Stability studies of the components of a prototype penicillinase (beta-lactamase)-linked ELISA kit.

Penicillinase (beta-lactamase) enzyme-linked immunosorbent assay (ELISA) for various reproductive hormones developed in the laboratory were found to have wide applicability in the fertility check clinic of the Institute. A need was thought to transform these assays into ready-to-use kit forms. Therefore, prototype ELISA kits for these hormones were developed and stability of the individual component was ascertained at various temperatures (room temperature, 37 degrees C and 2-8 degrees C). Stability studies were conducted on previously validated assay for pregnanediol-3 alpha-glucuronide (PdG). The studies showed that immunosorbents (antibody coated plates) are stable at room temperature for a period of 2 weeks, at 37 degrees C for 1 week and at 2-8 degrees C for a period of 9 months when preserved after treatment with glycerol solution. The lyophilised conjugate, standard and immunoassay buffer, colour reagent, and its substrate were stable at 37 degrees C up to 1 week and at room temperature up to 2 weeks and at 2-8 degrees C for a period of 6 months, during which the stability was studied.

An indirect ELISA for urinary gonadotropins using immobilized human menopausal gonadotropin.

An indirect ELISA for the estimation of urinary gonadotropins is described. Human menopausal gonadotropin is adsorbed on a microtitre plate, where it serves as an immunosorbent. The residual antigonadotropin antibody is captured by the immunosorbent after reaction with the sample or standard and detected with enzyme-labelled antispecies antibody (antirabbit gamma-globulin-horse radish peroxidase). The assay developed here is rapid and satisfies usual validatory criteria expected from an immunoassay. Moreover, it obviates the need for extraction of samples with acetone, as shown by the close agreement between the respective lutropin or follitropin concentrations in extracted and unextracted urine samples.

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.

Importance of selection of separation system in the development of enzyme immunoassay: an experience with follicle stimulating hormone (FSH) assay.

An antiserum to follicle stimulating hormone (FSH) obtained as a gift from National Institute of Health (NIH), U.S.A. could not be adsorbed on microtitre ELISA plates, although two other FSH antisera raised in authors' laboratory could be adsorbed. A good precision profile for the FSH assay using these three antisera could be achieved with only one separation system viz. solid phase anti rabbit gamma globulin (ARGG), out of the five separation systems tried. The study suggests that a few antisera used for radio-immunoassay (RIA) purposes may not by themselves get adsorbed on plastic plates. However, they could be effectively used for ELISA purposes using solid phase second antibody.

A simple ELISA for detection of ovulation.

A rapid, simple, two-step test for the detection of ovulation has been developed. The test is based on ELISA of pregnanediol glucuronide, a metabolite of progesterone, in urine collected specifically over a period of 3 h. The test is completed in 20 min and the results are assessed visually by naked eye.

An enhanced chemiluminescent enzyme immunoassay for follicle stimulating hormone.

An enhanced chemiluminescent enzyme immunoassay for serum follicle stimulating hormone is described which involves sequential reaction of anti-follicle stimulating hormone antibody immobilised to the inside surface of an opaque microtitre plate with sample, monoclonal anti-alpha thyroid stimulating hormone antibody, and an anti-mouse IgG - horseradish peroxidase conjungate. Bound peroxidase activity was measured using a p-hydroxycinnamic acid enhanced chemiluminescent luminol-hydrogen peroxide reaction. The assay was sensitive (detection limit 0.01 mU/well) precise (intra-assay precision 2.5-8.1%, inter-assay precision 6.7-11.9%) and results obtained with this assay and a competitive radioimmunoassay were in good agreement (correlation coefficient 0.98).

A comparison of polyclonal antisera to pregnanediol 3 alpha glucuronide obtained from three different rabbits and their use in ELISA.

The variation in the estimates of urinary pregnanediol glucuronide obtained by ELISA with 12 polyclonal antisera has been assessed. Of the 12 antisera, eight gave values comparable with each other and could be used interchangeably in the ELISA. Thus a large stock of polyclonal antibody which gave consistent values was generated and can now be used in diagnostic ELISA over a long period of time. This procedure is more economical than the development of monoclonal antibodies for the same purpose.

An enhanced chemiluminescence enzyme immunoassay for serum oestradiol.

Oestradiol in serum was determined with a simple enhanced chemiluminescent enzyme immunoassay. The assay is based on oestradiol labelled with horseradish peroxidase and the IgG fraction of an oestradiol antiserum coated on a black polystyrene microtitre plate. The enzyme activity of bound label was determined using a p-hydroxycinnamic acid-enhanced chemiluminescent reaction. The assay was sensitive (1.8 fmol/well), precise (intra- and inter-assay CV 4-10% and 8-12%, respectively for sample concentration in the range 122-1330 pmol/L) and showed good agreement with conventional radioimmunoassays (r = 0.99).

