BL-09CT-100 : 100 determinations

Estrone, estradiol and estriol, which are produced in ovaries, placenta, testes, adrenals, liver and adipose tissue, are the main estrogenic steroids of clinical interest.During the menstrual cycle, estrone fluctuations are similar to those of estradiol. About 60% of daily estrogen production consists of estradiol, which arises from ovarian secretion, while 40%, in the form of estrone, results mainly from the conversion of androstenedione secreted by both adrenals and ovaries. Factors influencing the conversion of androstenedione to estrone are weight, age, liver function, heart failure and thyroid dysfunction.After menopause, as a result of the cessation of cyclic ovarian function, estradiol only originates from the adrenals and the peripheral conversion of estrone, and is therefore present in the plasma at very low concentrations. The major estrogen in the blood circulation is estrone, at levels that are, however, insufficient to prevent estrogen deprivation from target organs such as hypothalamus, pituitary, uterus, vagina and breasts.Disorders of the ovary and female reproductive tract may result in hyperestronemia in women with polycystic ovary syndrome, or with ovarian tumors.

Radioimmunoassay is based on the ability of a limited quantity of antibody to bind a fixed amount of radiolabelled antigen(¹²⁵I-Ag). The percentage of bound radiolabelled antigen is inversely related to the concentration of unlabelled analyte in the sample. Separation of the bound and free radiolabelled antigen is necessary in order to determine the quantity of unlabelled antigen. The Bio-Line Estrone kit utilizes the coated tubes methodology. The quantity of unlabelled antigen in an unknown sample is then determined by comparing the remaining radioactivity in the coated tubes with data established using known standards in The same assay system.

3. Materials provided for 100 tests:

1. Estrone standards & control: 7 vials containing each 1.0 ml.

Standards range: 0-1500 pg/ml. Refer to vial labels for accurate standards & control concentrations.

2. ¹²⁵I-Estrone tracer: 1 vial (violet solution) containing 51 mL. Activity < 4ìCi or 148 kBq.

3. Wash solution concentrate: 1 vial of 2 ml of concentrate, to be diluted into 250 ml NaCl 9 ‰ and stored at 4° C.

4. Coated tubes: 2 x 50 tubes, coated with anti-

Anti-Estrone antiserum (Rabbit).

Sera should be separated from blood cells immediatly after collection. Sera are stable for at least 7 days at 4° C and for longer periods of time when stored frozen.

5. Assay procedure:

Bring reagents to room temperature and mix before use. Label tubes for total counts (Tc), standards, control sera and unknowns.

1. Pipet 100 ìl of standards, samples and controls into the corresponding tubes.

2. Add 500 ìl of Estrone Tracer solution (violet) to each tube.

3. Mix well, cover and incubate 120 minutes at room temperature, with horizontal shaking at 250 rpm.

4. Aspirate (or decant). Add 1 ml of wash solution to each tube, except Tc. Aspirate or decant.

5. Record the counts per minute (cpm) for each tube. Count all tubes for one minute.

8. Calculation of results:

Determine the average counts for each set of duplicate tubes. Divide this value by the average net counts of the B0, and multiply by 100 to yield the % B/B0

Plot % B/B0 for each standard vs its concentration in ng/ml on semi-log graph paper. The concentration of Estrone in the unknown samples may be read directly from the standard curve.

Range (pg/ml)

Male 20 - 80

Female

Foll. phase 10 - 75

Mid cycle 70 - 185

Lut. phase 40 - 120

Postmenop. < 80

Each laboratory should analyze normal samples to establish its own normal ranges.

1. Specificity: The relative percent of cross-reactivity by weight of Estrone and various related compounds was evaluated for the antibody used in this assay. Cross-reactivities are expressed as the amount of Estrone required to reduce the binding of ¹²⁵I-Estrone by 50%, relative to the amount of a related compound required to do the same.

Cross-reactivity of x = 100 x conc. Estrone /conc. compound  at 50% B/B₀

Compound x Cross-reactivity (%)

Estrone 100 %

17-á-Estradiol 0.2 %

17-â-Estradiol < 0.08 %

Estriol < 0.08 %

Equiline 5 %

2. Sensitivity: The lowest detectable concentration of Estrone that can be reliably distinguished from zero with this kit has been evaluated to be less than 12.5 pg/ml.

3. Precision and reproducibility: Within and between assay variations of two serum samples are mentioned in the following table:

                                            Sample 1             Sample 2

Mean                                    76 pg/ml             267 pg/ml

Within assay variation         6.8 %         7.2 %

Between assay variation     8.9 %         10.9 %

4. Linearity: The results obtained when diluting a serum with elevated Estrone concentration with an Estrone-free serum are summarized in the following table:

Dilution factor     Expected values     Experimental values

1:1                     130 pg/ml                         -

1:2                     65 pg/ml                         64 pg/ml

1:4                     32 pg/ml                         30 pg/ml

1:8                     16 pg/ml                         15 pg/ml

2. Dehertogh, R., Journal of Steroid Biochemistry 4:75, 1973.

3. De Jongh, F.H., et al., Acta Endocrinologica 77:575: 1974.

4. Doerr, P., Acta Endocrinologica 81:655, 1976.

5. Emmet, Y., et al., Acta Endocrinologica 69:567, 1972.

6. Fotherty, K., In: Hormones in Blood, 3rd ed., Vol. 3,

Gray, C.H. & V.H.T. James eds., Academic Press, 1979.

10. Longcope, C., et al., Steroids 20:439; 1972.

12. Moore, P.H., et al., Steroids 199: Aug. 1972

13. Reed, M.J., et al., Cancer Research 43:3940, 1983.

17. Vermulen, A., J. Clin. Endocr. Metab. 42:247, 1976.