Detection, isolation and characterization of cell free HLA A and B antigens from human amniotic fluid

Cell-free HLA A and B antigens, paternal and maternal, have been detected in human amniotic fluids obtained from women at 16-18 weeks' gestation. Fractionation of amniotic fluid samples on sephadex showed that HLA A and B antigen activity was mainly in the 40,000 to 70,000 mol. wt. fractions. Lentil lectin sepharose 4B affinity chromatography of 30,000 to 70,000 sephadex molecular weight fraction of amniotic fluids isolated proteins which gave 2 bands, M and N, on discontinuous polyacrylamide gel electrophoresis (disc PAGE without SDS). SDS disc PAGE of the LcH bound glycoproteins recovered from amniotic fluids indicated 4 main protein bands approximately 12,900, 32,000, 52,600 and 78,500 mol. wt. Schiff's stain of the SDS gels showed that all the proteins except the 12,900 mol. wt. polypeptide contained carbohydrate. It was suggested that the 12,900 and 32,000 mol. wt. proteins obtained on disc PAGE gels represented the 2 subunits of the HLA A and B antigen molecule, i.e., beta 2-microglobulin and the allospecific heavy chains, similar to the papain-solubilized moiety of membrane antigens. In the absence of SDS the 2 polypeptides migrate in polyacrylamide gels as 1 protein band M, representing the intact (undissociated) antigen molecules. Enzyme linked immunosorbent assays were proposed for the estimation of cell-free HLA antigens.