Elution purification and partial characterization of immunoglobulin-G bound to normal and pathological human placentae

Abstract

The unexpected failure of the mother to immunologically reject the foetus is partly thought to result from immunological properties of the placentae and other regulatory factors, which include immunoglobulin G (IgG). These antibodies exist on placentae as symmetric and asymmetric forms with the latter having only one antigen binding arm, cannot precipitate antigen hence incapable of effector functions. The aim of this study was to elute, purify, partially characterize and estimate the relative amounts of symmetric and asymmetric IgG molecules bound to normal and pathological placental trophoblast. Term placentae were obtained from delivery suite of Bielefeld Institute of Reproductive Medicine Germany and antibodies eluted with 0.5M Glycine buffer pH 2.5. These antibodies were purified by gel filtration on sephadex G200 and subsequently by affinity chromatography on Protein A sepharose column. Total IgG bound to placentae was estimated by Lowry et al. (1951) protein determination method while relative amounts of symmetric and asymmetric IgG estimated by further purification of Protein A eluates on Con-A sepharose column Re-association of eluate antibodies with isologous and third party placental microvesicle antigens was analyzed by Enzyme Linked Irnmunosorbent ,: Assay (ELISA). There was a wide variation in total IgG bound to normal term placentae ranging from as low as 60 to as high as 580IJ.g while calcified placentae contained between 20-1801J.g of IgG. Analysis of eluate by SDS-P AGE---Showed distinctively the heavy (55kDa) and light (22kDa) chains of IgG suggestigg that placental IgG has a relative molecular weight of 154kDa. Similar observations were made following gel filtration on "sephadex G-200 column. Total asymmetric IgG averaged 60% in normal placentae and 40% in pathological placentae and the reverse was true of symmetric IgG molecules. Calcification of placentae was associated with a lowplacental (mean 505g) and baby weight (mean 2980g). as compared to normal placental (mean 710g) and baby (mean 3400g) weights. Analysis by SDS-PAGE showed that asymmetric IgG had a higher concentration of light chain antibodies (26 kDa LCAb) while symmetric IgG had high concentration of heavy chain antibodies (53kDa HCAb). Placental IgG reassociated strongly with isologous and third party acidified placental microvesicles antigen suggesting that this antigen IS commonly expressed on human placentae. Trypsin digestion of placental microvesicles for 5, 20 and 45 minutes respectively led to the release of two peptides of relative molecular weight (23kDa and 53 kDa) with the latter resistant to further digestion.