Testosterone ELISA Kit

Catalog No. ABIN504793

Product Details Testosterone ELISA Kit

Target: Testosterone

Reactivity: Human

Detection Method: Colorimetric

Method Type: Competition ELISA

Application: ELISA

Purpose: Competitive Enzyme Immunoassay (TYPE 7): The essential reagents required for a enzyme immunoassay include antibody, enzyme-antigen conjugate and native antigen. Upon mixing biotinylated antibody, enzyme-antigen conjugate and a serum containing the native antigen, a competition reaction results between the native antigen and the enzyme-antigen conjugate for a limited number of antibody binding sites. The enzyme activity in the antibody bound fraction is inversely proportional to the native antigen concentration. By utilizing several different serum references of known antigen concentration, a dose response curve can be generated from which the antigen concentration of an unknown can be ascertained.

Analytical Method: Quantitative

Components: A. Testosterone Calibrators (1ml/vial) . Seven vials of serum reference for Testosterone at concentrations of 0 (A), 0. 1 (B), 0. 5 (C), 1. 0 (D), 2. 5 (E), 5. 0 (F) and 12. 0 (G) in ng/ml Store at 2-8°C. A preservative has been added. The calibrators can be expressed in molar concentrations (nM/L) by multiplying by 3. 47. For example: 1ng/ml x 3. 47 equal 3. 47 nM/LB. Testosterone Enzyme Reagent (1. 0 ml/vial). One vial of TestosterOne (Analog)-horseradish peroxides (HRP) conjugate in a protein stabilizing matrix with green dye. Store at 2-8°C. C. Steroid Conjugate Buffer (7. 0 ml/vial). One vial of reagent contains buffer, red dye, preservative, and binding protein inhibitors. Store at 2-8°C. D. Testosterone Biotin Reagent (6. 0 ml). One bottle of reagent contains anti-Testosterone biotinylated purified rabbit IgG conjugate in buffer, yellow dye and preservative. Store at 2-8°C. E. Streptavidin Coated Plate (96 wells). One 96-well microplate coated with 1. 0 µg/ml streptavidin and packaged in an aluminum bag with a drying agent. Store at 2-8°C. F. Wash Solution Concentrate (20ml). One vial containing a surfactant in buffered saline. A preservative has been added. Store at 2-30°C. G. Substrate A (7ml/vial). One vial contains tetramethylbenzidine (TMB) in buffer. Store at 2-8°C. H. Substrate B (7ml/vial). One vial contains hydrogen peroxide (H2O2) in buffer. Store at 2-8°C. I. Stop Solution (8ml/vial). One vial contains a strong acid (1N HCl). Store at 2-30°C. J. Product Instructions: Note 1: Do not use reagents beyond the kit expiration date. Note 2: Opened reagents are stable for 60 days when stored at 2-8°C. Note 3: Above reagents are for a single 96-well microplate.

Material not included: 1. Pipette capable of delivering 10µl, 50µl, and 100µl volumes with a precision of better than 1. 5%. 2. Dispenser(s) for repetitive deliveries of 0. 100ml and 0. 350ml volumes with a precision of better than 1. 5%. 3. Adjustable volume (200-1000µl) Dispenser(s) for conjugate. 4. Microplate washer or a squeeze bottle (optional). 5. Microplate Reader with 450nm and 620nm wavelength absorbance capability. 6. Absorbent Paper for blotting the microplate wells. 7. Plastic wrap or microplate cover for incubation steps. 8. Vacuum aspirator (optional) for wash steps. 9. Timer. 10. Quality control materials.

Application Details

Application Notes: Precautions: All products that contain human serum have been found to be non-reactive for Hepatitis B Surface Antigen, HIV 1&2 and HCV Antibodies by FDA required tests. Since no known test can offer complete assurance that infectious agents are absent, all human serum products should be handled as potentially hazardous and capable of transmitting disease. Good laboratory procedures for handling blood products can be found in the Center for Disease Control / National Institute of Health, Biosafety in Microbiological and Biomedical Laboratories, 2nd Edition, 1988, HHS Publication No. (CDC) 88-8395.

Sample Volume: 10 μL

Plate: Pre-coated

Reagent Preparation:

1. Working Enzyme Reagent - Stable for 1 year: Measuree 0. 7 ml of Testosterone Enzyme Reagent and add to the vial containing Steroid Conjugate Buffer. Store at 2-8°C. 2. Wash Buffer: Dilute contents of wash solution to 1000ml with distilled or deionized water in a suitable storage container. Diluted buffer can be stored at room temperature (20-27°C) for up to 60 days. 3. Working Substrate Solution - Stable for 1 year. Pour the contents of the amber vial labeled Solution A into the clear vial labeled Solution B. Place the yellow cap on the clear vial for easy identification. Mix and label accordingly. Store at 2 - 8 °C. Note: Do not use the working substrate if it looks blue.

Sample Collection: The specimens shall be blood, serum or plasma in type and the usual precautions in the collection of venipuncture samples should be observed. For accurate comparison to established normal values, a fasting morning serum sample should be obtained. The blood should be collected in a plain redtop venipuncture tube or (for plasma) in evacuated tube(s) containing heparin. Allow the blood to clot for serum samples. Centrifuge the specimen to separate the serum or plasma from the cells. Samples may be refrigerated at 2-8°C for a maximum period of five days. If the specimen(s) cannot be assayed within this time, the sample(s) may be stored at temperatures of -20 °C for up to 30 days. Avoid repetitive freezing and thawing. When assayed in duplicate, 0. 020ml of the specimen is required.

