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within a week or so of the estimated date of conception. SPECIMEN COLLECTION AND HANDLING to get the most reliable and consistent results. 3. VISUAL COLOR TEST FOR EARLY PREGNANCY Catalog No. MYM/US-V_HCG For In Vitro Diagnostic Use Only NAME AND INTENDED USE The MYM hCG HP-501 is designed for in vitro qualitative detection of Human Chorionic Gonadotropin (hCG) in patient’s urine and serum. It is a visual early pregnancy test. (For in vitro diagnostic use) PRINCIPLE OF THE ASSAY Urine specimen UBI HCG- MYM is a solid phase enzyme-linked immunosorbent assay (ELISA) based on the Collect urine in a container without any preservative. The preferred specimen is the first morning urine because it usually has the maximum concentration of hCG. Specimen that cannot be assayed immediately must be stored at 2-8oC for 24 hrs. or frozen up to six months. Avoid repeated freezing and thawing of urine sample. sandwich principle. The wells are coated with a monoclonal antibody directed toward a unique antigenic site on the hCG molecule. The samples are incubated in the wells with enzyme conjugate, which is a second monoclonal antibody, directed toward a different regions of hCG molecule and conjugated with horseradish peroxidase, to form a sandwich complex bound to the well. Unbound conjugate are then washed off with tap water. The amount of bound peroxidase is proportional to the concentration of the hCG present in the sample. Upon addition of the substrate and chromogen, the intensity of blue color developed is proportional to the concentration of hCG in the sample. Serum Specimen Collect blood by venipuncture. Allow it to clot and separate the serum by centrifugation at room temperature. If sera cannot be assayed immediately, they can be stored at 2-8C for a week or frozen for up to six months. Avoid repeated freezing and thawing of serum sample. Chorionic Gonadotrpopin (hCG) is a gycoprotein hormone normally produced by the placenta during pregnancy. The hormone is present in blood and urine around seven to thirteen days following implantation of the fertilized ovum. hCG increases in concentration at a geometric rate, reaching a peak near the end of the first trimester. High concentrations are observed throughout pregnancy. After parturition, concentration declines rapidly, and becomes undetectable within a few days. Structurally intact hCG molecules consist of two non-covalently linked poly-peptide subunits, the alpha and beta chain subunits. The alpha subunit is similar to that of other glycoprotein hormones, such as Thyroid Stimulating Hormone (TSH), Luteinizing Hormone(LH), and Follicle Stimulating Hormone(FSH). The differences in the amino acid sequences of the beta subunits of the respective hormones accounts for their target organ specificities and immunochemical distinctiveness. Monoclonal antibodies produced from hybridomas represent an essentially unlimited supply of homogeneous antibody of precisely defined specificity. Monoclonal antibodies which react with unique sites on the beta chain of the hCG molecule are essential for differentiation between hCG and LH, FSH and TSH. Assay for the beta subunit has permitted the detection of pregnancy ASSAY PROCEDURE 1. 2. 3. 4. 5. 6. 7. 8. 9. SUMMARY AND EXPLANATION OF TEST WARNING AND PRECAUTION MYM kit is designed for in vitro use only. The Reference and Negative Controls are formulated in a salt-protein matrix. These Controls can be applied to both urine or serum specimens. The components in this kit are intended for use as an integral unit . The components of different lots should not be mixed. MATERIALS PROVIDED 1. 2. 3. 4. 5. STORAGE AND STABILITY Store the kit at 2-8C in a refrigerator. Keep MAGIWEL in a dry bag with 7. 8. desiccants. The unopened reagents are stable until expiration of the kit. Solution A and Solution B should be colorless; if solutions turn blue, it must be replaced. Do not expose test reagents to strong light during storage or usage. Secure the desired number of coated wells in the holder. Mark data sheet with sample identification. Dispense 50 uL of urine or serum or controls into appropriate wells. Dispense 50 uL of enzyme conjugate into each wells Incubate for 5 minutes or longer at room temperature. Remove incubation mixture by flicking the plate into sink. Rinse the wells 5 times with running tap water. Dispense 50 ul of Solution A and 50 ul of Solution B into each well. Incubate 5 minutes or longr at room temperature. Observe blue color development and mark data. (UBI HCG MAGIWEL Cat No. HP-501) 6. Use a new disposable pipet tip for each specimen. Microwell Strips: Anti-hCG monoclonal antibodies coated wells. Enzyme Conjugate (6 mL): Anti-hCG monoclonal antibody conjugated to horseradish peroxidase. Solution A : Buffer solution containing peroxide. Solution B: Tetramethybenzidine. Negative Control (0.75 mL): Saltprotein matrix with 0.01% proclin. Cut off control(0.75 mL): Calibrated to 25 mIU hCG/mL with preservative (0.01% proclin). Positive control: Calibrated to 100 mIU hCG/mL with preservative (0.01% proclin). Well Holder: For securing individual wells. PREPARATION FOR ASSAY 1. 