Rh Antigen and Hapten. Nature of Antigen and Its Isolation from Erythrocyte Stroma

Abstract

The Rh factor in erythrocyte stroma can be demonstrated by serum inhibition technique. The stroma was prepared from human red cells after centrifugation and removal of the plasma. Leucocyte layer cells from 10 to 100 bleedings were pooled and hemolyzed by diluting with 10 vol. of dist. water at 5[degree]C. The soln. was fed through a Sharpies strpercentrifuge and the sediment stored at 2oC at pH 7 to 8 or dried by lyophylization. Suspensions of dried material were made in 10% conc. in isotonic saline or phosphate buffer pH 7.7, ionic strength 0.2. Serial dilutions of 10% suspensions were incubated at 37[degree] for 1/2 hr. with an equal amt. of constant strength (1-20 dilution) of an anti Rh0 serum (titer 1-500). The supernatant was theh incubated for 1/2 hr. with an equal amt. of a 2% suspension of Rh + cells. The tubes were centrifuged for 1 min. at 800 r.p.m. and agglutination was detd. by microscopic observation. The titer of the stroma was read as the highest dilution of the 10% suspension which inhibited the constant strength of the anti Rh0 serum. Stroma prepared from Rh + cells exhibited inhibition titers of 1-4 to 1-16 while stroma From Rh[long dash]cells showed no inhibition. The Rh factor in stroma is destroyed by incubation at 56[degree] for 5 min. Destruction took place in moisture in the absence of O2. The post hemblytic residue (stroma) was separated into 2 fractions: elinin, which was sedimented at pH 7 to 8, and stromatin, which precipitates at pH 5.5 to 6. Elihin, which comprised about 35% of stroma, is 40-50% lipids extractable with 3:1 alcohol ether and has N content of 9.1% and P, 1%. Stromatin is 12% N and 0.42% P. The samples so far prepd. of these 2 fractions are not yet to be considered free of each other and perhaps not homogeneous within themselves. Only elinin contained the factor inhibiting anti-Rh serum and had 5 times the amt. of A and B factor as compared to stromatin. Ether-soluble material inhibiting the anti-Rh serum in a dilution of 1-128 has been extracted from elinin. The ether-soluble factor is not inactivated by exposure to 56[degree] for 60 min.