Estriol ELISA Kit

Catalog No. ABIN649067

This Colorimetric ELISA kit is designed for the quantitative measurement of Human Estriol.

Quick Overview for Estriol ELISA Kit (ABIN649067)

Target: Estriol

Reactivity: Human

Detection Method: Colorimetric

Method Type: Competition ELISA

Application: ELISA

Product Details Estriol ELISA Kit

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. u-Estriol Calibrators (1ml/vial). Six vials of serum reference for unconjugated estriol at concentrations of 0 (A), 0. 4 (B), 2. 0 (C), 5. 0 (D), 15 (E), and 30. 0 (F) 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. 45. For example: 1ng/ml x 3. 45 equal 3. 45 nM/LB. u-Estriol Enzyme Reagent (6. 0 ml/vial). One vial of Estriol (Analog)-horseradish peroxides (HRP) conjugate in a protein stabilizing matrix with red dye. Store at 2-8°C. C. u-Estriol Biotin Reagent (6. 0 ml). One bottle of reagent contains anti-unconjugated Estriol biotinylated purified rabbit IgG conjugate in buffer, blue 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 A (7ml/vial). One vial contains tetramethylbenzidine (TMB) in buffer. Store at 2-8°C. G. Substrate B (7ml/vial). One vial contains hydrogen peroxide (H2O2) in buffer. Store at 2-8°C. H. Stop Solution (8ml/vial). One vial contains a strong acid (1N HCl). Store at 2-30°C. I. 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. 2. 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 heparanised plasma in type and taken with the usual precautions in the collection of venipuncture samples. The blood should be collected in a redtop (with or without gel additives) venipuncture tube 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 unconjugated estriol 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 unconjugated estriol 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 unconjugated estriol 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 calibrator, 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 (25µl) of the appropriate serum reference, control or specimen into the assigned well. 3. Add 0. 050 ml (50µl) of the U-Estriol 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 U-Estriol 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. The results should be read within 30 minutes of adding the stop solution. Note: Dilute the sample, suspected of concentrations higher than 30ng/ml, by diluting 1:2 and/or 1:5 with unconjugated estriol 0 ng/ml calibrator or male patient sera with a known low value for estriol. Multiply the result by the dilution factor of 2 or 5 as required to obtain the concentration of the sample.

Storage: 4 °C/-20 °C

Target Details for Estriol

Target: Estriol

Target Type: Hormone

Background: Summary and Explanation of the test: The last few years have seen the development of screening for fetal Down Syndrome by measurement of multiple markers in maternal blood (1). Although amniocentesis has been widely available for more than 40 years it can only be selectively used to diagnose the disorder because of the hazard to fetus. Of most employed for differential diagnosis the commonly used procedures are AFP, hCG, free beta-HCG and unconjugated estriol. (2) Unconjugated estriol in the serum of pregnant women originates almost exclusively from precursors in the fetus, via the placenta. (3) The clinical evidence shows that in uncomplicated pregnancies, the production of estriol increases steadily throughout the last trimester, however, in pregnancies complicated by placental insufficiency the synthesis of estriol decreases rapidly. For many years the most commonly used method for monitoring estriol synthesis (as an index to fetal stress) has been to measure estriol and estriol conjugates in a 24 hr urine sample(4). However, changes in renal clearance and diurnal variations can make the results of these determinations suspect. In recent years investigators have found the determinations of unconjugated estriol in pregnancy plasma as an alternative to the urinary assay to be a better marker of fetal stress(6). Abnormally low levels of estriol in a pregnant woman may indicate a problem with the development in the child. Levels of estriol in non-pregnant women do not change much after menopause, and levels are not significantly different from levels in men (7). Unconjugated estriol ELIA Kits use a specific anti-estriol antibody, and do 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 Estriol 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 Estriol concentration. Intended Use: The Quantitative Determination of Unconjugated (Free) Estriol 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.