The preparation and properties of a hapten-cellulose antibody adsorbent

The significance of erythrocyte antigen site density

“…METHODS Antisera. The immunizing antigen was prepared by coupling diazotized sulfanilic acid with edestin by methods previously described (5). Young adult-New Zealand albino rabbits were immunized by intravenous injection of 5 mg of sulfanilic acid-azo-edestin (S-Ed) three times a week for two courses of 4 wk separated by a 7 wk period.…”

“…As the purified rabbit antibodies were tested at 3.10 (IgG) and 2.55 (IgM) mg of protein/ml, significant (over 1%) contamination of either fraction would have been detected. Precipitating antibody was measured by a quantitative micro method (8) using bovine serum albumin-sulfanilic acid (S-BSA) (5). Fluorescein-labeled purified IgG anti-S was prepared by conjugating 3.1 mg of protein with 1.0 mg of fluorescein isothiocyanate for 1 hr at pH 8.0 and removing the unbound dye by Sephadex G-25 gel filtration.…”

The significance of erythrocyte antigen site density

“…METHODS Antisera. The immunizing antigen was prepared by coupling diazotized sulfanilic acid to edestin by methods previously described (4). Young adult New Zealand albino rabbits were immunized by intravenous injection of 5 mg of sulfanilic acid-azo-edestin three times a week for two courses of 4 wk separated by a 7 wk period.…”

“…sulfanilic acid-cellulose immunoadsorbent (4,6). The antibody was eluted with the specific hapten (0.1 M sulfanilic acid, and 1.0 M NaCI, pH 7.3), and dialyzed against six changes of borate-saline.…”

“…Buffer solutions. The composition of the 10% boratesaline, pH 7.85 (4), the VBS (13, alternative procedure), the hypotonic phosphate-saline EDTA (8), and the isotonic phosphate-saline (3) buffers have been previously described. Calcium and magnesium were omitted from the VBS for all studies other than immune hemolysis.…”