An interlaboratory study was performed in six laboratories to prove the validation of the ELISA method developed for quantitative determination of beta-lactoglobulin (BLG) in foods. The ELISA kit used for this study is based on rabbit polyclonal antibody. In-house validation of the kit did not produce false-positive results or cross-reactivity in a broad range of food matrixes containing no milk proteins. All participants obtained the BLG kit with a standard operational procedure, the list of the samples, samples, and a protocol for recording test results. The study included 14 food samples (extruded breakfast cereals, bread, two soy desserts, butter, chicken ham, chicken meat, wheat flour, long grain rice, jelly, two whey drinks, crackers, and bitter chocolate) and six spiked samples (two rice, two wheat flour, and two chicken meat). Nine samples of food matrixes containing no milk proteins showed BLG content lower than the first standard (0.15 mg/kg). Two samples of food matrixes with no milk proteins revealed BLG content higher than standard 3 (1.5 mg/100 g) and standard 4 (5.0 mg/100 g). Three food samples containing milk were tested as positive, and all spiked samples were evaluated as positive. The statistical tests (Cochran, Dixon, and Mandel) and analysis of variance were used to evaluate the interlaboratory study results. Repeatability and reproducibility limits, as well as LOQ (0.22 mg BLG/kg) and LOD (0.07 mg BLG/kg), for the kit were calculated.

A collaborative study in 10 laboratories was performed to validate an ELISA method developed for the quantitative determination of peanut protein in foods. The ELISA kit used for this study is based on rabbit polyclonal antibody. This kit does not produce any false-positive results or cross-reactivity with a broad range of peanut-free food matrixes. All participants obtained the peanut ELISA kit with standard operational procedures, a list of samples, the samples, and a protocol for recording test results. The study included 15 food samples. Three food matrix samples of zero peanut content showed peanut protein content lower than the first standard (0.10 mg/kg). Three samples with peanut declared as an ingredient revealed peanut protein content outside the calibration curve (absorbance was above the highest standard) in all laboratories, and three samples had the peanut content reported either above the highest standard or within the calibration curve, depending on the laboratory. Six samples with peanut declared as an ingredient gave the peanut protein content within the calibration curve. Only these six samples, together with a positive control sample (CS2), were used for statistical evaluation. The statistical tests (Cochran, Grubbs, and Mandel) and analysis of variance were used for the evaluation of the collaborative study results. Repeatability and reproducibility limits, as well as an LOQ (LOQcollaborative 0.22 mg peanut proteins/kg) and an LOD (LODcollaborative 0.07 mg peanut proteinslkg) for the kit were calculated.

Abstract:Within the framework of the research project ELISA methods for the quantitative determination of allergenic substances in foodstuff and raw materials were developed. ELISA kits for allergenic proteins of milk (casein, beta-lactoglobulin and BSA) egg white proteins and mustard proteins were validated and collaborative studies were performed to prove the validation of the ELISA methods developed. Various methods of extraction were tested. The parameters as a limit of detection, as a limit of quantification, robustness, repeatability and accuracy were determined. A broad range of zero matrices for allergens were tested as well. The ELISA kits are suitable for the determination of allergens according to EU legislation Directive 2005/26/EC and Directive 2006/142/EC in the laboratories focused on this topic.