The conjugation of testosterone with horseradish peroxidase and a sensitive enzyme assay for the conjugate

• Published in: Steroids Vol. 29; no. 5; p. 701
• Main Authors: Rajkowski, K M, Cittanova, N, Desfosses, B, Jayle, M F
• Format: Journal Article
• Language: English
• Published: United States 01-05-1977
• Subjects: Aminosalicylic Acids - pharmacology; Ammonia - pharmacology; Drug Stability; Enzyme Activation; Enzyme-Linked Immunosorbent Assay; Horseradish Peroxidase - metabolism; Hydrogen Peroxide - pharmacology; Hydrogen-Ion Concentration; Kinetics; Microchemistry; Peroxidases - metabolism; Protein Binding; Testosterone - analysis
• ISSN: 0039-128X
• DOI: 10.1016/0039-128X(77)90021-6

The formation of a horseradish peroxidase-testosterone conjugate for the enzyme-linked immunoassay of testosterone was investigated, using tritiated testosterone to follow the reaction. The formation of testosterone-3-(carboxymethyl) oxime-peroxidase by the mixed anhydride method was found to give a conjugate of high enzymatic activity and with three molecules of testosterone per molecule of peroxidase. The optimum conditions for the assay of peroxidase activity were studied and an assay capable of measuring 1 to 5 ng of the conjugate developed; the standard curve being virtually linear. The stability of the conjugate in solution and the effect of lyophilisation on enzymatic activity are also described. The peroxidase-testosterone conjugate was suitable for enzyme-linked immunoassay and the quantities measurable with the peroxidase assay covered the range necessary for a plasma testosterone assay. The stability of the conjugate was such that no particular precautions were necessary for its storage.