AFP, hCG, uE3 ELISA Kit

Catalog No. ABIN649089

Product Details

Target: AFP, hCG, uE3

Reactivity: Human

Detection Method: Colorimetric

Method Type: Competition ELISA

Application: ELISA

Purpose: Immunoenzymometric assay (TYPE 3 for hCG - AFP): The essential reagents required for an immunoenzymometric assay include high affinity and specificity antibodies (enzyme and immobilized), with different and distinct epitope recognition, in excess, and native antigen, biotinylated (AFP/HCG) antibody. After adding biotinylated antibody, the enzyme-labeled antibody and a serum containing the native antigen, reaction results between the native antigen and the antibodies, without competition or steric hindrance, to form a soluble sandwich complex. 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. Calibrators (6 x 1 ml/vial) (dried-1ml). Reconstitute each vial with 1ml of distilled or deionized water. The reconstituted calibrators are stable for one year at 2-8°C. B. AFP Enzyme Reagent (13ml/vial): One vial contains enzyme labeled antibody, biotinylated monoclonal mouse IgG in buffer, dye, and preservative. Store at 2-8°C. CHCG Enzyme Reagent (13ml/vial): One vial contains enzyme labeled antibody, biotinylated monoclonal mouse IgG in buffer, dye, and preservative. Store at 2-8°C. D. uE3 Biotin Reagent (6 ml/vial). One vial contains biotin labeled specific biotinylated affinity purified rabbit IgG in buffer, dye, and preservative. Store at 2-8°C. E. uE3 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. F. Streptavidin Coated Microplate (2 x 96 wells). One 96-well microplate coated with streptavidin and packaged in an aluminum bag with a drying agent. Store at 2-8°C. G. Wash Solution Concentrate (20 ml). One vial contains a surfactant in buffered saline. A preservative has been added. Store at 2-30°C. H. Sample Diluent (75 ml). One vial contains normal human serum free of hCG stabilized with preservatives. I. Substrate A (2 x 7ml/vial). One bottle contains tetramethylbenzidine (TMB) in buffer. Store at 2-8°C. J. Substrate B (2 x 7ml/vial). One bottle contains hydrogen peroxide (H2O2) in buffer. Store at 2-8°C. K. Stop Solution (2 x 8 ml/vial). One bottle contains a strong acid (1N HCl). Store at 2-30°C. L. 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 192 well kit see table on last page for 96 well kit.

Material not included: 1. Pipette (s) capable of delivering 25µl, 50µl, 100 (l volumes with a precision of better than 1. 5%. 2. Dispenser(s) for repetitive deliveries of 0. 100ml and 0. 300ml volumes with a precision of better than 1. 5%. 3. Microplate washers or a squeeze bottle (optional). 4. Microplate Reader with 450nm and 620nm wavelength absorbance capability. 5. Absorbent Paper for blotting the microplate wells. 6. Plastic wrap or microplate cover for incubation steps. 7. Vacuum aspirator (optional) for wash steps. 8. Timer. 9. 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 licensed reagents. 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. The diluted wash buffer can be stored at room temperature (20-27°C) for up to 60 days. 2. Patient Sample Preparation: For HCG patient samples (first trimester), dilutions should be made as follows: Place 0. 5 ml of Sample Diluent into a test tube and add 25 (l of patient sample. Vortex to mix. (Dilution 1:21). Remove 25ml of (1:21) dilution and dispense into another test tube containing 1. 0 ml of Sample Diluent (1/41) (Final Dilution 1:861). Assay the 1:861 dilutions and multiply the results by the dilution factor 861. If hCG from normal populations are to be run, no dilutions are required (unless the patient's hCG is suspected to be greater than 250mIU/ml). 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 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 -20 °C or cooler for up to 30 days, in smaller aliquots. Avoid repetitive freezing and thawing. When assayed in duplicate, 0. 125ml of the specimen is required for all three (3) parameters.

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 (as is and dilutions) 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 and specimens (diluted for hCG) into the assigned well. (For AFP and hCG): 3a. Add 0. 100 ml (100µl) of the AFP Enzyme Reagent or HCG Enzyme Reagent to each well. It is very important to dispense all reagents close to the bottom of the coated well. (For uE3): 3b. Add 0. 050 ml (50µl) of the U-Estriol Enzyme Reagent to all wells. Swirl the plate gently for 20-30 seconds to mix the contents. 3c. Add 0. 050 ml (50 (l) of the U-Estriol Antibody biotin reagent to all the wells. 4. Swirl the microplate gently for 20-30 seconds to mix and cover. 5. Incubate 60 minutes at room temperature. 6. Discard the contents of the microplate by decantation or aspiration. If decanting, tap and blot the plate dry with absorbent paper. 7. 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. 8. 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. 9. Incubate at room temperature for 15 minutes. 10. Add 0. 050ml (50µl) of stop solution to each well and mix gently for 15-20 seconds. Always add reagents in the same order to minimize reaction time differences between wells. 11. 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.

