1. Antibodies prepared in the rabbit to penicilloyl-rabbit serum albumin or to penicilloyl-bovine serum albumin were demonstrated by immunodiffusion and absorption methods to be apparently directed exclusively against the penicilloyl moiety. The antibodies could precipitate heavily substituted penicilloyl polylysine, as well as each other, with "reactions of identity". All could mutually deplete detectable antibodies by cross-absorption.

2. Small haptens containing both fluorescein and the penicilloyl group were synthesized. They were also capable of completely absorbing the antipenicilloyl antibodies from rabbit antiserum, as evidenced by immunodiffusion tests. The haptens were used in fluorescence polarization tests with gamma globulin from normal and immunized rabbits, and from normal and allergic humans. The rabbit antipenicilloyl gamma globulins in concentrations as low as 5 to 10 µg of protein/ml could significantly increase the polarization of the haptens. Normal gamma globulin had no effect at the highest concentrations tested, 1200 µg/ml. In all tests, rabbit and human antibody reacted similarly.

3. Fluorescence polarization titration curves for both human and rabbit antipenicilloyl gamma globulins were analyzed by computer and the antibody concentrations, avidities, and heterogeneity constants were determined. For the human antibody, the latter two values were 3.0 x 107 M–1 and 0.78, while for the rabbit, they were 8.7 x 106 M–1 and 0.71. The data were employed to estimate the limit of sensitivity of fluorescence polarization for detecting antipenicilloyl antibodies. Under the conditions employed, this value was roughly 0.4 µg antibody/ml.

4. When the whole rabbit sera were tested for penicilloyl antibodies by fluorescence polarization, both normal (preimmune) and immune sera revealed striking and equivalent increases in polarization with the penicilloyl haptens. This non-specific binding was shown to be due at least in part to serum albumin. Indications were obtained that it might be significantly reduced by increasing the pH or the salt concentration of the medium, or by addition of certain anions.

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