An enzyme immunoassay for serum and urinary levonorgestrel in human and non-human primates

A microtiter plate enzyme immunoassay (EIA) is reported for the measurement of levonorgestrel (LNG) in serum and urine samples of human and non-human primates, and the results are compared to data obtained by radioimmunoassay (RIA). Rabbit polyclonal antibodies were raised against the bovine serum albumin conjugate of the 3-O-carboxymethyl oxime (CMO) derivative of LNG. The enzyme label was produced by the conjugation of horseradish peroxidase to LNG at the 3-position by the same CMO bridge used for the immunogen. The assay requires 2.5 hours to perform using 2.2-azino-di-(3-ethylbenzthiazoline sulfonic acid) diammonium salt as the chromogenic substrate. Serum (100 microliters) is extracted with petroleum ether prior to assay, whereas urine samples (25 microliters) are diluted and measured directly. The sensitivity of the assay is 0.25 pg/well with a 50% displacement of label at 7.5-9.5 pg and a linear response through 250 pg/well. Minimum levels of 8.7 and 10.0 pg/ml can be detected in serum and urine samples, respectively. Changes in serum LNG concentrations were measured in women and non-human primates following LNG implantation or injection. In the non-human primate study, serum LNG concentrations began to rise rapidly following i.m. injection of LNG, with peak levels occurring on days 3 to 5, then decreasing to approximately 25-35% of peak levels for the duration of the study. Circulating concentrations of 1.86 +/- 0.18 ng/ml LNG were reached in women the first week post-insertion of Norplant implants and decreased by 50% at 7-10 days, 75% after 14-21 days, followed by a steady decrease during the next 60-70 days to constant low levels that exhibited a high individual variation. Correlation coefficients of EIA and RIA results were 0.988 for human serum, 0.926 for human urine, and 0.972 for non-human primate serum.