Storage: 4 °C/-20 °C

Target Details

Target: AFP, hCG, uE3

Background: Summary and Explanation of the test: Monitoring of HCG, AFP and uE3 concentrations, at regular intervals, are considered to be very important to determine the fetal well-being. The collective information provided by these three assays (Triple Screen) provides the clinician with the comprehensive picture of the development of a healthy fetus and the health of the mother. Any anomaly seen during the first trimester can be corrected unless it is caused by some genetic abnormality. We provide the clinician with a single tool to monitor all three analytes, using 125 µl of patient serum (50µl for AFP, 50µl for uE3 and 25 µl for hCG), in a single 75 minutes combination assay. Alpha-Fetoprotein (AFP) is a glycoprotein with a molecular weight of 70 kDA. AFP is normally produced during fetal development by the hepatocytes, yolk sac and to a lesser extent by the gastrointestinal tract. Serum concentrations reach the highest level at twelve weeks of gestation. This peak level gradually decreases to less than 25 ng/ml after one year of postpartum. Thereafter, the levels reduce further to less than 10 ng/ml. The presence of abnormally high AFP concentrations in pregnant women is considered a risk marker for open neural tube defects (ONTDs). Elevated levels of AFP are found in patients with primary hepatoma and yolk sac-derived germ tumors. AFP is the most useful marker for the diagnosis and management of hepatocellular carcinoma. Human chorionic gonadotropin (hCG) concentration increases dramatically in blood and urine during normal pregnancy. hCG is secreted by placental tissue, beginning with the primitive trophoblast, almost from the time of implantation, and serves to support the corpus luteum during the early weeks of pregnancy. HCG or hCG similar glycoproteins can also be produced by a wide variety of trophoblastic and nontrophoblastic tumors. The measurement of hCG, by assay systems with suitable sensitivity and specificity has proven great value in the detection of pregnancy and the diagnosis of early pregnancy disorders. According to the literature, serum and urine concentrations of biologically active (non-nicked) hCG is detectable as early as 10 days after ovulation, reaching 100 mIU/ml by the first missed period. It rises exponentially in the first trimester, doubling almost every 48 hours to a peak (50,000 to 200,000 mIU/ml) by the end of the first trimester. Then a gradual decline is observed reaching approximately one fifth of the peak and remains at this level until term. 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 plasma during pregnancy 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)The AccuBind Pregsafe (Triple Screen) measures not only AFP, but beta-HCG and unconjugated Estriol (uE3) as well. The test is more accurate and screens for additional genetic disorders. Generally speaking the combination test will identify greater than 60% of the babies with Down Syndrome and 80-90% of the babies with neural tube defects. This option had not been available, especially in developing countries, with conventional testing like ultrasound alone. (11)In this method, the combination calibrator (containing different levels of AFP, HCG and E3), patient specimen or control is first added to a streptavidin coated well. Biotinylated monoclonal and enzyme labeled antibodies (directed against distinct and different epitopes of AFP and HCG) are added and the reactants mixed. Reaction between the various analyte specific antibodies and native analyte forms a sandwich complex that binds with the streptavidin coated to the well. In the case of uE3 an E3 analog coupled with HRP (Enzyme) is added followed by specific biotinylated E3 antibody. A competition occurs between labeled E3 and the native E3 for a limited number of sites on the antibody. After the completion of the required incubation period, the excess enzyme labeled antibody or analog is washed off via a wash step. Addition of a suitable substrate produces color, In HCG and AFP the intensity of the color is directly proportional to the concentration while in E3 it is inversely proportional to the concentration of the analyte. The employment of several serum references of known levels of hCG, AFP and E3 permits the construction of a dose response curve of activity and concentration. From comparison to the dose response curve, an unknown specimen's concentration can be interpolated. Intended Use: The Quantitative Determination of Alpha-Fetoprotein (AFP), Chorionic Gonadotropin (hCG) and Unconjugated Estriol (uE3) Concentration in Human Serum by a Microplate Immunoenzymometric assay. Q. C. Parameters: In order for the assay results to be considered valid the following criteria should be met:For AFP & HCG the absorbance (OD) of calibrator F should be greater than 1. 3. For uE3 the absorbance of calibrator A should be greater than 1. 3. 3. Four out of six quality control pools should be within the established ranges.