DHEA-S  Coated Tubes   

Direct ¹²⁵I-Radioimmunoassay kit for the quantitative determination of serum DHEA-S.

for in vitro diagnostic use only

1. Summary and background of the test:

The most important plasma adrenal C19-androgens are Dehydroepiandrosterone Sulfate (DHEA-S), Dehydroepiandrosterone (DHEA) and Androstenedione. In normal young adults, DHEA-S circulates at a concentration of approximately ten times that of Cortisol, and at least hundred times that of DHEA, which makes it the steroïd of greatest concentration in human plasma(1). The adrenal androgens are synthesized from cholesterol by adrenal cortex and can be metabolized to physiologically more potent androgens like testosterone and its 5-alpha reduced metabolite dihydrotes-tosterone. The increased secretion of adrenal androgens and the associated early signs of sexual maturation are called andrenarche. Adults with hyperprolactinemia have increased secretion of androgens (2).Moreover, pituitary gonadotropins do not appear to be involved in the production of adrenal androgens. It appears, therefore, that the prolactin and ACTH control adrenal androgens secretion even if most studies (3) suggest that DHEA and, to a lesser extent, androstenedione, are the adrenal androgens most responsive to ACTH stimulation. During pregnancy, sine formation of estrogen in the placenta is dependent on circulating C19-steroids, it is possible to evaluate the placental clearance of maternal plasma DHEA-S through placental estradiol formation (4). The determination of DHEA-S should be considered in:

-premature andrenarche

-congenital adrenal hyperplacia

-Cushing's syndrome

-hirsutism

DHEA-S determination can be considered as a useful marker in the follow-up of hyperandrogenic women.

It should also be kept in mind that age changes and sex differences can modify serum DHEA-S concentrations throughout adulthoud (5-6).

2. Principle of the test :

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 increasing 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 DHEA-S 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:

Kit contains sufficient reagents for 100 determinations.

1. DHEA-S human serum based standards & control: 8 vials containing each 500 ìl except Zero 1ml.

a. Control: 1.8 ± 0.4 ìg/ml.

b. Standards: 0-0.1-0.5-1-2-4-8 ìg/ml.

2. ¹²⁵I-DHEA-S tracer: 1 vial (red solution) containing 52 ml. Activity < 5ìCi or 185 kBq.

3. Coated tubes: 2 x 50 tubes, coated with Anti-DHEA-S antiserum (Rabbit).

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

Sera should be separated from blood cells immediately after collection. Sera are stable for at least 7 days at 4^o 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 20 ìl of standards, samples and controls into the corresponding tubes.

2. Add 500 ìl of tracer solution (red) to each tube. Mix well (sideways shaking of whole rack).

3. Mix well, cover and incubate 45 minutes at 37° C.

4. Aspirate (or decant). Wash twice, adding 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.

 

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

% B/B₀ = cpm (Stds, Controls or unknowns) x 100/cpm (B₀)

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

9. Expected Values:

newborn             1600-3690

adult male:

-Prepubescent     110-620

-24-40 years     1910-3380

-65-85 years     170-910

adult female

- Prepubescent     150-650

-Adult                 780-3430

-Term Pregnancy 230-1220

-Postmenoposal    120-660

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

Conversion factor: 1 ng/ml = 2.6 nmol/l

10. Specific performance characteristics:

1. Specificity:

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

Cross-reactivity of x = 100 x conc. DHEA-S/conc. compound at 50% B/B_o

Compound x Cross-reactivity (%)

DHEA-S 100 %

DHEA < 5 %

Progesterone < 3 %

Estradiol < 0.50 %

Cortisol < 0.50 %

Testosterone < 0.50 %

Estrone < 0.50 %

17-OH-Progesterone < 0.05 %

17-OH-Pregnenolone < 0.05 %

Corticosterone < 0.05 %

Dihydrotestosterone < 0.05 %

Androstenedione < 0.05 %

2. Sensitivity:

The lowest detectable concentration of DHEA-S that can be reliably distinguished from zero with this kit has been evaluated to be £ 15 ng/ml.

3. Precision and reproducibility:

Assays variations of two serum samples are mentioned in the following table.

                                    Sample 1     Sample 2

Mean                             783 ìg/m        3613ìg/ml

Within assay variation         6.6 %        5.7 %

Between assay variation     7.1 %         6.9 %

4. Linearity:

The results obtained when diluting a serum with elevated DHEA-S concentration with a DHEA-S- free serum are summarized in the following table

Dilution factor Expected values Experimental values

1:1                     3602 ìg/ml                 -

1:2                     1801 ìg/ml             1755 ìg/ml

1:4                     901 ìg/ml                 897 ìg/ml

1:8                     450 ìg/ml                 439 ìg/ml

1:16                     225 ìg/ml               217 ìg/ml

1:32                     113 ìg/ml                 106 ìg/ml