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| GENTAUR • Belgium : + 32 2 7325688 • France : 01 43250150 • Italy : 02 36006593 • Germany : +49 241 6085 13140 | |
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CEA ELISA KIT (REF: EK-38) / 475 Euro The CEA ELISA system provides a direct in vitro quantitative determination of human carcinoembryonic antigen (CEA) in human serum in the range of 0-100 ng/ml. Each kit contains materials sufficient for 96 determinations permitting the construction of one standard curve and the assay of 39 unknowns in duplicate.Introduction Carcinoembryonic antigen (CEA) is a cell-surface glycoprotein with a molecular weight of 180-200kD, that occurs in high levels in colon epithelial cells during embryonic development. Levels of CEA are significantly lower in colon tissue of adults, but can become elevated when inflammation or tumours arise in any endodermal tissue, including the gastrointestinal tract, respiratory tract, pancreas and breast. An overexpression of CEA protein has been detected in a variety of adenocarcinomas, including gastric, pancreatic, small intestine, colon, rectal, ovarian, breast, cervical and non-small-cell lung cancers. CEA is also expressed by epithelial cells in several non-malignant disorders, including diverticulitis, pancreatitis, inflammatory bowel disease, cirrhosis, hepatitis, bronchitis and renal failure and also in heavy smokers. Therefore CEA should not be regarded as a tumour-specific marker for the screening of general population for undetected cancers. However, the determination of CEA levels provides important information about patient prognosis, recurrence of tumours after surgical removal and effectiveness of therapy. 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 streptavidin coated 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 and the capture antibody - at room temperature for 1 hour, 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 CEA in patient samples is read off a calibration curve constructed by plotting binding values against a series of calibrators containing known amount of CEA. Contents of the kit 1. 1 bottle CONJUGATE (12ml), ready to use, containing anti-CEA antibodies in buffer with 0,01 % merthiolate.2. 7 vials STANDARD (7 x 0,5 ml), ready to use, containing (S0-S6) 0; 2,5; 5; 10; 25; 50; 100 ng/ml CEA (WHO 1st IS 73/601 Int.Std.) in human serum with 0.1% NaN3.3. 2 vials CONTROL SERUM, low (CI) and high (CII). Lyophilised human serum with 0.1% NaN3. The concentration of the control serum is specified in the quality certificate enclosed. See Preparation of reagents . 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) with 0.01 % merthiolate.. See Preparation of reagents. 7. 1 bottle STOP REAGENT (6 ml) 1M sulfuric acid, ready to use. 8. 1 bottle SAMPLE DILUENT (5 ml) with 0,1% NaN3 , ready to use. Plate map Quality certificate Pack leaflet Plastic foil Materials, tools and equipment required Precision pipettes with disposable tips (25, 50, 100, 200 and 300 µl), vortex mixer, shaker, plastic foil, absorbent tissue, ELISA photometer. Recommended tools and equipment Repeating pipettes, multi-channel ELISA pipettes, ELISA microplate washer. 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. Samples with a CEA concentration higher than that of the most concentrated standard should be diluted with sample dilutent and reassayed. Preparation of reagents, storage The actual expiry date is given on the package label and in the quality certificate. After opening store the reagents at 2-8°C for up to 60 days. 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 for up to 60 days. Add 500 µl distilled water to the lyophilised control serum. Mix gently with shaking or vortexing (foaming should be avoided). Ensure that complete dissolution is achieved, and allow the solution to equilibrate at room temperature for at least 20 minutes. Store at 2-8° for up to 60 days. Assay procedure (For a quick guide , refer to Table 1.) 1. Equilibrate reagents and samples to room temperature before use. Homogenize all reagents and samples by gentle mixing to avoid foaming. 2. Label the plate map for duplicates of each standards (S0-S6), control serums (C1,C2) and samples (Mx). 3. Pipette 25 µl each of standards, control serums and samples into the appropriate wells. 4. Pipette 100 µl of conjugate solution into each well by the multi-channel pipette. 5. Cover the plate by the enclosed foil, place it on the shaker. Incubate the plate for 1 hour, shaking (300 RPM) at room7. Table 1. Assay Protocol, Pipetting Guide (all volumes in microlitres)
