Studies with radioiodinated cold agglutinins of ten patients

Chronic Hemolytic Anemia due to Cold Agglutinins: the Mechanism of Resistance of Red Cells to C′ Hemolysis by Cold Agglutinins*

“…The methods used for observing C' hemolysis by cold agglutinin and for preparing and employing radioiodinated cold agglutinin have been previously described (2).…”

“…Additional antisera with activity for flue-or flu-globulins prepared by injection of these individual fractions of fl-globulins into rabbits were available and confirmed the activity of the anti-fl-globulin serum described above. 2 Preparations of 'I-labeled anti-fl-globulin serum. The method of Horejsi and Smetana (11) was used to frac- 1 Antipolio immune globulin was used.…”

“…The method of Horejsi and Smetana (11) was used to frac- 1 Antipolio immune globulin was used. 2 Antisera for fle-and fl,.-globulins were obtained Through the courtesy of Dr. John Boyer, Cleveland. tionate the rabbit anti-,f-globulin serum. The gamma globulin so obtained was dialyzed against 0.15 M NaCl and 0.20 M borate buffer, pH 8.0, overnight at 50C.…”

“…From the 131I activity of the original preparation of tagged protein using 1.0 x 106 as the molecular weight of the antibody, we calculated that 40,000 molecules of cold agglutinin per cell were present when 2.5%o hemolysis of the control cells occurred, whereas 112,000 molecules were present when 2.5% hemolysis of the cells previously coated with C' globulin occurred (2). These ,data indicate that several times the concentration of cold agglutinin must be adsorbed to the surface of red cells coated with C' globulins compared to uncoated cells to produce equal hemolysis.…”

“…It must be added that the concentration of globulin in the two systems was probably not comparable, since it has been computed that there are approximately 500,000 antigen sites for the cold agglutinin and less than one-tenth that number for anti-D antibody (2,27). Probably many more molecules of C' globulin can be attached to red cells by cold agglutinin than the number of yG molecules represented by maximum concentration of D antibody.…”