Haasnoot Willem, Smits Nathalie G E, Kemmers-Voncken Anniek E M, Bremer Maria G E G
RIKILT-Institute of Food Safety, PO Box 230, 6700 AE Wageningen, The Netherlands.
J Dairy Res. 2004 Aug;71(3):322-9. doi: 10.1017/s0022029904000317.
Two monoclonal antibodies (MAb) raised against bovine kappa-casein were developed and applied in an automated optical biosensor (Biacore 3000) to create easy and fast direct and inhibition biosensor immunoassays (BIA) for the detection of cows' milk in the milk of ewes and goats. With both assay formats, low limits of detection (<01%) and fast run times (around 5 min) were obtained. For sample preparation, milk was diluted in buffer (direct assay) or in an antibody-containing buffer (inhibition assay) only. For quantitative analysis, calibrants of cows' milk in ewes' or goats' milk were used. Advantages of the direct BIA are: the single reagent format (biosensor chip immobilized antibodies only); the use of small amounts of antibodies (2 microg for >350 tests); and the wide measurement range (0.1 to 10% cows' milk). Despite these advantages, the inhibition BIA (using kappa-casein immobilized on the chip) was preferred because of the possible application of non-purified Mab, the higher responses, the higher sensitivity at relevant low percentages of cows' milk and its robustness (>800 cycles per chip).
制备了两种针对牛κ-酪蛋白的单克隆抗体（MAb），并将其应用于自动光学生物传感器（Biacore 3000），以创建简便快速的直接和抑制生物传感器免疫分析方法（BIA），用于检测母羊和山羊奶中的牛奶。两种分析形式均获得了低检测限（<0.1%）和快速运行时间（约5分钟）。对于样品制备，牛奶仅在缓冲液（直接分析）或含抗体的缓冲液（抑制分析）中稀释。为了进行定量分析，使用了母羊或山羊奶中牛奶的校准物。直接BIA的优点包括：单一试剂形式（仅生物传感器芯片固定化抗体）；使用少量抗体（2微克可进行>350次测试）；以及宽测量范围（0.1%至10%牛奶）。尽管有这些优点，但抑制BIA（使用固定在芯片上的κ-酪蛋白）更受青睐，因为其可应用非纯化的MAb、响应更高、在相关低比例牛奶时灵敏度更高且具有耐用性（每个芯片>800个循环）。
