The red cells for the Rh antibody tests were used within 3 days after drawing except for the ** f cells, which had been glycerolized and stored at -- 20 ` C for approximately 1 year. These cells were thawed at 37 ` C for 30 min and were deglycerolized by alternately centrifuging and mixing with descending concentrations of glycerol solutions (20, 18, 10, 8, 4 and 2%). The cells were then washed three times with saline and resuspended to 2% in saline.

Blood samples were allowed to clot at room temperature for 3 hr, centrifuged and the serum was removed. The serum was measured volumetrically and subsequently dialyzed in the cold for at least 24 hr against three to four changes, approximately 750 ml each, of ``starting buffer.'' This buffer, pH 8.6, was 0.005 M in ** f and 0.039 M in tris (hydroxymethyl) -- aminomethane (Tris). After dialysis the sample was centrifuged and the supernatant placed on a ** f cm column of DEAE-cellulose equilibrated with starting buffer. The DEAE-cellulose, containing 0.78 mEq of N/g, was prepared in our laboratory by the method of Peterson and Sober (7) from powdered cellulose, 100 -- 230 mesh. The small amount of insoluble material which precipitated during dialysis was suspended in approximately 5 ml of starting buffer, centrifuged, resuspended in 2.5 ml of isotonic saline and tested for antibody activity.

The chromatography was done at 6 ` C using gradient elution, essentially according to Sober and Peterson. The deep concave gradient employed (fig. 2) was obtained with a nine chambered gradient elution device (``Varigrad,'' reference (8)) and has been described elsewhere. the other, a shallow concave gradient (Fig. 1), was produced with a so-called ``cone sphere'' apparatus, the ``cone'' being a 2 -- liter Erlenmeyer flask and the ``sphere,'' a 2 -- liter round-bottom flask. Each initially contained 1700 ml of buffer; in the sphere was starting buffer and in the cone was final buffer, 0.50 M in both ** f and Tris, pH 4.1.