2. Before beginning the test, bring all samples and reagents to room temperature (20-25C) and gently mix. Have all reagents and samples ready before the start of the assay. Once the test has begun it must be performed without any interruptions RESULTS AND INTERPRETATION Negative Results No color indicates a nondetectable amount of hCG in the sample. Positive Results Blue color the same as or more intense than the Cut off Control (25 mIU/mL) indicates presence of hCG in the sample. Indeterminate Result Less blue color than the 25 mIU hCG/mL Cut off Control indicates hCG concentration less than 25 mIU/mL. Another sample taken from the patient at least 48 hours later should be reassayed side by side in the repeat assay. The Cut off, Positive and Negative Controls run with patient sample establish the validity of a test. The control concentrations of hCG were arbitrarily selected. To be positive or negative for pregnancy. Conditions such as hydatidiform mole, choriocarcinoma, trophoblastic or nontrophoblastic neoplasms may be associated with the presence of hCG in the sample. EXPECTED VALUES AND LIMITATIONS hCG is normally not detected in the urine or serum of healthy men and nonpregnant women by UBI HCG MAGIWEL. The urine hCG concentrations of pregnant women begin to rise as soon as the first week following implantation, reach 40 mIU/mL ten days after concenption, and reach a maximum concentration (in excess of 100,000 mIU/mL) during the latter part of the first trimester of pregnancy. During the first six weeks of pregnancy, serum hCG concentrations have a doubling time of approximately 2 days. Following delivery hCG concentrations rapidly decrease and usually return to normal in several days. High levels of hCG may persist after hCG injections in patients undergoing infertility therapy. HCG may be present in very low levels in ectopic pregnancies while conditions like choriocarcinoma, trophoblastic or nontrophoblastic neoplasms, or hydatidiform mole may result in high concentrations of hCG. PERFORMANCE CHARACTERISTICS Sensitivity The analytical sensitivity of the MAGIWEL test has been set at 25 mIU/mL based on the 1st international Standard (WHO 1st IRP) Interference The addition of 200 μg/ml of each of the following compounds does not interfere with MAGIWEL test for hCG: acetaminophen, ascorbic acid, acetylsalicylic acid, atropine, gentisic acid, methadone, and phenothiazine. Abnormally elevated levels of protein, ketones, bilirubin or hemoglobin do not affect the essay. Accuracy Analysis of Urine Samples: Urine samples from known non-pregnant subjects (N=30) are spiked with known amountsof hCG to a final concentration of 12.5, 25 and 50 mIU/mL. HCG Added (mIU/mL) Number of Assay Positive Negative Indeterminate Sensitivity(%) 0 12.5 25 50 30 0 30 0 30 3 2 25 30 28 0 2 93 30 30 0 0 100 Analysis of Serum Samples: Serum samples from known non-pregnant subject (N=20) are spiked with known amounts of hCG to a concentration of 12.5, 25, 50 mIU/mL. Specificity 250 mIU/mL hFSH 1,000 mIU/mL hTSH 1,000 IU/mL Negative Negative Negative HCG Added (mIU/mL) Number of Assay Positive Negative Indeterminate Sensitivity(%) 0 20 0 20 0 12.5 25 50 20 0 20 0 20 19 0 1 95 20 20 0 0 100 Qualitative EIA Quantitative EIA 100 100 100 44 44 44 56 56 56 Each laboratory should utilize internal controls at several levels to monitor assay performance. The control should be treated as unknown. Results obtained should be in agreement with the assigned value of the control. The controls can be obtained from commercially available sources, but should not contain sodium azide as preservative. REFERENCE 1. 3. 4. Correlation with EIA Methods Serum samples from 100 females are analyzed by MAGIWEL visual test and with other accepted EIA methods as following: UBI MAGIWEL Quality Control 2. The immunological specificity of MAGIWEL test was determined from cross reaction with known quantities of purified preparation of Luteinizing Hormone (hLH), Follicle Stimulating Hormone (FSH) and Thyroid Stimulating Hormone (hTSH). hLH Methods Number of Assay No.of Samples with hCG > 25 mIU/mL No.of Samples with hCG < 25 mIU/mL 5. Lenton, E. L. Neal and R. Sulaimlan. Plasma concentrations of human gonadotropin from time of implantation until second week of pregnancy. Fertility and Sterility 37: 773, 1982. Braunstein, G.D. First-trimester chorionic gonadotropin measurements as an aid in the diagnosis of early pregnancy disorders Am. J. Obst. Gynec. 131:25, 1978. Kosasa, T.S. Measurement of human chorionic gonadotropin. J. Reprod. Med. 26:201, 1981. Catt, K.J., Dufau, M.L., and Vaitukaitis, J.L., Appearance of hCG in Pregnancy plasma following the initiation of implantation of the blastrocyst. J.Clin.Endocr. Metab. 40:537, 1975. Uotila, M., Ruoslahti, E., and Engvall, E., Two-site sandwich enzyme immunoassay with monoclonal antibodies to human alpha fetoprotein. J. Immunol Methods 42: 11, 1981. 6. Braunstein, G.D., Rasor,J., Adler,D.,Danzer,H.,and Wade,M.E.Serum human chorionic gonadotropin levels throughout normal pregnancy. Am.J.Obstet.Gynecol. 126:678, 1976. 7. Batzer,F. Hormonal evaluation of early pregnancy. Fertility and Sterility 34:1, 1980. 8. Dipietro, D.L., Ectopic pregnancy: interpreting HCG levels. Laboratory Management 19:11, 1981 9. Engvall, E., and Perlmann, P., Enzyme linked immunosorbent assay (ELISA) quantitative assay of immunoglobulin. Immunochemistry 8:871, 1971. 10. Bangham, D.R. and Storring, P.L. Standardization of human choironic gonadotropin, hCG subunits, and pregnancy test. Lancet 1:390, 1982. Mar 2006 (2)