Comparison of Androstenedione-3-Oxime-BSA Antibody with Androstenedione-6-Carboxymethylthioether-BSA Antibody for Radioimmunoassay of Androstenedione

Two different antisera by androst-4-ene-3, 17-dione (Δ4-A-dione) -3-oxime-BSA and Δ4-A-dione-6-Carboxymethylthioether (CMTE) -BSA were made. The specificity and affinity of these antisera were satisfactory.Δ4-A-dione-3-oxime and Δ4-A-dione-6-CMTE were synthesized by the method in current use. Δ4-A-dione-3-oxime-BSA and Δ4-A-dione-6-CMTE-BSA were prepared by the mix-anhydride method.Throughout two or three months several rabbits were immuninized with these different antigens. Both antisera showed high affinity and specificity for Δ4-A-dione.When considered from these steroid radicals and side chains of Δ4-A-dione-3-oxime and Δ4-A-dione-6-CMTE, the specificity of these two antibodies showed different results.Antiserum produced from the latter showed a higher specificity than that produced fromthe fo rmer against other steroids because the latter reserved 3-and 17keto radicals without destruction. From these results it seemed that the preservation of 3- or 17-keto radicals was important in preparing the antigen ofΔ4-A-dione-3-oxime antiserum.Both antisera had no great affinity with 3- or 17-α hydroxy steroids. Whether the structure of the A ring is 4-ene or 4-ane and A/B configuration is 'trans' or 'cis' was not an important factor on the affinity of steroid with these antisera.For the assay, 20,000 diluted antiserum ofΔ4-A-dione-6-CMTE-BSA was used. 0.2 ml of plasma was diluted with 0.2 ml of distilled water. For recovery calculation 1,000 dpm of tritiated Δ4-A-dione was subsequentry added. This sample was extracted by 1.5 ml of dichloromethane twice. The dried sample was applied to the microcolumn of a Sephadex LH-20 and eluted by hexan benzene methanol (85 : 10 : 5).One third of this elute was used for the measuring of recovery. 10,000 dpm of tritiated Δ4-A-dione was added to the remainder and it was used for radioimmunoassay.The accuracy and precision of this method were acceptable. Blank value, recovery, and normal range of male plasma were within the limits of admission.