Dehydroepiandrosterone sulfate (DHEA-S) ELISA Kit

Sample

Serum, Plasma, Urine, Saliva, Fecal Extracts, Tissue Culture Media

Detection Method

cELISA

Intended Use

The Dehydroepiandrosterone sulfate Immunoassay kit uses a speciffcally generated antibody to measure Dehydroepiandrosterone sulfate (DHEA-S) in serum, plasma, urine, and saliva samples, and in fecal extracts.

Contents of Kit

1. Coated Clear 96 Well Plate
Clear plastic microtiter plate(s) coated with donkey anti-sheep IgG.
2. Dehydroepiandrosterone sulfate (DHEA-S) Standard 70 μL
Dehydroepiandrosterone sulfate at 1,200 ng/mL in a special stabilizing solution.
3. Dehydroepiandrosterone sulfate (DHEA-S) Antibody 3mL
A sheep polyclonal antibody speciffc for dehydroepiandrosterone sulfate
4. Dehydroepiandrosterone sulfate (DHEA-S) Conjugate 3mL
A dehydroepiandrosterone sulfate-peroxidase conjugate in a special stabilizing solution.
5. Assay Buffer Concentrate 28mL
A 5× concentrate that must be diluted with deionized or distilled water.
6. Wash Buffer Concentrate 30mL
A 20X concentrate that must be diluted with deionized or distilled water.
7. TMB Substrate 11mL
8. Stop Solution 5mL
A 1M solution of hydrochloric acid. CAUSTIC.
9. Plate Sealer

Storage

All components of this kit should be stored at 4°C until the expiration date of the kit.

Precision

Intra Assay Precision
Three human samples were diluted with Assay Buffer and run in replicates of 20 in an assay. The mean and precision of the calculated DHEA-S concentrations were:

Inter Assay Precision
Three human samples were diluted with Assay Buffer and run in duplicates in eighteen assays run over multiple days by four operators. The mean and precision of the calculated DHEA-S concentrations were:

Sensitivity

Sensitivity was calculated by comparing the OD's for eighteen wells run for each of the B0 and standard #5. The detection limit was determined at two (2) standard deviations from the B0 along the standard curve.
Sensitivity was determined as 90.9 pg/mL.
The Limit of Detection for the assay was determined in a similar manner by comparing the OD's for twenty runs for each of the zero standard and a low concentration human serum sample.
Limit of Detection was determined as 75.6 pg/mL.

General Description

Dehydroepiandrosterone sulfate, C₁₉H₂₈O₅S, (5-androsten-3ß, 16a-diol-17-one sulfate, DHEA-S) is the major C19 steroid secreted by the adrenal cortex, and is a precursor in testosterone and estrogen biosynthesis. It is produced by the addition of a sulfate group to dehydroepiandrosterone (DHEA), catalyzed by the sulfotransferase enzymes, SULT1A1 and SULT1E1, which also produce estrone sulfate from estrone. DHEA sulfate can also be back-converted to DHEA through the action of steroid sulfatase. DHEA-S has relatively low androgenic activity due to the 17-ketone group rather than hydroxyl group. However the bioactivity of DHEA-S may be high due to its high serum concentrations at 100-1,000-fold higher than testosterone or DHEA and its weak afffnity for sex-hormone binding globulin.

The physiological role of DHEA-S is not well deffned, with serum levels being high in the fetus and neonates, low during childhood and increased during puberty. DHEA-S levels decline during the third decade of life. DHEA-S, unlike DHEA and other steroids, does not show a signiffcant diurnal or day-to-day variation. DHEA-S levels are not increased due to ACTH administration and do not change signiffcantly during the normal menstrual cycle. DHEA-S has a lower metabolic clearance rate than DHEA.
Since DHEA-S is primarily produced by the adrenal glands, it is useful as a marker for adrenal function. Adrenal tumors, cancers, and hyperplasia can lead to the overproduction of DHEA-S. While elevated levels may not be noticed in adult men, they can lead to amenorrhea and visible symptoms of virilization. These changes vary in severity and may include a deeper voice, hirsutism, male pattern baldness, muscularity, acne and enlargement of the Adam's apple. Women with polycystic ovary syndrome tend to have elevated levels of DHEA-S. Excess levels of DHEA-S in children can cause precocious puberty in boys; and ambiguous external genitalia, excess body hair, and abnormal menstrual periods in girls.
1. Dorfman, RI and Shipley, RA., Androgens, J. Wiley and Sons, NY, 1956, 116-128.
2. Pang, S. and Riddick, L., "Pediatric Endocrinology, A Clinical Guide, 2nd Ed.", F. Lifshitz (Ed.), Marcel Dekker, Inc. New York, 1990, 259-291.
3. de Peretti, E. and Forest, MG., "Pattern of plasma dehydroepiandrosterone sulfate levels in humans from birth to adulthood: evidence for testicular production", J. Clin. Endocrinol. Metab., 1978, 47:572-577.
4. Lashansky, G., et. al., "Normative data for adrenal steroidogenesis in a healthy pediatric population: age- and sex-related changes after adrenocorticotropin stimulation", J. Clin. Endocrinol. Metab., 1991, 73:674-686.
5. Zumoff, B., et. al., "Sex differences in the twenty-four-hour mean plasma concentrations of dehydroisoandroster one (DHA) and dehydroisoandrosterone sulfate (DHAS) and the DHA to DHAS ratio in normal adults", J. Clin. Endocrinol. Metab., 1980, 51:330-333.
6. Pang S., "Late-onset adrenal steroid 3 beta-hydroxysteroid dehydrogenase deffciency. I. A cause of hirsutism in pubertal and postpubertal women", J. Clin. Endocrinol. Metab., 1985, 60:428-39.

Standard Curve

Average the duplicate OD readings for each standard and sample. Create a standard curve by reducing the data using the 4PLC fitting routine on the plate reader, after subtracting the mean OD's for the NSB. The sample concentrations obtained, calculated from the %B/B0 curve, should be multiplied by the dilution factor to obtain neat sample values.