AFP, hCG, uE3 CLIA Kit

Produktnummer ABIN504762

Das Human AFP, hCG, uE3 ELISA Kit (ABIN504762) ist ein Chemiluminescent ELISA Kit zur Detektion von Human AFP, hCG, uE3.

Kurzübersicht für AFP, hCG, uE3 CLIA Kit (ABIN504762)

Target: AFP, hCG, uE3

Reaktivität: Human

Nachweismethode: Chemiluminescent

Methodentyp: Sandwich ELISA

Applikation: ELISA

Produktdetails

Verwendungszweck: 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.

Analytische Methode: Quantitative

Produktmerkmale: The Quantitative Determination of Alpha-Fetoprotein (AFP), Chorionic Gonadotropin (hCG) and Unconjugated Estriol (uE3) Concentration in Human Serum by a Microplate Chemiluminescence assay (For Research Use Only)

Anwendungsinformationen

Applikationshinweise: 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.

Plattentyp: Pre-coated

Protokoll:

Specimien Collection and Preparation:

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_x001E_8oC for a maximum period of five (5) days. If the specimen(s) cannot be assayed within this time, the sample(s) may be stored at _x001E_20oC 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.

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. 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 25(l 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 patients hCG is suspected to be greater than 250mIU/ml). 3. Working Signal Reagent Solution - Store at 2 - 8(C. Determine the amount of reagent needed and prepare by mixing equal portions of Signal Reagent A and Signal Reagent B in a clean container. For example, add 1 ml of A and 1ml of B per two (2) eight well strips (A slight excess of solution is made). Discard the unused portion if not used within 36 hours after mixing. If complete utilization of the reagents is anticipated, within the above time constraint, pour the contents of Signal Reagent B into Signal Reagent A and label accordingly. .

Test Procedure:

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 (25l) of the appropriate serum reference, con_x001F_trol and specimens (diluted for hCG) into the assigned well. (For AFP and hCG): 3a. Add 0.100 ml (100l) of the AFP Tracer Reagent or HCG Tracer 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 (50l) of the U-Estriol Tracer 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 45 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 350l of wash buffer (see Reagent Preparation Section), decant (tap and blot) or aspirate. Repeat two (2) additional times for a total of five (5) washes. An automatic or manual plate washer can be used. Follow the manufacturers 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 (2) additional times. 8. Add 0.100 ml (100l) of working signal solution to all wells (see Reagent Preparation Section). Always add reagents in the same order to minimize reaction time differences between wells. 9. Incubate at room temperature for five (5) minutes. 10. Read the RLUs (Relative Light Units) in each well in a microplate luminometer for at least 0.2 seconds/ well. The results can be read within 30 minutes of adding the signal solution.

Beschränkungen: Nur für Forschungszwecke einsetzbar

Handhabung

Antigendetails

Target: AFP, hCG, uE3

Hintergrund: 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. Monobind provides the clinician with a single tool to monitor all three analytes, using 125 (l of patient serum (50(l for AFP, 50(l for uE3 and 25(l 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 100mIU/ml by the first missed period. It rises exponentially in the first trimester, doubling almost every 48 hours to a peak (50,000 to 300,000mIU/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 Monobind AccuLite( 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 > 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 (Tracer) 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 light, In HCG and AFP the intensity of the light 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.