Sánchez Lourdes, Pérez María D, Puyol Pilar, Calvo Miguel, Brett Gary
Tecnología de los Alimentos, Facultad de Veterinaria, Zaragoza, Spain.
J AOAC Int. 2002 Nov-Dec;85(6):1390-7.
Eight laboratories participated in a collaborative study to evaluate an enzyme-linked immunosorbent assay (ELISA) to determine soy, pea, and wheat proteins in pasteurized or ultra-high temperature (UHT) milk powders. To perform this assay, polyclonal antibodies for soy, pea, and wheat proteins were obtained from rabbit sera. Collaborators received calibration standards composed of milk powder containing 0-8% (w/w) vegetal protein in total protein and blind test samples containing approximately 1, 2, and 5% (w/w) vegetal protein. An indirect competitive ELISA was performed with a kit prepared by a participating laboratory; the kit contained plates coated with soy, pea, or wheat proteins, the corresponding specific antisera, enzyme-labeled second antibody, and substrate solution. Test samples and calibrants were extracted with phosphate-buffered saline, pH 7.4, containing 0.05% Tween and assayed with the ELISA kits. The degree of adulteration was affected by the type of heat treatment applied to the samples. The estimated percentage of vegetal protein addition was close to the theoretical value for pasteurized samples but much lower for UHT samples. For pasteurized samples, intralaboratory relative standard deviations ranged from 5 to 22% and interlaboratory relative standard deviations ranged from 14 to 34%.
八个实验室参与了一项合作研究，以评估一种酶联免疫吸附测定法（ELISA），用于测定巴氏杀菌或超高温（UHT）奶粉中的大豆、豌豆和小麦蛋白。为了进行该测定，从兔血清中获得了针对大豆、豌豆和小麦蛋白的多克隆抗体。合作者收到了校准标准品，其由总蛋白中含有0 - 8%（w/w）植物蛋白的奶粉组成，以及含有约1%、2%和5%（w/w）植物蛋白的盲测样品。使用参与实验室制备的试剂盒进行间接竞争ELISA；该试剂盒包含包被有大豆、豌豆或小麦蛋白的板、相应的特异性抗血清、酶标记的二抗和底物溶液。测试样品和校准品用含有0.05%吐温的pH 7.4磷酸盐缓冲盐水提取，并用ELISA试剂盒进行测定。掺假程度受样品所采用的热处理类型影响。对于巴氏杀菌样品，添加植物蛋白的估计百分比接近理论值，但对于超高温样品则低得多。对于巴氏杀菌样品，实验室内相对标准偏差范围为5%至22%，实验室间相对标准偏差范围为14%至34%。
