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| GENTAUR • Belgium : + 32 2 7325688 • France : 01 43250150 • Italy : 02 36006593 • Germany : +49 241 6085 13140 | |
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EK-80ACE051201 AFP ELISA KIT (REF: EK-80) The AFP ELISA system provides direct quantitative in vitro determination of human alpha-foetoprotein (AFP) in human serum. AFP can be assayed in the range of 0-100 IU/ml using 100 µl serum samples.Introduction Alpha-fetoprotein (AFP) is a glycoprotein with a molecular mass of 65000. It is normally produced in large amounts by the foetal liver. AFP level increases progressively and reaches a peak at the 30th week in the maternal serum, thereafter, it dec-reases gradually. An elevated maternal serum AFP is associated with the neural tube defect (spina bifida) and placental abnormalities, whereas decreased AFP levels in both maternal serum and amniotic fluid are related to foetal chromosomal abnormalities (Down-syndrome). Patients affected by liver carcinoma, testicular or ovarian teratocarcinoma present very high AFP levels. Although less frequently, an increased AFP concentration may also be observed in gastric, breast and bronchial tumours. To cover the very large range of concentration associated with the great variety of pathologic processes, the current microtiter plate immunoassay (ELISA) is a simple and powerful diagnostic tool for the detection of AFP in the range 0 - 100 IU/ml. Principle of method The technology uses two high affinity monoclonal antibodies in an immunometric assay system. The two antibodies react simul-taneously 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 standard solid-phase ELISA system the reaction is carried out in a microtiter plate (12 strips) which acts as the binder of sandwich complex. In the present product standards and samples are incubated with the conjugate which contains the horse radish 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 AFP in patient samples is read off a calibration curve constructed by plotting binding values against a series of calibrators containing known amount of AFP. Contents of the kit 1 . 1 vial CONJUGATE (12 ml), ready to use, containing anti-AFP antibodies in buffer with blue dye 0,01 % merthiolate and 2 mg/ml chloracetamide (CAA).2. 5 vials STANDARD (1 ml per vial), containing 0 (S0), 10 (S1), 25 (S2), 50 (S3), 100 (S4) IU/ml AFP (WHO 1st IRP 72/225) in serum with 0.1% Kathon CG.(1 IU/ml = 1.21 ng/ml) 3. 1 vial CONTROL SERUM. 1.0 ml human serum with 0.1% Kathon CG. The concentration of the control serum is specified in the quality certificate enclosed. 4. 1 vial 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 vial STOP REAGENT (6 ml) 1M sulfuric acid Plate map Cover stick Quality certificate Pack leaflet 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, disposable plastic reagent-trays (separate for each reagent), 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 IU/ml AFP, the sample can be diluted 10-fold with S0 standard 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.) 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 standard (S0-S4), control serum (C) and samples (Sx). 3. Pipette 100 µl each of standards, control serum 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,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 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:S1-4/Mx (OD) –S0(OD) B/Bmax (%) = ---------------------------------- x 100 S4 (OD) –S0(OD), S0 is the zero-binding, or, non-specific binding (NSB), Mx= sample. 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 0.08 IU/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. 1) Hook effect There is no high dose " hook effect " up to the AFP concentration of 5000 IU/ml . Specificity The monoclonal antibodies used in this IRMA kit are specific for AFP. No cross reactivity was found with human albumin. 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 AFP. 86 ± 7,62 % (mean ± SD) was obtained for 12 serum pools. Dilution test (linearity) A serial dilution of 12 individual serum samples was carried out with the zero-standard. The equation for the expected (x) against the measured (y) concentration was: y=0.9737 x – 0,2657, R = 0.9538, n= 12 Expected Values Healthy male: 0.47 – 5.39 IU/ml. Healthy female: 0.37 – 4.41 IU/ml At 16-week gestation: 30 IU/ml (1 MOM) Hepatocarcinoma: >100 IU/ml Hepatitis: <100 IU/ml It is recommended that each laboratory determine a reference range for its own patient population, since this may vary in different laboratories or regions. Procedural notes 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 | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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