ISO 21572:2013 pdf download.Foodstuffs — Molecular biomarker analysis — Protein-based method
8 Procedure
8.1 General Storage conditions and shelf-life of lateral flow strips, antibodies, conjugates, substrates, etc. shall be clearly specified by the provider. Use appropriate laboratory equipment with low protein binding capacity (e.g. polypropylene tubes) to prevent protein adsorption during the whole procedure. For the use of this International Standard, general requirements of quality assurance for laboratories shall be observed (e.g. concerning calibration of apparatus, double determination, blanks, use of reference materials, preparation of calibration curves). Carefully clean all equipment coming into direct contact with the sample to prevent contamination. See ISO/IEC 17025 for more information.
8.2 Preparation of sample solution Once a representative sample is obtained, specific sample preparation procedures may be found in the annexes. Grind samples as specified in the method before test portions are taken, if necessary. Powders/flour might have swelling properties and may require more extraction solution if a manufacturer’s method does not specify this information. If the sample is not immediately used, follow your laboratory’s procedure for storage (e.g. –20 °C or below). Laboratory samples containing high amounts of fat may be nonhomogeneous and a larger test sample should be extracted. If applicable, instructions may be found in the annexes. Weigh an appropriate amount (as specified in the relevant annex) of a representative test sample for analysis to create a test portion for extraction. Add extraction solution and homogenize or mix.
8.3 Extraction Use an extraction procedure suitable for the matrix. Details of appropriate conditions for the extraction/dilution of the test portions, controls and reference materials are provided in Annex A for ELISA and Annex B for lateral flow strips. Care should be taken to use extraction procedures validated for the matrix. Extracted samples should be immediately used or treated as specified in the procedure for storage.
8.4 Preparation of calibration curves, positive controls and reference materials For the preparation of calibration curves, positive controls and reference materials for Annex A, it is recommended to use matrix matched reference materials or reference materials that have been validated for the matrix. Calibration curves are not routinely required for qualitative application such as lateral flow strips, however, positive and negative controls can be prepared at the discretion of the analyst.
8.5 Assay procedure For a quantitative test, select the required number of wells, (e.g. in ELISA) for the test portion(s) to be analysed, including blanks, measurement standards, and add each of them at minimum in duplicate, properly diluted to the assay. For a qualitative test or semiquantitative test, select the required number of test (e.g. lateral flow strips or ELISA) needed for the test portions to be analysed. The stability of the final signal can vary. Read the results in a timely manner as specified in the annexes. According to the method chosen, follow the instructions of each method for sample analyses, including blanks and measurement standards (if necessary). Allow the reaction to occur at a specified temperature range and time. If necessary, terminate the reaction according to the method described in the relevantannex. For example, if ELISA method requires acquiring data on a spectrophotometer, perform this step. In the case of qualitative tests, generally these are interpreted visually, follow the kit instructions.
9 Interpretation and expression of results
9.1 General The parameters to interpret vary depending on whether the assay is qualitative, semiquantitative or quantitative. For quantitative methods, the coefficient of variation (CV) of optical density values resulting from replicate measurements of a sample test solution, in general, should not exceed 15 %. The CV of calculated concentrations resulting from replicate measurements of a sample test solution, in general, should not exceed 20 %. If the CV limit is exceeded, the analyses should be repeated on freshly prepared sample test solution.
To establish a CV, in this case, at least three determinations shall be carried out (e.g. values from three microtitre wells). Negative results shall be reported as “negative at the limit of detection” and the limit of detection shall be reported. Positive results below the limit of quantification shall be reported as “positive above the limit of detection, but below the limit of quantification”. The limits of quantification and detection shall be reported.ISO 21572 pdf download.ISO 21572-2013 pdf download
