1. The preparation is described of a deep red protein dye, R-salt-azo-benzidine-azo-crystalline egg albumen, which contains no more than traces of protein with the original egg albumen specificity.
2. Based on previous publications of the writers, a quantitative method is given for the micro estimation of precipitin in the antisera to the dye. The method gives the actual weight of precipitin and may be applied to the determination of the maximum amount of precipitable antibody in any antiserum.
3. Data are given (1) on the influence of the period between the final injection and the bleeding on the precipitin content of rabbit antisera to the azo protein; (2) on the magnitude of the antibody response following the injection of multiple doses of the antigen varying within wide limits; (3) on the variations in the precipitin content of the sera of rabbits given successive courses of antigen injections; and (4) on the stability of antisera stored in the cold.
4. Four antisera were obtained in which over 100 times as much precipitin was recovered as the amount of antigen injected. This supplements the growing mass of evidence against the theory that specific antigen fragments are actually incorporated into the antibody molecule.