The effect of formalin and periodic acid on the Rho (D) red cell antigen at 37°C for 30 min was studied with incomplete I131 anti-Rho (D). Pretreatment of the Rho (D) positive red cell with formalin resulted in a decrease in the amount of I131 bound to the red cell which was directly proportional to the formol concentration up to 0.66 M. Periodic acid to a concentration of 0.0008 M produced a marked decrease in the ability of red cells to bind anti-Rho (D) which was not directly proportional to the concentration of periodic acid. The antibody able to bind to the formalin-treated cells was stable to hemolysis, whereas the small quantity of antibody able to bind to the periodic acid-treated cell was bound poorly as judged by the inability to recover the cell bound I131 in the stroma.

The Rho (D) antigen exposed by papain was unaffected by pretreatment of the red cell with formalin. Periodate treatment of the intact red cell produced losses of from 20 to 90% of the Rho (D) which would normally be exposed by papain. The susceptibility of the enzyme-exposed Rho (D) antigen to inactivation with formalin was similar to that of the Rh antigen on the untreated red cell, whereas the enzyme-exposed antigen was more resistant than the Rho (D) antigen on the untreated cell to periodic acid inactivation. Quantitative absorption studies indicate that the I131 bound to the red cells following the various treatments represents specific anti-Rho (D).

The results are discussed in terms of the mechanism of action of formalin and periodic acid on the Rho (D) antigen and in terms of the nature of the Rho (D) antigen exposed by proteolysis.

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