摘要
目的优化蛋白芯片检测β-乳球蛋白(β-Lg)的技术条件。方法用芯片点样仪将β-乳球蛋白的一种抗体点样于三维基片上,用其另一种抗体作为检测抗体,以Cy3标记的羊抗作为二抗进行检测。以双抗夹心法对β-Lg抗原进行检测。结果β-Lg单克隆鼠抗66#被确定为点样探针,选择接触式点样进行芯片点样,点样数在42~92点之间可出现较好的点样一致性;β-Lg固定探针的浓度选择0.5 mg/mL,其检测抗体的稀释度为1∶2000;1%无蛋白封闭液被优选为封闭剂;β-Lg检测下限与生物检测限分别为17.54和55.31 ng/mL。根据β-Lg的S型曲线确定了其线性范围并建立了对β-Lg具有最佳决定系数(R^2=0.9993)的回归方程与标准曲线。结论本研究优化了牛乳中β-Lg检测的蛋白芯片检测条件,建立了定量检测牛乳β-Lg的蛋白芯片平台。
Objective To optimize the conditions of protein chip assay for bovine milk β-Lactoglobulin（ β-Lg）. Methods A microarrayer was used for printing anti-β-Lg as antibody I on each 3-dimensional-slide,another antis β-Lg antibody was used as detection antibody II and goat antibody coupled to Cy3 was used as antibody III. The standard β-Lg was detected by double antibody sandwich technique. Results Mouse monoclonal β-Lg antibody66# was chosen as the probe and contact printing as the printing method. The range between 42 and 92 spots was chosen as the basic printing condition.The concentration of β-Lg probes was 0. 5 mg / mL. The β-Lg detection antibody titre was 1∶2000. One percent no protein blocking solution was choosen as the blocking buffer. The lower detection limit and the biological detection limit of β-Lg were 17. 54 ng / m L and 55. 31 ng / m L respectively. The linear range was determined according to the S type curve of β-Lg and the best fitting models and standard curve were established for β-Lg（ R^2=0. 9993）. Conclusion The study optimizes conditions of a quantitative analysis system for measurement of β-Lg with protein chip,thus establishing the protein chip platform for quantitative detection of bovine milk β-Lactoglobulin.
作者
马欣欣
殷继永
孙静
黄建
朴玮
李瑾
陈頔
苑晓琳
霍军生
Ma Xinxin Yin Jiyong Sun Jing Huang Jian Piao Wei Li Jin Chen Di Yuan Xiaolin Huo Junsheng(National Institute for Nutrition and Health, Chinese Center for Diease Control and Prevention,Beijing 100050, China)
出处
《卫生研究》
CAS
CSCD
北大核心
2017年第1期78-83,共6页
Journal of Hygiene Research
基金
中国疾病预防控制中心青年科研基金(No.2015A202)
