A "study in depth" is reported concerning the case of hemolytic disease of a group B infant born to a group O mother, whose group A twin was apparently unaffected. It was shown that the hemolytic disease of the affected infant was due to a specific anti-B antibody. The study included parallel examinations of the antibodies in the sera of the three individuals.

The specificity of the B-anti-B reaction was demonstrated in vitro and in vivo. The powerful anti-B antibody of the mother had no effect on the group A twin, in whose serum anti-B was present in large amounts.

In vitro, studied by the usual technics of cross absorption the maternal and the fetal anti-A and anti-B serum antibodies behaved as if strictly specific. By applying successive absorption, elution and neutralization techniques, however, it was possible to demonstrate additional cross-reacting antibodies in the maternal serum which could be separated from one another and from the specific anti-A and anti-B antibodies. From the erythrocytes of the normal group A twin such cross-reacting antibody could be eluted.

The cross-reacting anti-B antibody, isolated in pure form in eluates, could be shown to be loosely attached to group A erythrocytes without producing visible agglutination reactions while after elution from A cells it did visibly agglutinate group B cells. It could be eluted from A cells, absorbed by fresh A cells and reeluted while retaining its anti-B effect. It was neutralized by group B saliva only. A separate anti-A antibody with similar properties was eluted from B cells and specifically neutralized by group A saliva.

A partial affinity of these antibodies for heterologous erythrocytes but not for specific soluble substances was thus demonstrated. These findings support neither the linkage hypothesis of cross reactions between anti-A and anti-B nor the C-anti-C hypothesis of hemolytic disease. They are in keeping with the view that group O sera contain variable complexes of anti-A and anti-B antibodies, composed of multiple fractions with different partial specificities. It is suggested that the occurrence or non-occurrence of cross-reacting antibodies found in sera of group O mothers whose infants develop hemolytic disease is best explained on this basis. It is further stressed that the demonstration of an antibody in mother or child in ABO hemolytic disease does not necessarily indicate its pathologic significance.

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