Free Chorionic Gonadotropin ELISA Kit
F-BetaCG
Reaktivität: Human
Colorimetric
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- Target
- Free Chorionic Gonadotropin (F-BetaCG)
- Reaktivität
- Human
- Nachweismethode
- Colorimetric
- Applikation
- ELISA
- Verwendungszweck
- Immunoenzymometric sequential assay (TYPE 4): 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. In this procedure, the immobilization takes place during the assay at the surface of a microplate well through the interaction of streptavidin coated on the well and exogenously added biotinylated monoclonal anti-fbeta-hCG antibody. Upon mixing monoclonal biotinylated antibody, and a serum containing the native antigen, reaction results between the native antigen and the antibody, forming an antibody-antigen complex. Simultaneously the biotin attached to the antibody binds to the streptavidin coated on the microwells resulting in a complete immobilization of the complex. Excess enzyme is washed off via a wash step. A suitable substrate is added to produce color measurable with the use of a microplate spectrophotometer. The enzyme activity on the well is directly proportional to the native free 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.
- Bestandteile
- A. fbeta-hCG Calibrators (1ml/vial). Six vials of references fbeta-hCG Antigen at levels of 0 (A), 2 (B), 5 (C), 10 (D), 25 (E) and 50 (F) mIU/ml. Store at 2-8°C. A preservative has been added. Note: The calibrators, human serum based, were calibrated using a reference preparation, which was assayed against the WHO 1PstP IRP (75/551). Conversion to mass units equal One mIU/ml is equivalent to 1 ng/ml B. f alpha hCG Biotin Reagent (13ml/vial): One vial containing biotin labeled monoclonal mouse IgG in buffer, dye, and preservative. Store at 2-8°C. C. f alpha hCG Enzyme Reagent (13ml/vial): One vial containing Enzyme (HRP) labeled monoclonal mouse IgG in buffer, dye, and preservative. Store at 2-8°C. D. Streptavidin Coated Plate (96 wells). One 96-well microplate coated with streptavidin and packaged in an aluminum bag with a drying agent. Store at 2-8°C. E. Wash Solution Concentrate (20 ml). One vial containing a surfactant in buffered saline. A preservative has been added. Store at 2-30°C. F. Substrate A (7ml/vial). One bottle containing tetramethylbenzidine (TMB) in buffer. Store at 2-8°C. G. Substrate B (7ml/vial). One bottle containing hydrogen peroxide (HB2BOB2B) in buffer. Store at 2-8°C. H. Stop Solution (8m/vial). One bottle containing 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.
- Benötigtes Material
- 1. Pipette capable of delivering 25µl and 50µl 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. 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.
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- Applikationshinweise
- 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.
- Aufbereitung der Reagenzien
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- Wash Buffer: Dilute contents of wash concentrate to 1000ml with distilled or deionized water in a suitable storage container. Store at room temperature 20-27 °C for up to 60 days. 2. Working Substrate Solution: 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.
- Probennahme
- The specimens shall be blood, serum 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 without additives or anti-coagulants. Allow the blood to clot for samples. Centrifuge the specimen to separate the serum 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.
- Ergebnisberechnung
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A dose response curve is used to ascertain the concentration of fbeta-Chorionic Gonadotropin 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 fbeta-hCG concentration in mIU/ml on linear graph paper (do not average the duplicates of the serum references before plotting). 3. Draw the best-fit curve through the plotted points. 4. To determine the concentration of fbeta-hCG 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 mIU/ml) from the horizontal axis of the graph (the duplicates of the unknown may be averaged as indicated).
- Beschränkungen
- Nur für Forschungszwecke einsetzbar
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- Handhabung
- 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 (25µl) of the appropriate serum reference, control or specimen into the assigned well. 3. Add 0. 100 ml (100µl) of the f(hCG Biotin Reagent to each well. It is very important to dispense all reagents close to the bottom of the coated well. 4. Swirl the microplate gently for 20-30 seconds to mix and cover. 5. Incubate 30 minutes at room temperature. 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 the fbetahCG Enzyme Reagent to each well. DO NOT SHAKE THE PLATE AFTER ENZYME ADDITION 9. Cover and incubate 30 minutes at room temperature. 10. Discard the contents of the microplate by decantation or aspiration. If decanting, blot the plate dry with absorbent paper. 11. Add 300µl 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. Followthe 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. 12. 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. 13. Incubate at room temperature for 15 minutes. 14. 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. 15. 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.
- Lagerung
- 4 °C/-20 °C
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- Target
- Free Chorionic Gonadotropin (F-BetaCG)
- Andere Bezeichnung
- Chorionic Gonadotropin (Beta-Subunit) (Free) (fbeta-hCG)
- Hintergrund
- Summary and Explanation of the test: 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 potential diagnosis of early pregnancy disorders. Free beta-hCG subunit testing has improved the diagnostic probability of abnormal pregnancy/disease states (1). Patients with trophoblastic diseases produce ordinary and irregular forms of hCG, e. G. nicked hCG, hCG missing the beta-subunit C-terminal segment, hyperglycosylated hCG and free beta subunit. On the other hand common epithelial tumors of the urogenital tract frequently express the free beta-Subunit of hCG with no concomitant expression of its heterodimer partner, the common alpha subunit of the glycoprotein hormone. While most hCG assays do a very good job of monitoring the normal pregnancies, still there needs to be a system of differential diagnosis of ovarian tumors, epithelial tumors and trophoblastic malfunctions. That is where determination of free alpha subunit, free alpha subunit, nicked hCG and nonnicked hCG etc are of individual value. Although fbeta-hCG normally constitutes less than 1% of the total hCG concentrations in normal pregnancy, it constitutes a significant part (as much as 26% of hCG) in trophoblast disease (2,3). There is also increasing evidence that free beta subunit may be better than total hCG measurement in assessing Down's Syndrome (4). In this method, fbeta-hCG calibrator, patient specimen or control is first added to a streptavidin coated well. Biotinylated monoclonal antibody (specific for fbeta-hCG) is added and the reactants mixed. Reaction between the fbeta-hCG antibody and native fbeta-hCG forms complex that binds with the streptavidin coated to the well. The excess serum proteins are washed away via a wash step. Another enzyme labeled monoclonal antibody specific to f alpha hCG is added to the wells. The enzyme labeled antibody binds to the f alpha hCG already immobilized on the well through its binding with the biotinylated monoclonal antibody. Excess enzyme is washed off via a wash step. A color is generated by the addition of a substrate. The intensity of the color generation is directly proportional to the concentration of the f alpha hCG in the sample. The employment of several serum references of known free alpha Chorionic Gonadotropin levels permits the construction of a dose response curve of activity and concentration. From comparison to the dose response curve, an unknown specimen's activity can be correlated with free alpha Chorionic Gonadotropin concentration. Intended Use: The Quantitative Determination of Free Beta (beta) Chorionic Gonadotropin Subunit Concentration in Human Serum by a Microplate Immunoenzymometric assay. Q. C. Parameters: Maximum Absorbance (Calibrator F) equal greater than1. 3Maximum Absorbance (Calibrator A) equal Ulower thanU 0. 1
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