Titration studies with IgG anti-A antibodies revealed similar activity at both 4 and 37 °C. The saline activity of IgG anti-A antibodies generally increased as the papain titre increased with both cord and adult A, and A2 cells. Sera with papain titres >256 had saline titres in the range 32-256 at 37 °C. Estimations of the IgG anti-A content of ‘immune’ anti-A sera made by titration following partial neutralization revealed a decreasing quantitation of the results with increasing saline IgG anti-A titre, as shown by comparison to the 2-ME method.

Using monoclonal antibodies to C3 it has been shown that the red blood cells of patients with cold haemagglutinin disease carry on their cells C3d,g (α-2D-globulin) rather than C3d. C3d,g seems to be the final product of in vivo C3 activation in fluid phase and on red cells. The cleavage of C3dg to C3d and C3g does not appear to occur in vivo either in the fluid phase or on red cell bound C3bi. In vitro C3-coated red cells prepared by antibody or low ionic strength techniques produce cells with C3d and C3bi as the predominant C3 fragment, whereas the Fruitstone technique in which coating occurs by the alternative pathway has principally C3b. The activity of C3 cleaving enzymes in whole serum is strongly influenced by the ionic conditions of the serum.