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| GENTAUR • Belgium : + 32 2 7325688 • France : 01 43250150 • Italy : 02 36006593 • Germany : +49 241 6085 13140 | |
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ßhCG ELISA KIT (REF: EK-76) / 475 Euro The human chrorion gonadotrophin ( βhCG) ELISA system provides a direct quantitative determination of βhCG in human serum. βhCG can be assayed in the range of 0-100 mIU/ml using 100 µl serum samples. Each kit contains materials sufficient for 96 determinations permitting the construction of one standard curve and the assay of 42 unknowns in duplicate.Introduction Human Chorionic Gonadrotrophin (hCG) is a glycoprotein with a molecular weight of 38000, secreted by the placenta. Like other glycoprotein hormones (hLH, hTSH and hFSH), ßhCG contains two different subunits, an α- and a β-chain, linked by noncovalent bindings. The primary structures of the α subunits of these hormones are virtually identical, while their β subunits, responsible for the immunological and biological specificity, are different. Thus a specific determination of ßhCG can only be made by the determination of its β component. The measured ßhCG content results almost exclusively from intact ßhCG molecules but there can be a contribution, albeit a usually negligible fraction of the total, from the free βhCG subunit.ßhCG appears in the sera of pregnant women five days after the implantation of blastocyst and its concentration continually increases until the third month of the pregnancy. The maximum concentration can reach values up to 100 IU/ml. Then the hormone level drops to 25 IU/ml and stays around this value until the last trimester. Elevated ßhCG concentrations are frequently seen in the case of trophoblastic and nontrophoblastic neoplasia, and choriocarcinoma. Ectopic hormone production can also be found in the metastatic breast cancer and with tumours of the liver, stomach, lung, and uterus often results in the elevated ßhCG concentration both in men and in non pregnant women. The current sandwich ELISA system is particularly designed for the direct determination of neoplastic βhCG, whilst gestational βhCG levels can be measured after the pre-dilution of patient sera.Principle of method This technology uses two high affinity monoclonal antibodies in an immunometric assay system. The two antibodies react simultaneously with the antigen present in standards or samples This reaction leads to the formation of a capture antibody - antigen - signal antibody complex, also referred to as a "sandwich". In the solid-phase ELISA system the reaction is carried out in a microtiter plate which acts as the binder of sandwich complex. In the present product standards and samples are incubated with the conjugate which contains the horseradish peroxidase (HRPO) labelled antibody at 37 oC for 2 hours, then washed repeatedly. After the addition of a ready-to-use tetramethyl-benzidine (TMB)/peroxide substrate the signal is measured in an ELISA photometer at 450 nm wavelength. The concentration of antigen is directly proportional to the optical density measured in the wells. The unknown concentration of ßhCG in patient samples is read off a calibration curve constructed by plotting binding values against a series of calibrators containing known amount of ßhCG. Contents of the kit 1 . 1 bottle CONJUGATE (12 ml), ready to use, containing anti- ßhCG antibodies in buffer with blue dye and 0,01 % merthiolate és 2 mg/ml chloracetamide (CAA).2. 5 vials STANDARD (1 ml per vial), ready to use, containing 0 (S0), 10 (S1), 25 (S2), 50 (S3), 100(S4) mIU/ml ßhCG (WHO 1st IS 99/688) in serum with 0.1% Kathon CG.(1 IU/ml = 1.21 ng/ml) 3. 1 vial CONTROL SERUM (1.0 ml), ready to use, human serum with 0.1% Kathon CG. The concentration of the control serum is specified in the quality certificate enclosed.4. 1 bottle SUBSTRATE (25 ml) ready to use, in brown plastic bottle. Do not expose to direct light!5. 1 piece MICROTITER PLATE, ready to use. 12 strips, packed in an air-tight foil.6. 1 bottle WASH BUFFER CONCENTRATE (20 ml), 0.01 % merthiolate.. See Preparation of reagents.7. 1 bottle STOP REAGENT (6 ml) 1M sulfuric acid.8. 1 bottle DILUTION SERUM (15 ml) with 0.1% Kathon CG.Plate map Quality certificate Pack leaflet Plastic cover Materials, tools and equipment required Precision pipettes with disposable tips (50, 100, 200 and 300 µl), distilled water, vortex mixer, shaker, plastic foil, absorbent tissue, ELISA thermostate, ELISA photometer Recommended tools and equipment Repeating pipettes, multi-channel ELISA pipettes Specimen collection and storage Serum samples can be prepared according to common procedures used routinely in clinical laboratory practice. Samples can be stored at 2-8 °C if the assay is carried out within 24 hours, otherwise aliquots should be prepared and stored deep frozen (-20°C). Frozen samples should be thawed and thoroughly mixed before assaying. Repeated freezing and thawing should be avoided. Do not use lipemic, hemolyzed or turbid specimens. If in an initial assay the serum sample is found to contain more than 100 mIU/ml βhCG, the sample can be diluted 1000-fold with dilution serum and reassayed as described in Assay Procedure.Preparation of reagents, storage Add the wash buffer concentrate (20 ml) to 600 ml distilled water to obtain 620 ml wash solution. Upon dilution store at 2-8°C until expiry date. Store the rest of reagents between 2-8°C after opening. At this temperature each reagent is stable until expiry date. The actual expiry date is given on the package label and in the quality certificate. Assay procedure (For a quick guide , refer to Table 1.) Table 1. Assay Protocol, Pipetting Guide (all volumes in microlitres)
