Several human pathologic sera containing high titered cold agglutinins were studied to determine whether the serologic activity ascribed to an "incomplete cold antibody" could be separated from the "complete" cold agglutinin activity. Separation was not achieved by physicochemical methods, including zone electrophoresis, density gradient ultracentrifugation, and anion exchange chromatography. Both activities were susceptible to destruction by mercaptans. Neither activity could be differentially absorbed from the sera. Using "Bombay" and I-negative ("i") red cells, a difference in specificity of the two activities for the H or I antigen of human erythrocytes could not be demonstrated. The simplest interpretation of these findings is that there is only one antibody involved, the cold agglutinin, and that the serologic manifestation usually attributed to an additional "incomplete cold antibody", i.e. the production of a positive antiglobulin reaction of the "non-γ-globulin" type, results from an interaction of complement components with the cold agglutinin-erythrocyte complex.
Three of these cold agglutinating sera were unreactive with I-negative erythrocytes, in keeping with the reported anti-I specificity of these antibodies. A fourth serum retained moderate, though greatly reduced, activity against these cells, and the interpretation of this finding is discussed.
The anti-H specificity of the incomplete cold antibodies in normal human sera was confirmed by their failure to sensitize "Bombay" erythrocytes. This was in sharp contrast to the excellent reactivity of the pathologic sera with these cells, demonstrating that pathologic cold agglutinins are unrelated to the incomplete cold antibodies present in most normal sera.