Excretion of estrone-3-glucuronide in urine in spontaneous and induced ovulatory cycles.

Response of the ovary to ovulation-inducing agents, such as clomiphene citrate and gonadotropin, was assessed using estimations of urinary estrone-3-glucuronide (E1G) by an ELISA test. Baseline data collected over 58 cycles from regularly menstruating women were used as reference. Further, the pattern of E1G excretion was compared with ultrasound assessment of follicular growth. In ovulatory cycles, a good correlation is seen between follicular growth and follicular function. However, when cystic follicles develop after treatment, the correlation is poor: the follicle increases in size without a corresponding increase in E1G excretion. It is suggested that ultrasound examination and hormonal pattern should be used concurrently to assess the response of the ovary to ovulation-inducing agents.

Improvement in the antibody binding characteristics of microtitre wells by pretreatment with anti-IgG Fc immunoglobulin.

A preliminary coating of the surface of polystyrene microtitre wells with anti-IgG Fc immunoglobulin improves the binding capacity of the wells for antibody and leads to a marked improvement in the reproducibility of immunoassays carried out in the wells. In an enhanced chemiluminescent enzyme immunoassay for oestradiol the binding capacity for oestradiol of wells precoated with anti-IgG Fc immunoglobulin and then coated with anti-oestradiol immunoglobulin was more than twice that of wells coated directly with anti-oestradiol immunoglobulin. Intra-assay precision in the precoated wells ranged from 1.3 to 4.0% compared to 2.9 to 6.7% (0-3200 fmol oestradiol) in wells which had not been precoated.

Application of penicillinase linked ELISA of pregnanediol glucuronide for detection of ovulation and assessment of corpus luteal function.

A penicillinase linked enzyme immunoassay was developed for the estimation of pregnanediol-3 alpha-glucuronide (PdG) in urine. The immunoassay satisfied all the validity criteria and was used in detecting ovulation and in the assessment of corpus luteal function (CLF) during spontaneous or induced cycles. Reference values were established by estimating PdG levels in daily early morning urine samples during 31 menstrual cycles obtained from 17 regularly menstruating women. A PdG value of 1.7 micrograms/mg creatinine (micrograms/mgC) (90th Centile of follicular phase) in any MLP (mid-luteal phase) sample was considered as indicating ovulation. A value of 4.6 micrograms/mgC (20th centile of MLP) was considered to be evidence of sufficient CLF. When this approach was applied to 20 infertile cases, detection of the occurrence of ovulation/anovulation was made correctly in 19 out of 20 cases (95%). Accuracy was poor (55.6%) when the aim of the diagnosis was corpus luteal deficiency. Higher accuracy (88.9%) for corpus luteal deficiency/corpus luteal adequacy was obtained when the sum of PdG concentrations in three MLP samples were taken into consideration. A total of 13.8 micrograms/mgC (thrice the 20th centile for MLP) indicated probable corpus luteal deficiency, and values above this limit were considered to indicate corpus luteal adequacy.

Antacid does not reduce the bioavailability of oral contraceptive steroids in women.

The bioavailability of contraceptive steroids was studied in 12 women who were given an antacid and a contraceptive pill simultaneously. They were given a single pill containing ethinyl estradiol (EE2) 30 micrograms in combination with either norethisterone (NET) acetate 1 mg (n = 6), or levonorgestrel (LNg) 150 micrograms, (n = 6). Blood samples were collected up to 24 hours. Four weeks later the same pill was administered along with a single tablet of antacid (magnesium trisilicate 0.5 g and dried aluminum hydroxide 0.25 g) and blood samples were collected as before. Serum levels of NET, LNg and EE2 were measured by radioimmunoassay. No effect of antacid administration on bioavailability of any contraceptive steroid was observed as judged by peak levels and areas under concentration-time curve (AUC). Incidentally, significantly higher serum concentrations of EE2 were observed when it was administered in combination with NET than with LNg. The possible reasons for this finding are discussed.

PIP: The serum levels of both the estrogen and progestin in 2 different oral contraceptives were analyzed by radioimmunoassay (RIA) for 24 hours after taking a single contraceptive pill with or without an antacid. 6 Indian women not taking any other medication took a 30 mcg ethinyl estradiol (EE) and 1 mg norethisterone (NET) pill, and 6 others took a 30 mcg EE with 150 mcg levonorgestrel (LNg) pill at 9 AM. 4 wks later they took the same steroid pill after chewing a tablet of antacid containing magnesium trisilicate .5 g and aluminum hydroxide .25g (Gelusil, Warner). Serum RIA levels and areas under the curves were identical, when each group served as its own control, except that EE levels were significantly higher in the NET combination. The reasons for this discrepancy, whether due to pill excipients, radioimmunoassay cross reactions or biological factors such as sex hormone binding globulin activities, were unexplained. Apparently, self-dosing with this antacid will not affect bioavailability of oral contraceptive steroids.