Calculation of results The calculation is illustrated using representative data. Data obtained should be similar to those shown in Table 2. Manual calculation Calculate the average OD for each pair of duplicates. Subtract the mean NSB (non specific binding) from all (standards and samples) mean OD-s. Draw the standard curve on a lin-lin graph paper by plotting calculated OD of each standard level (ordinate) against the respective concentration (abscissa). Obtain sample values by interpolation of sample OD on the standard curve. Data evaluation using normalized binding For computerised calculations and/or quality assessment normalised specific binding values, rather than OD values are used. Specific binding values can be calculated for each standard and sample according to the following equation: S1-4/Mx (OD) –S0 (OD) B/Bmax (%) = ---------------------------------- x 100 S4 (OD) –S0 (OD), S0 is the zero-binding, or, non-specific binding (NSB), Mx = samples. Table 2. Typical assay data
00,511,522,50102030405060708090100 Figure 1: A typical standard curve (Do not use to calculate unknown samples!) Characterization of assay Typical assay parameters Bo/Bmax < 5 % Sensitivity For the analytical sensitivity 1.5 mIU/ml has been obtained by assaying 15 replicates of the zero standard. The sensitivity has been determined as the concentration corresponding to the sum of the mean OD and its double standard deviation. For the functional sensitivity 4 mIU/ml has been obtained by measuring low-level sera in 15 independent run. This value is defined as the concentration intercept at 22 % CV of the inter-assay imprecision curve. Hook effect There is no high dose " hook effect " up to the ßhCG concentration of 33000 mIU/ml . Specificity Cross-reactivities of monoclonal antibodies used in this assay are 0.18%, 0.37 %, and 0.05 % against hTSH, hLH, and hFSH, respectively. Precision 6 patient samples were assayed in 15 replicates to determine intra-assay precision. Values obtained are shown below.
Reproducibility To determine inter-assay precision 6 patient samples were measured in duplicates in 15 independent assays by 2 operators using different kit batches. Values obtained are shown below.
Recovery Recovery was defined as the measured increase expressed as per cent of expected increase upon spiking serum samples with known amount of βhCG. 91 ± 11.6 % (mean ± SD) was obtained for 16 serum pools. Dilution test (linearity) A serial dilution of 12 individual serum samples was carried out with the zero-standard. The equation of linear regression obtained for the expected (x) against the measured (y) concentration was: y=0.9737 x – 0,2657, R = 0.9538, n= 12 Expected Values Healthy adults: . 5 mIU/ml Gestation, week-16: 33000 mIU/ml (1 MoM) It is recommended that each laboratory determine a reference range for its own patient population, since this may vary in different laboratories or regions. Pathological values tumour > 20 mIU/ml The results obtained should only be interpreted in the context of the overall clinical picture. None of in vitro diagnostic kits can be used as the one and only proof of any disease or disorder. Procedural notes 1) The wash procedure is critical. Insufficient washing will result in poor precision and falsely elevated absorbance readings. 2) The TMB substrate is sensitive to certain handling and storage conditions. Please note the following precautions: -TMB is very light sensitive and direct expsosure to light (especially sunlight) should be avoided. -Do not leave the cap off of the storage bottle for prolonged periods of time. -To avoid contaminating the entire bottle of substrate, never pipette directly from the substrate bottle. Always pour just the necessary volume of substrate into a separate container for use. -Discard the excess TMB substrate after use. Additional information Components from various lots or from kits of different manufacturers should not be mixed or interchanged. Precaution Biohazard Human blood products used in the kit have been obtained from healthy human donors. They were tested individually by using approved methods (EIA, enzyme immunoassay), and were found to be negative, for the presence of both Human Immunodeficiency Virus antibody (Anti-HIV-1) and Hepatitis B surface Antigen (HBsAg). Care should always be taken when handling human specimens to be tested with diagnostic kits. Even if the subject has been tested, no method can offer complete assurance that Hepatitis B Virus, Human Immunodeficiency Virus (HIV-1), or other infectious agents are absent. Human blood samples should therefore be handled as potentially infectious materials. Chemical hazard Components contain Kathon CG as an antimicrobial agent. The Kathon CG present in each pack is 6.5 mg.
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