Calculation of results The calculation is illustrated using representative data. Data otained should be similar to those shown in Table 2. Manual calculation Calculate the average OD for each pair of duplicates. Subtract the mean NSB from all (standars 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 A.U. values are used. Specific binding values can be calculated for each standard and sample according to the following equation: S1-6/C1-2/Mx(OD)–S0(OD) B/Bmax (%) = -------------------------------------x 100 S6 (OD) – S0 (OD) S0 is the zero-binding, or, non-specific binding (NSB). Table 2. Typical assay data
Figure 1: A typical standard curve (Do not use to calculate unknown samples) Characterization of assay Typical assay parameters Bo/Bmax < 5% Specificity No cross reactivity with NCA can be detected in normal physiological levels. Sensitivity The analytical sensitivity or minimum detectable limit is calculated by the interpolation of the mean counts of zero standard plus 2 standard deviation from the standard curve. Determination was carried out using 15 replicates of zero standard response. The value of analytical sensitivity is 0,4 ng/ml. The functional sensitivity is a measure of the CEA concentration that is significantly different from zero as determined by the inter-assay precision profile (22 % CV). The value of functional sensitivity is: 1,3 ng/ml. Precision 4 human serum pool samples were assayed in 10 replicates to determine intra-assay precision. Values obtained are shown below
Reproducibility To determine inter-assay precision 5 human serum sample pools were measured in duplicates in 15 independent assays by 5 operators using different kit batches. Values obtained are shown below:
Linearity – dilution test Individual human serum samples were diluted with the sample diluent of the KIT. The diluted samples were measured according to KIT protocol.
00,511,522,50102030405060708090100hCEA concentration ng/mlOD 450 nm Recovery Recovery was defined as the measured increase expressed as per cent of expected increase upon spiking serum samples with known amount of CEA. The average per cent recover for 5 serum pools spiked with CEA at 3 levels was: 91,.3 ± 4,04%, with a range of 85,4 % to 98,8%. Hook effect There is no high dose "Hook effect" up to the CEA concentration of 30000 ng/ml. Expected Values In 95% of healthy subjects, CEA levels are usually <5.0 ng/ml. For individuals who smoke normal CEA levels are usually <10.0ng/ml. It is recommended that each laboratory determine a reference range for its own patient population. The results obtained should only be interpreted in the context of the overall clinical picture. None of the in vitro diagnostic kits can be used as the one and only proof of any disease or disorder. Procedural notes A thorough understanding of this package insert is necessary for successful use of the kit. Reliable results will only be obtained by using precise laboratory techniques and accurately following the package insert. Use a clean disposable pipette tip for each reagent, Standards, Control or specimen. Pipetting of samples should not extend beyond 10 minutes to avoid assay drift. Always add reagents in the same order to minimize reaction time differences between wells. wash procedure is critical. Insufficient washing will result in poor precision and falsely elevated absorbance readings. Avoid microbial contamination of reagents, especially of the Conjugate solution and TMB substrate: -Do not leave the cap off of the storage bottle for prolonged periods of time. -Never pipette directly from the bottle. Always pour just the necessary volume into a separate container for use then discard the excess after use. The results should be read within 30 minutes of adding the Stop solution. Heterophilic antibodies such as human anti-mouse antibodies (HAMA) are frequently found in the serum of human objects. Those antibodies can cause severe interference in many immunodiagnostic procedures. This assay has been designed to minimize that kinds of interfernce. Nevertheless, complete elimination of this interference from all patient specimens cannot be guaranteed. A test result that is inconsistent with the clinical picture and patient history should be interpreted with caution. 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 sodium azide and merthiolate antimicrobial agent. Dispose of waste by flushing with copious amount of water to avoid build-up of explosive metallic azides in copper and lead plumbing. The total azide present in each pack is 13 mg. Caution! The stop reagent is corrosive. Avoid contact with it because may cause skin irritations and burns.
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