Rapid-hCG ELISA Kit

Catalog No. ABIN649054

Product Details

Target: Rapid-hCG

Reactivity: Human

Detection Method: Colorimetric

Method Type: Sandwich ELISA

Application: ELISA

Purpose: Immunoenzymometric assay: The essential reagents required for an immunoenzymometric assay include high affinity and specificity antibodies (signal and capture), with different and distinct epitope recognition, in excess, and native antigen. In this procedure, the calibrator, control or patient sample is added to the wells coated with anti HCG antibody. HCG from the sample binds to the Anti-HCG (MoAb) on the wells. Subsequently an enzyme labeled Anti-HCG is added to the wells. HCG from the sample forms a sandwich between the two antibodies. Excess enzyme and sample is removed via a wash step. The enzyme activity in the antibody-bound fraction is directly proportional to the native antigen concentration. By utilizing several different serum references of known antigen values, a dose response curve can be generated from which the antigen concentration of an unknown can be ascertained. A suitable substrate is added to the wells to generate color in varying intensity depending upon the concentration of HCG in the wells. The intensity of the color in the sample can be visually compared to the known calibrators to obtain qualitative results or the color development can be read with the help of a microplate spectrophotometer to obtain semi-semi-quantitative results.

Analytical Method: Quantitative

Components: A. hCG Calibrators (1ml/vial) (Lyophilized). Five vials, of references for hCG Antigen at levels of 0(A), 25(B), 50(C), 100(D) and 250(E) mIU/ml. Store at 2-8°C. Reconstitute each vial with 1. 0ml of distilled or deionized water. Note: The calibrators, human serum based, were calibrated using a reference preparation, which was assayed against the WHO 3rd IS (75/537). B. Anti-hCG Enzyme Conjugate (13 ml/vial). One vial, containing enzyme labeled affinity purified Goat Anti-HCG (IgG) in buffer, dye, and preservative. Store at 2-8°C. C. Anti-HCG Coated Micropla(96 wells). One 96-well microplate coated with Anti-HCG (MoAb-IgG) and packaged in an aluminum bag with a drying agent. Store at 2-8. D. Substrate Reagen(14 ml/vial). One bottle, containing tetramethylbenzidine (TMB) and H2O2 in a buffer. Store at 2-8°C. E. Wash Solution Concentrate (20ml). One vial containing a surfactant in buffered saline. A preservative has been added. Store at 2-30°C (see Reagent Preparation Section). F. Stop Solution (8 ml/vial). One bottle, containing 1N HC l. Store at 2-8°C. 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. Pipets capable of delivering 25µl, 50µl and 100µl volumes with a precision better than 1. 5%. 2. Plastic wrap or microplate cover for incubation steps. 3. Microplate Reader with 450nm and 620nm wavelength absorbance capability. (For Semi-Semi-quantitative Interpretation Only). 4. Absorbent Paper for blotting the microplate wells. 5. Timer. 6. Quality control materials. 7. Urine collection containers.

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.

Sample Volume: 25 μL

Plate: Pre-coated

Reagent Preparation:

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.

Sample Collection: 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. Centrifuge the specimen to separate the serum from the cells. Urine Sample: Collect urine sample in a clean container. For most accurate results it is advisable to collect first morning urine sample. 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. 05 ml of the specimen is required.

Calculation of Results:

A dose response curve is used to ascertain the concentration of human chorionic gonadotropin in unknown specimens. 1. Record the absorbance obtained from the printout of the microplate reader. 2. Plot the absorbance for each serum reference versus the corresponding hCG concentration in mIU/ml on linear graph paper. 3. Draw the best-fit curve through the plotted points. 4. To determine the concentration of hCG for an unknown, locate the average absorbance of 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. Note: Computer data reduction software designed for IEMA/ ELISA assays may also be used for the data reduction.

Restrictions: For Research Use only

Handling

Handling Advice: Before proceeding with the assay, bring all reagents, references controls and samples to room temperature (20-27°C). 1. Format the microplate wells for each serum reference, control and patient specimen to be assayed. Replace any unused microwell strips back into the aluminum bag, seal and store at 2-8°C. 2. Dispense pipette 0. 025 ml (25µl) of the appropriate serum reference, control or specimen into the assigned well. 3. Add 100µl of hCG-Enzyme Conjugate solution to all wells. 4. Swirl the microplate gently for 5-10 seconds to mix and incubate at room temperature for 10 minutes. 5. Discard the contents of the microplate by decantation or aspiration. If decanting, blot the plate dry with absorbent paper. 6. 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 used, fill each well to the top by squeezing the container. Avoiding air bubbles. Decant the wash and repeat two additional times. 7. Add 100µl of substrate solution to all the wells. DO NOT SHAKE THE PLATE AFTER SUBSTRATE ADDITION. 8. Incubate at room temperature for 5 minutes. 9. For Qualitative results see Interpretation of Results Section. 10. For Semi-quantitative results go to stepNumber 11 below. 11. Add 50µl of stop solution to each well and gently mix. 12. Read the absorbance in each well at 450nm using a reference wavelength of 620-630nm to minimize well imperfections) in a microplate reader. Note: The results should be read within 30 minutes of adding the stop solution.

Storage: 4 °C/-20 °C

Target Details

Target: Rapid-hCG

Background: 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 diagnosis of early pregnancy disorders. Mishell et al , reported data obtained by immunologic measurements of hCG in urine and serum obtained throughout pregnancy. Values for serum or urine (voided in the morning) were similar. There was a rapid rise in the hCG levels after conception that peaked between 60-80 days of pregnancy, with a mean value of 100,000 mIU/ml, followed by a gradual fall to 20,000 mIU/ml by day 120. In this method, hCG calibrator, patient specimen or control is first added to the antibody coated well. Monoclonal enzyme labeled antibody (directed against distinct and different epitopes of hCG) is added and the reactants mixed. Reaction between the various hCG antibodies and native hCG forms a sandwich complex that binds with the antibody coated to the well. After the completion of the required incubation period, the enzyme-Chorionic Gonadotropin antibody bound conjugate is separated from the unbound enzyme-Chorionic Gonadotropin conjugate by aspiration or decantation. The activity of the enzyme present on the surface of the well is quantitated by reaction with a suitable substrate to produce color. The employment of several serum references of known Chorionic Gonadotropin levels permits 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 Chorionic Gonadotropin concentration. Intended Use: The Visual Qualitative or Spectrophotometric Semi-Semi-quantitative Determination of hCG in Human Serum or Urine by a Microplate Immunoenzymometric assay for early detection of pregnancy. Q. C. Paramters: In order for the assay results to be considered valid the following criteria should be met. The absorbance (OD) of calibrator E should be greater than 1.0. 2. Four out of six quality control pools should be within the established ranges.