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.

Two IgG-type cold auto-agglutinins causing transiently occurring haemolytic anaemia in children are described. The antibodies were limited to the IgG fraction obtained by DEAE cellulose chromatography and were resistant to 2-mercaptoethanol treatment. One auto-agglutinin reacted ‘incompletely’ (in the en-zyme test), the other reacted ‘completely’ and its reactivity was furthermore abolished by treatment of red cells with proteolytic enzymes, but not with neuraminidase. Because of the inactivation of the corresponding antigen on red cells by proteolytic enzymes, this receptor belongs to the Pr complex, but it can be distinguished from Pr(1)/Pr(2), through its resistance to neuraminidase. Therefore a new symbol ‘Pr(a)’ is proposed. Based on these findings an immunochemical classification for antigens corresponding to cold auto-antibodies is given and a new nomenclature of the antigens which are inactivated by proteases is proposed depending on their determination or nondetermination by neuraminic acid.