Hemagglutination tests with protease- and neuraminidase(RDE)-treated red cells are essential to demonstrate anti-Pr cold agglutinins, since RDE and proteases inactivate the Pr antigens in contrast to the I/i antigens. RDE treatment of red cells,however, reveals the T antigen which reacts with anti-T present in anti-Pr sera. Furthermore,anti-I, also present in anti-Pr sera, shows increased reactions with RDE- and proteasetreated red cells. Therefore, T-anti-T and I-anti-I reactions mask the loss of anti-Pr activity against RDE- and protease-treated red cells when low-titer anti-Pr sera are studied. Absorption of anti-Pr sera with RDE-treated red cells removes anti-T and anti-I, leaving anti-Pr cold agglutinins which only react with untreated red cells, not with RDE- or protease-treated red cells. Anti-I contaminating anti-Pr in warm eluates from untreated red cells or stroma can be also removed by absorption with enzyme-treated red cells. By eliminating T-anti-T interference from sera, it was possible to show that all Pr determinants known are determined by N-acetylneuraminic acid.

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