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Experimental and results

The insoluble material which precipitated during dialysis against starting buffer always showed intense agglutinin activity, regardless of the blood group of the donor.

With either of the gradients described, chromatography on DEAE-cellulose separated agglutinins of the ABO series into at least three regions ( Figs. 1 and 2 ): one of extremely low anionic binding capacity, one of low anionic binding capacity and one of high anionic binding capacity.

These have been labeled Regions 1, 2, and 4, respectively, in Fig. 1.

When the early part of the gradient was flattened, either by using the gradient shown in Fig. 2 or by allowing the `` cone-sphere '' gradient to become established more slowly, Region 2 activity could sometimes be separated into two areas ( donors P. J. and R. S., Fig. 1 and E. M., Fig. 2 ).

The latter procedure gave rise to a small active protein peak ( Region 1a ) between Regions 1 and 2.

In 2 of 15 experiments on whole serum a region of agglutinin activity with intermediate anionic binding capacity was detected ( Region 3, Fig. 1 ).

Moreover, after concentration using negative pressure dialysis, agglutinin activity could sometimes be detected in the region designated 2a ( donors P. J., D. A., and J. F., Fig. 1 ).