Nyquist-Battie Cynthia, Frank Laura E, Lund Deanna, Lim Daniel V
Department of Public Health, University of North Florida, Jacksonville, Florida 32224, USA.
J Food Prot. 2004 Dec;67(12):2756-9. doi: 10.4315/0362-028x-67.12.2756.
Sandwich enzyme-linked immunosorbent assay, especially when coupled with biosensor technology, is a simple methodology that can rapidly screen juices for Escherichia coli O157:H7 contamination. However, sampling directly from apple juice and ciders has been postulated to reduce immunoassay sensitivity. In fluorescence sandwich enzyme-linked immunosorbent assays using commercially available polyclonal or monoclonal antibodies, sampling pasteurized apple juice spiked with E. coli O157:H7 compared to spiked phosphate-buffered saline shifted the range of detection. The spiked apple juice range of detection was 10(4) to 10(6) CFU/ml, whereas that for spiked phosphate-buffered saline was 10(6) to 10(8) CFU/ml, representing a hundredfold difference in sensitivity. Apple juice also increased background fluorescence intensity (P < 0.001) while reducing the net fluorescence intensity per CFU (P < 0.001). The addition of the polymer polyvinylpyrrolidone to apple juice significantly improved assay performance by increasing sensitivity and net fluorescence intensity per CFU and by reducing background fluorescence. Adjusting pH of apple juice from 3.9 to 7.4 improved assay performance but not to the degree seen with phosphate-buffered saline or polyvinylpyrrolidone-treated apple juice samples. The apple juice polyphenol, epicatechin, reduced net fluorescence intensity in a concentration-dependent manner, a change that was reversed by polyvinylpyrrolidone. Taken all together, these results suggest that polyvinylpyrrolidone can improve detection of O157:H7 in juices by reducing the effect of polyphenols on fluorescence sandwich enzyme-linked immunosorbent assay performance.
夹心酶联免疫吸附测定法，尤其是与生物传感器技术结合使用时，是一种简单的方法，可快速筛查果汁中是否存在大肠杆菌O157:H7污染。然而，有人推测直接从苹果汁和苹果酒中采样会降低免疫测定的灵敏度。在使用市售多克隆或单克隆抗体的荧光夹心酶联免疫吸附测定中，与加标的磷酸盐缓冲盐水相比，对加标了大肠杆菌O157:H7的巴氏杀菌苹果汁进行采样会改变检测范围。加标苹果汁的检测范围为10⁴至10⁶CFU/ml，而加标磷酸盐缓冲盐水的检测范围为10⁶至10⁸CFU/ml，灵敏度相差百倍。苹果汁还增加了背景荧光强度（P < 0.001），同时降低了每CFU的净荧光强度（P < 0.001）。向苹果汁中添加聚合物聚乙烯吡咯烷酮可通过提高灵敏度和每CFU的净荧光强度以及降低背景荧光来显著改善测定性能。将苹果汁的pH从3.9调节到7.4可改善测定性能，但改善程度不如磷酸盐缓冲盐水或聚乙烯吡咯烷酮处理的苹果汁样品。苹果汁中的多酚表儿茶素以浓度依赖的方式降低净荧光强度，这种变化可被聚乙烯吡咯烷酮逆转。综上所述，这些结果表明聚乙烯吡咯烷酮可通过降低多酚对荧光夹心酶联免疫吸附测定性能的影响来提高果汁中O157:H7的检测率。
