Antibodies to 1,2-naphthoquinone.

SUMMARY We have prepared an antibody against 1,2-naphthoquinone. This was accomplished by conjugating the quinone to bovine gamma-globulin. The conjugation product showed a visible light absorption peak of 445 m/u. The globulin-quinone antigen was used to produce rabbit antibodies shown to be specific for 1,2-naphthoquinone. The antibodies could be demonstrated by tanned cell hemagglutination and by passive cutaneous anaphylaxis. Quinone conjugated to human serum served as antigen in the former test. In passive cutaneous anaphylaxis, the quinone itself provoked the reac tion. However, experimental evidence suggests an in vivoconjugation of the compound with guinea pig serum. The extent of cross-reaction with related naphthoquinone de rivatives could be determined quantitatively by inhibition of hemagglutination. The possible value of this type of serological reagent for studying the process of carcino-genesis is discussed. Landsteiner established the fact that small molecules could act as immunizing antigens when chemically coupled to protein molecules (6). He demonstrated the union of antibodies against such antigens with the low molecular weight haptens by means of precipitation or inhibition tests. We be came interested in the production of antibodies against 1,2-naphthoquinone. This compound is of importance because of its possible implication in the carcinogenic action of beta-naphthylamine. It has the further property of conjugating spontaneously to proteins without diazotization or other chemical manipulations. Naphthoquinone was conjugated to bovine proteins and used for injection of rab bits. The specificity of the antibody so produced was determined. Technics for the production and study of this antibody are presented in this paper. MATERIALS AND METHODS Solutions of haptens and their quantitation.†" Stock solutions of chemical compounds were pre pared in isotonic saline solution. Adjustments in pH to physiological ranges for use in immunologi-cai procedures were made with l N NaOH and HC1. Concentrations were determined in cases of soluble material by weight of dry chemical before preparing the solution. A different procedure was employed for 1,2-naphthoquinone, which is only slightly soluble in water; a solution was prepared from the dry powder furnished by the National Aniline Com pany. The compound was placed in phosphate-buffered saline, pH 7.7.1 One part of powder by volume was agitated with 3 parts of buffer over night at 37° C. Solutions in the range of 200 mg. per cent quinone were obtained after insoluble ma terial was removed by centrifugation at low speed. Absorbencies at 445 m/i were measured on the Beckman Model DU Spectrophotometer. The …