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.

Clinical performance and endocrine profiles with contraceptive vaginal rings containing a combination of estradiol and D-norgestrel.

Contraceptive vaginal rings (CVRs) impregnated with a combination of estradiol and d-norgestrel were studied in 39 women. The rings were inserted as three weeks "in" and one week "out" schedule to allow withdrawal bleeding. Ovulation was inhibited in all cycles and promptly resumed on completion of the treatment cycles. Bleeding control was excellent. Incidence of breakthrough bleeding was 2.11 per cent in 273 cycles studied, with 89 per cent of the cycles within the acceptable range (25 - 35 days). Levels of estradiol after an initial rise were maintained between 50-100 pg/ml and, d-norgestrel levels were relatively constant between 1.8-3 ng/ml during the period while the ring remained in situ. Clinical acceptance was good. The main problem encountered was of spontaneous expulsion of the ring in the toilet, which might be due to the squatting toilet habits of Indian women and laxity of vaginal wall.

PIP: Contraceptive vaginal rings (CVRs) impregnated with a combination of estradiol and d-norgestrel were studied in 39 women. The rings were inserted on a 3 weeks "in" and 1 week "out" schedule to allow withdrawal bleeding. Ovulation was inhibited in all cycles and promptly resumed on completion of the treatment cycles. Bleeding control was excellent. Incidence of breakthrough bleeding was 2.11% in 273 cycles studied, with 89% of the cycles within the acceptable range (25-35 days). Levels of estradiol after an initial rise were maintained between 50-100 pg/ml and, d-norgestrel levels were relatively constant between 1.83 ng/ml during the period while the ring remained in situ. Clinical acceptance was good. The main problem encountered was of spontaneous expulsion of the ring in the toilet, which might be due to the squatting toilet habits of Indian women and laxity of the vaginal wall.

A study of interaction of a low-dose combination oral contraceptive with anti-tubercular drugs.

Low-dose combination contraceptive (containing norethisterone acetate 1 mg and ethinyl estradiol 30 micrograms) was administered to women receiving concurrent therapy with either Rifampicin or "triple" antitubercular treatment consisting of paraaminosalicylic acid (PAS), isonicotinic acid hydrazide (INH) and streptomycin. Plasma levels of norethisterone (NET) and ethinyl estradiol (EE), PAS and INH were measured and the area under curve (AUC) was calculated for NET and EE. Rifampicin treatment (9 women) caused a statistically significant reduction of the plasma NET levels as well as the AUC of NET. In this group of women, though a trend for reduction in EE levels was observed in individual subjects, it was not statistically significant. Out of 7 regularly menstruating women on Rifampicin therapy, 2 showed a premenstrual rise of plasma progesterone (P) levels (> 4 ng/ml) suggesting an ovulatory cycle and 3 experienced menstrual irregularities. In contrast, plasma levels of NET and EE as well as their AUCs were not altered in 8 women receiving "triple" antitubercular therapy. Only one woman out of 8, had menstrual irregularity and all women had P levels in the anovulatory range. Furthermore, oral contraceptive treatment did not alter the plasma levels of PAS and INH.

Influence of antisera on the estimations of norethisterone and levonorgestrel.

PIP: The specificity of 6 different antisera for norethisterone (NET) and 10 different antisera for levonorgestrel (LNG) were studied. These antisera had differing degrees of cross-reactivities with the metabolites of the respective steroids. Antisera obtained from the same animal revealed a trend towards higher cross-reactions with the length of immunization period. Animal variation appeared to be a major factor in deciding the specificity of antisera. Using these antisera radioimmunoassays for NET and LNG were standardized; this fulfilled the required criteria for reliability. When NET and LNG concentrations were determined in pooled plasma which was obtained from women using oral contraceptives containing the respective steroids, it was found that the variation in the cross-reactivities of the metabilites with the antiserum used had negligible effect on NET estimates but did influence the LNG estimates.^ieng

Capillary and venous blood estrogen in pregnancy.

Total estrogens in the third trimester of pregnancy were measured in matched samples of venous and capillary blood serum. A close correlation was observed between capillary blood estrogens (CBE) and venous blood estrogens (VBE). After establishing norms for CBE during the third trimester of normal pregnancy, the parameter was assessed for its use in high-risk pregnancy in comparison with VBE. In 96.6% of the 59 high-risk pregnancy cases studied, the conclusions drawn about the fetoplacental unit on the basis of CBE and VBE were similar.