Estradiol (17-Estradiol) ELISA Kit

Catalog No. ABIN649066

Product Details

Target: Estradiol (17-Estradiol)

Reactivity: Human

Detection Method: Colorimetric

Method Type: Competition ELISA

Application: ELISA

Purpose: Delayed Competitive Enzyme Immunoassay (TYPE 9): The essential reagents required for a enzyme immunoassay include antibody, enzyme-antigen conjugate and native antigen. Upon mixing the biotinylated antibody with a serum containing the antigen, a reaction results between the antigen and the antibody. 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. Estradiol Calibrators (1ml/vial) . Seven vials of serum reference for estradiol at concentrations of 0 (A), 20 (B), 100 (C), 250 (D), 500 (E), 1500 (F) and 3000 (G) in pg/ml. Store at 2-8°C. A preservative has been added. The calibrators can be expressed in molar concentrations (nM/L) by multiplying by 2. 72. For example: 1pg/ml x 3. 67equal 3. 67 pM/LB. Estradiol Enzyme Reagent (6. 0 ml/vial). One vial of Estradiol (Analog)-horseradish peroxides (HRP) conjugate in a protein-stabilizing matrix red with dye. Store at 2-8°C. C. Estradiol Biotin Reagent (6. 0 ml). One bottle of reagent contains anti-estradiol biotinylated purified rabbit IgG conjugate in buffer, green dye and preservative. Store at 2-8°C. D. 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. E. Wash Solution Concentrate (20ml). One vial contains a surfactant in buffered saline. A preservative has been added. Store at 2-30°C. F. Substrate Reagent (12ml/vial). One bottle contains tetramethylbenzidine (TMB) and hydrogen peroxide (H2O2) in buffer. Store at 2-8°C. G. Stop Solution (8ml/vial). One vial contains a strong acid (H2SO4). Store at 2-30°C. H. 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 25ml and 50ml 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.

Plate: Pre-coated

Reagent Preparation:

1. 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. Note: Do not use the working substrate if it looks blue.

Sample Collection: The specimens shall be blood, serum or heparanised plasma in type and taken with the usual precautions in the collection of venipuncture samples. For accurate comparison to establish normal values, a fasting morning serum sample should be obtained. The blood should be collected in a redtop (with or without gel additives) venipuncture tube(s) or for plasma use 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. 050ml of the specimen is required.

Calculation of Results:

A dose response curve is used to ascertain the concentration of estradiol 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 estradiol concentration in pg/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 estradiol 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 pg/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. 025 ml (25 µl) of the appropriate serum reference, control or specimen into the assigned well. 3. Add 0. 050 ml (50µl) of the Estradiol Biotin Reagent to all wells. 4. Swirl the microplate gently for 20-30 seconds to mix. 5. Cover and incubate for 30 minutes at room temperature. 6. Add 0. 050 ml (50µl) of Estradiol Enzyme Reagent to all wells. Add directly on top the reagents dispensed in the wells. 7. Swirl the microplate gently for 20-30 seconds to mix. 8. Cover and incubate for 90 minutes at room temperature. 9. Discard the contents of the microplate by decantation or aspiration. If decanting, blot the plate dry with absorbent paper. 10. 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. 11. Add 0. 100 ml (100µl) of 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. 12. Incubate at room temperature for 20 minutes. 13. 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. 14. Read the absorbance in each well at 450nm (using a reference wavelength of 620-630nm. The results should be read within 30 minutes of adding the stop solution. Note: Dilute the samples suspected of concentrations higher than 3000pg/ml 1:5 and 1:10 with estradiol 0 pg/ml calibrator or male patient serum pools with a known low value for estradiol.

Storage: 4 °C/-20 °C

References

Abdi, Salehnia, Hosseinkhani: "Kit ligand decreases the incidence of apoptosis in cultured vitrified whole mouse ovaries." in: Reproductive biomedicine online, Vol. 30, Issue 5, pp. 493-503, (2015) (PubMed).

Target Details

Target: Estradiol (17-Estradiol)

Background: Summary and Explanation of the test: Measurement of estradiol in serum or plasma is considered to be the most reliable way to assess its rate of production. Estradiol (17beta-estradiol) is a steroid hormone (molecular weight of 272. 3 daltons), which circulates predominantly protein-bound. In addition to estradiol, other natural steroidal estrogens include estrone, estriol and their metabolites. Natural estrogens are hormones secreted principally by the ovarian follicles and also by the adrenals, corpus luteum, and placenta and, in males, by the testes. Exogenous estrogens (natural or synthetic) elicit, to varying degrees, all the pharmacologic responses usually produced by endogenous estrogens. Estrogenic hormones are secreted at varying rates during the menstrual cycle throughout the period of ovarian activity. During pregnancy, the placenta becomes the main source of estrogens. At menopause, ovarian secretion of estrogens declines at varying rates. The gonadotropins of the anterior pituitary regulate secretion of the ovarian hormones, estradiol and progesterone, hypothalamic control of pituitary gonadotropin production is in turn regulated by plasma concentrations of the estrogens and progesterone. This complex feedback system results in the cyclic phenomenon of ovulation and menstruation. Estradiol determinations have proved of value in a variety of contexts, including the investigation of precocious puberty in girls and gynecomastia in men. Its principal uses have been in the differential diagnosis of amenorrhea and in the monitoring of ovulation induction. This kit uses a specific anti-estradiol antibody, and does not require prior sample extraction of serum or plasma. Cross-reactivity to other naturally occurring and structurally related steroids is low. The employment of several serum references of known estradiol concentration permits construction of a graph of activity and concentration. From comparison to the dose response curve, an unknown specimen's activity can be correlated with estradiol concentration. Intended Use: The Quantitative Determination of Estradiol 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 pg/ml should be greater than 1.3. 2. Four out of six quality control pools should be within the established ranges.