Usha M. Joshi

A sensitive and specific enzymeimmunoassay for serum testosterone

A very sensitive enzymeimmunoassay for testosterone was developed using testosterone-penicillinase conjugate and an antibody to testosterone-3-(O-carboxymethyl)oxime-bovine serum albumin. The specificity of the assay was demonstrated by the fact that estradiol-17 beta, estrone, estriol, progesterone, 17 alpha-hydroxy-progesterone, dehydroepiandrosterone, androstenedione, cortisol and cortisone were ineffective in crossreacting with testosterone while dihydrotestosterone was 8 times less crossreactive as compared to testosterone. The minimum detectable amount of testosterone was 10-15 pg per assay tube. Intra-assay and inter-assay coefficients of variation for samples containing 0.3-6ng/ml of testosterone were 6-8% and 8-10%, respectively. A high degree of correlation (r = 0.97) was observed between serum testosterone values obtained by enzymeimmunoassay and radioimmunoassay. The levels of testosterone in the sera of normal men and women and those in hypogonadal males following stimulation with human chorionic gonadotropin determined by this enzymeimmunoassay appear similar to those reported by other investigators.

Penicillinase as a marker in enzyme-linked immunosorbent assays for steroid hormones.

Using penicillinase (E.C. 3.5.2.6) as marker and microtitre ELISA plates for immunoadsorption, sensitive and specific assays for two steroid hormones viz. testosterone in plasma and estrone-3-glucuronide in urine were developed. The entire assay can be performed in 4 h and requires simple laboratory equipment such as an incubator and a colorimeter. The enzyme labelled steroids have a shelf life of more than 3 years.

Effect of steroidal contraceptives on antibody formation in the human female

Abstract The antibody response to tetanus toxoid was studied in female subjects receiving various forms of steroidal contraceptives as compared with that in control subjects using conventional contraceptives and an IUCD. It was observed that the immune response in groups using steroidal contraceptives was considerably lower than that of the control.

Diurnal variations of serum testosterone levels in the male bonnet monkey (Macaca radiata)

Diurnal variations in serum testosterone levels were studied in six adult maleMacaca radiata. Though the testosterone levels fluctuated considerably during the 24-hr period, a distinctive diurnal pattern characterized by lower concentrations during day hours and higher concentrations during night hours was observed.

Secretion of oestrogens into saliva during pregnancy in the human.

A study was devised to determine whether large quantities of estrogens are present in saliva of pregnant women and whether fetal growth could be assessed by this parameter. Unconjugated estrogens were collected from women during late pregnancy and estimated by radioimmunoassay in serum and saliva. Both parameters showed a steady rise with advancing pregnancy and there was a correlation between estrogen levels in both serum and saliva within 1 week of delivery and birth weight. Serum estrogens and salivary estrogens show a parallel. A pregnant laboratory technician could lead to a blank of 4-20 pg in estrogen assay by contamination of a tube by 100 mgl of her saliva. Assessment of the use of salivary estimations may be useful during high risk pregnancy.

Some characteristics of the normal menstrual cycle of the bonnet monkey (M. radiata)

Sixty-two menstrual cycles were observed in five bonnet monkeys over a period of 12 months. The menstrual cycles ranged in length from 22 to 35 days, although cycles of from 26 to 29 days were most frequent. Menstrual flow occurred for two to seven days, the flow lasting for three to four days in the majority (82%) of the cycles. Serum estradiol-17β (E2) and progesterone (P) were measured by radioimmunoassay in two consecutive cycles in each monkey and correlated with changes in the quantity of cervical mucus secretion and spinnbarkeit. The day of the estradiol peak was considered as day 0. The E2 levels began to rise one to three days before the peak, returning to baseline levels within two days and thereafter remained at low levels. The serum P rose 2 days after the E2 peak, and maximum level was attained at 10 to 12 days after the E2 peak. The quantity of cervical mucus increased proportionately with estrogen. However, the quantity remained at maximum levels even after the decline of the serum E2 level. Similar trends were observed with the spinnbarkeit.

Diminution in humoral antibodies to tetanus toxoid after Ovulen therapy in mice.

Abstract Mice treated with Ovulen showed diminished immunological response to tetanus toxoid. The decrease in humoral antibody-titre increased with increasing doses of Ovulen.

Screening infertile women for the assessment of corpus luteal function and their response to therapy by ELISA of pregnanediol glucuronide

Corpus luteal function was assessed by estimating pregnanediol 3-alpha-glucuronide (PdG) in three midluteal-phase urine samples collected from 85 women attending the infertility clinic. The previously established cut off limits based on PdG estimations were useful in detecting anovulation in 23 cases, corpus luteal adequacy in 42 cases and corpus luteum deficiency (CLD) in 20 cases. In 8 women CLD could be corrected with 50 mg of clomiphene citrate (CC) therapy whereas 6 women required 100 mg of CC and 3 pregnancies were achieved. This rapid screening method is thus useful in segregating a large number of women according to their ovulatory status and in the subsequent treatment of CLD.

Phase I comparative clinical trial with subdermal implants — Bioabsorbable levonorgestrel or norethisterone pellet fused with cholesterol

The potential for antifertility effect of two bioabsorable pellets, one containing norethisterone (NET) and the other containing levonorgestrel (LNG) fused with cholesterol, was studied in a group of healthy, menstruating but sterilised women. The pellets weighed approximately 30 mg and contain 85% steroid and 15% cholesterol. A single NET pellet was inserted in 4 subjects, out of which 2 were also studied for steroid pattern in blood. After reaching peak levels within 48 hours, the plasma NET levels declined gradually within a fortnights time, and thereafter, ranged between 200 to 700 mg/ml up to 90 days post-insertion. After this period, occassional spurts of NET release were seen. Bleeding pattern was studied in 37 cycles; mid-luteal progesterone (P) estimation was done in 16 cycles, mid-cycle cervical mucus was studied in 27 cycles and post-coital test (PCT) in 7 cycles. Cycle length with pellet insertion was of 25 to 37 days duration except one cycle of 55 days duration. All cycles studied during the treatment were ovulatory (P greater than 5 ng/ml) and no consistent effect was observed on cervical mucus as well as on PCT. Thus, a single NET pellet does not seem to have a reliable contraceptive potential. A single LNG pellet was inserted in 8 subjects, and steroid patterns were studied in 6 women. Peak levels of LNG were reached within 24 hours of pellet insertion and the levels fell gradually in a weeks time. Thereafter, LNG levels ranged between 100-400 pg/ml up to 8 months. In contrast to the observation with NET pellets, only one subject showed frequent spurts of LNG release until the 4th month of treatment. Bleeding pattern was studied in 90 cycles, P was estimated in 52 cycles, mid-cycle cervical mucus studied in 55 cycles and PCT done in 26 cycles. Breakthrough bleeding (BTB) occurred on 15 occasions, 11 episodes being in 2 subjects. Cycle length varied between 21 to 42 days. The LNG pellet did not exert a consistent effect either on ovulation inhibition or cervical mucus or PCT. In their present form, therefore, a single LNG pellet also does not appear to have a reliable contraceptive effect.

Serum bile acids in women taking combination contraceptives

A longitudinal study in 60 women was undertaken to observe the changes, if any, in serum bile acids after taking oral combination pills containing either 50 or 30 microgram of ethinyl estradiol. The women were followed up to 12 months. Serum bile acids (cholyglycine conjugates) were estimated by radioimmunoassay. The combination contraceptives had no appreciable effect on serum bile acids.