Calculation of Results:

A dose response curve is used to ascertain the concentration of Testosterone in unknown specimens. 1. Record the absorbance obtained from the printout of the microplate reader. 2. Plot the absorbance for each duplicate serum reference versus the corresponding Testosterone concentration in ng/ml on linear graph paper (do not average the duplicates of the serum references before plotting). 3. Connect the points with a best-fit curve. 4. To determine the concentration of Testosterone for an unknown, locate the average absorbance of the duplicates for each unknown on the vertical axis of the graph, find the intersecting point on the curve, and read the concentration (in ng/ml) from the horizontal axis of the graph (the duplicates of the unknown may be averaged as indicated).

Restrictions: For Research Use only

Handling

Handling Advice: Before proceeding with the assay, bring all reagents, serum references and controls to room temperature (20-27°C). 1. Format the microplates' wells for each serum reference, control and patient specimen to be assayed in duplicate. Replace any unused microwell strips back into the aluminum bag, seal and store at 2-8°C. 2. Pipette 0. 010 ml (10µl) of the appropriate serum reference, control or specimen into the assigned well. 3. Add 0. 050 ml (50µl) of the working Testosterone Enzyme Reagent to all wells (see Reagent Preparation Section). 4. Swirl the microplate gently for 20-30 seconds to mix. 5. Add 0. 050 ml (50µl) of Testosterone Biotin Reagent to all wells. 6. Swirl the microplate gently for 20-30 seconds to mix. 7. Cover and incubate for 60 minutes at room temperature. 8. Discard the contents of the microplate by decantation or aspiration. If decanting, blot the plate dry with absorbent paper. 9. Add 350µl of wash buffer (see Reagent Preparation Section), decant (tap and blot) or aspirate. Repeat two additional times for a total of three washes. An automatic or manual plate washer can be used. Follow the manufacturer's instruction for proper usage. If a squeeze bottle is employed, fill each well by depressing the container (avoiding air bubbles) to dispense the wash. Decant the wash and Repeat two additional times. 10. Add 0. 100 ml (100µl) of working substrate solution to all wells (see Reagent Preparation Section). Always add reagents in the same order to minimize reaction time differences between wells. DO NOT SHAKE THE PLATE AFTER SUBSTRATE ADDITION. 11. Incubate at room temperature for 15 minutes. 12. Add 0. 050ml (50µl) of stop solution to each well and gently mix for 15-20 seconds. Always add reagents in the same order to minimize reaction time differences between wells. 13. Read the absorbance in each well at 450nm (using a reference wavelength of 620-630nm to minimize well imperfections) in a microplate reader. The results should be read within 30 minutes of adding the stop solution. Note: Dilute the samples suspected of concentrations higher than 12 ng/ml 1:5 and 1:10 with Testosterone 0 ng/ml calibrator or female patient sera with a known low value for testosterone.

Storage: 4 °C/-20 °C

Target Details for Testosterone

Target: Testosterone

Abstract: Testosterone Products

Target Type: Hormone

Background: Summary and Explanation of the test: Testosterone, (17alpha Hydroxy-4-androstene-3-one), a C19 steroid, is the most potent naturally secreted androgen1. In normal post pubertal males, testosterone is secreted primarily by the testes with only a small amount derived from peripheral conversion of 4-Androstene-3, 17-dione (ASD)2. In adult women, it has been estimated that over 50% of serum testosterone is derived from peripheral conversion of ASD secreted by the adrenal and ovary, with the remainder from direct secretion of testosterone by these glands In the male, testosterone is mainly synthesized in the interstitial Leydig cells and the testis, and is regulated by the interstitial cell stimulating hormone (ICSH), or luteinizing hormone (LH) of the anterior pituitary (the female equivalent of ICSH)3. Testosterone is responsible for the development of secondary sex characteristics, such as the accessory sex organs, the prostate, seminal vesicles and the growth of facial, pubic and auxiliary hair. Testosterone measurements have been very helpful in evaluating hypogonadal states. Increased testosterone levels in males can be found in complete androgen resistance (testicular feminization). Common causes of decreased testosterone levels in males include: hypogonadism, orchidectomy, estrogen therapy, Klinefelter's syndrome, hypopituitarism, and hepatic cirrhosis2-4. In the female, testosterone levels are normally found to be much lower than those encountered in the healthy male. Testosterone in the female comes from three sources. It is secreted in small quantities by both the adrenal glands and the ovaries, and in healthy women 50-60% of the daily testosterone production arises from peripheral metabolism of prohormone, chiefly androstenedione. Common causes of increased serum testosterone levels in females include polycystic ovaries (Stein-Leventhal syndrome), ovarian tumors, adrenal tumors and adrenal hyperplasia. Virilization in women is associated with the administration of androgens and endogenous overproduction of testosterone. There appears to be a correlation between serum testosterone levels and the degree of virilization in women, although approximately 25% of women with varying degrees of virilism have serum testosterone levels that fall within the female reference range. Intended Use: The Quantitative Determination of Total Testosterone Concentration in Human Serum or Plasma by a Microplate Enzyme Immunoassay. Q. C. Parameters: In order for the assay results to be considered valid the following criteria should be met: 1. The absorbance (OD) of calibrator 0 ng/ml should be greater than 1.3. 2. Four out of six quality control pools should be within the established ranges.