reject the fetus has been partly associated with the immunological properties of the placental trophoblast, production of blocking antibodies, down-regulation of the maternal immune system and the presence of the placental barrier among others. A complete understanding of the factors responsible for the success of the maternal-fetal relationship in human pregnancy might also carry a message, immunologically or otherwise, to the related areas of the graft-host relationship in transplantation and the tumor-host relationship in oncology. More so, the lack of an answer for immunologically compromised conditions such as recurrent spontaneous abortions highlights the need for onward research into possible ways of alleviating the psychological and economic stress among the sufferers. Although it is not clear at this time whether or not placental bound immunoglobulins may-provide ultimate treatment to immunologically compromised . pregnancies such as recurrent spontaneous abortions, characterisation of these - . antibodies and identification of the antigens to which they are bound on the placenta during pregnancy would be instrumental in advancing ways of managing

immunologically compromised pregnancies. On the other hand, their cognate antigens could be used in the development of contraceptive vaccines. This study sought to purify and partially characterise immunoglobulins bound on the goat placenta and to investigate the antibody-antigen interaction on the placenta. Goat placentae were obtained from goats kept at the Animal Physiology Department, University of Nairobi and the abattoirs within Nairobi. Placental microvesicles were prepared and antibodies eluted with 0.5M glycine buffer pH 2.5. Protein content was estimated by Bicinchoninic acid (BCA) assay. Purification was done by gel filtration on sephadex G-200, and by affinity chromatography on Protein G-sepharose column. Total IgG per placenta was determined by radial immunodiffusion (RID) while the molecular weight of eluate IgG was estimated by SDS-PAGE and gel filtration on sephadex G-200 column. Isoelectric pH (pI) of the eluate IgG was determined by isoelectric focussing on tube gels. Protein estimates showed a recovery in the range of 3-60%. This variation may be associated with differential susceptability of different placental membranes to acid digestion during the elution process. Gel filtration on sephadex G-200 showed a single protein peak. Purification on Protein-G sepharose column yielded pure IgG whose biological activity was further+confirmed by two-dimensional

immunodiffusion and RID. Total IgG obtainable from each mature placenta was estimated at between 4.231lg - i09.27l-fg. Further analysis by SDS-PAQE showed the heavy and the light chains of- placental IgG at (62.21kDa) and (45.2kDa)

respectively, while the systemic IgG was estimated at 62.2kDa (heavy chain) and 46.1kDa (light chain) respectively. These observations suggest that goat placental IgG is approximately 214.8kDa in molecular weight, which compares well with systemic IgG (216.8kDa). However placental IgG had a PI of 6.02 while the systemic IgG had a PI of 6.05. This slight difference in PI and molecular weight suggest that the functional differences between these two forms of IgG may be related to molecular organization rather than to size or primary structure. That the difference observed in pI may result from increased glycosylation in one of the two Fab arms of the placental IgG is an attractive hypothesis but this was not tested. Trypsin digestion of placental microvesicles (5,10,15,20, 30 and 45 minutes) led to the release of a 53kDa peptide similar to that reported in the human placentae, suggesting some degree of conservation in placental antigens for reproductive purposes. However, the interaction between placental IgG and its cognate antigen in placentae was not investigated due to lack of materials. This area presents an attractive focus for further investigation into the immunology of pregnancy. Based on these observations it is suggested that goat placental antigens and their antibodies. thereto may be useful as models for the study of maternal-foetal

interactions during pregnancy in human and other mammalian species.