JZUS - Journal of Zhejiang University SCIENCE

Journal of Zhejiang University SCIENCE B 2015 Vol.16 No.2 P.155-166

Determination of royal jelly freshness by ELISA with a highly specific anti-apalbumin 1, major royal jelly protein 1 antibody

Author(s): Li-rong Shen, Yi-ran Wang, Liang Zhai, Wen-xiu Zhou, Liang-liang Tan, Mei-lu Li, Dan-dan Liu, Fa Xiao
Affiliation(s): Zhejiang Key Laboratory for Agro-Food Processing, Fuli Institute of Food Science, Department of Food Science and Nutrition, Zhejiang University, Hangzhou 310058, China
Corresponding email(s): shenlirong@zju.edu.cn
Key Words: Freshness, Royal jelly, Major royal jelly protein 1 (MRJP1), Enzyme-linked immunosorbent assay (ELISA), High specific antibody

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Abstract: Major royal jelly protein 1 (MRJP1), designated apalbumin 1, has been regarded as a freshness marker of royal jelly (RJ). A MRJP1-specific peptide (IKEALPHVPIFD) identified by bioinformatics analysis of homologous members of the major royal protein family was synthesized and used to raise polyclonal anti-MRJP1 antibody (anti-SP-MRJP1 antibody). Western blot analysis showed that anti-SP-MRJP1 antibody only reacted with MRJP1 in RJ. In contrast, the previously reported antibody against recombinant MRJP1 (anti-R-MRJP1 antibody) reacted with other members of MRJP family in RJ. enzyme-linked immunosorbent assay (ELISA) using anti-SP-MRJP1 antibody demonstrated that MRJP1 content in RJ stored at 40 °C significantly degraded by 37.3%, 55.9%, 58.0%, 60.6%, 65.7%, 72.7%, and 73.1% at 7, 14, 21, 28, 35, 42, and 49 d, respectively, when compared with MRJP1 content in fresh RJ (0 d). Optical density analysis of MRJP bands from sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) profiles demonstrated that the degradation of MRJP1, MRJP2, MRJP3, and MRJP5 in RJ was strongly and positively correlated with the period of storage (P<0.0001). Our results indicated anti-SP-MRJP1 antibody was highly specific for MRJP1, and ELISA using the antibody is a sensitive and easy-to-use method to determine the freshness and authenticity of RJ.

用高度特异性王浆主蛋白1多克隆抗体ELISA法检测蜂王浆新鲜度

目的：为蜂王浆主蛋白1（MRJP1）的快速检测和鉴别提供科学依据，为蜂王浆的质量控制提供技术支持。
创新点：首次比较了MRJP1特异性多克隆抗体与MRJP1重组表达蛋白多克隆抗体对王浆主蛋白家族的免疫反应差异，验证了蜂王浆中MRJP1蛋白降解与保温时间的相关性，建立了以MRJP1作为蜂王浆新鲜度生物标志物的快速检测方法。
方法：通过蜂王浆主蛋白家族蛋白的氨基酸序列同源性分析，筛选出MRJP1的特异性多肽区域，进行人工合成，免疫兔子后取血清制备成特异性多克隆抗体。用蛋白质印迹法（Westernblot）检测了MRJP1特异性多克隆抗体与MRJP1重组表达蛋白多克隆抗体对王浆主蛋白家族的免疫反应。以新鲜蜂王浆为对照品，用MRJP1特异性抗体酶联接免疫吸附剂测定（ELISA）法和变性电泳胶灰度扫描法分别测定保温（40°C）7～49天的蜂王浆中MRJP1含量的变化，并进行了相关性分析。
结论：MRJP1的特异性抗体对MRJP1蛋白具有专一的免疫识别特性，可特异性地检测代表蜂王浆新鲜度的MRJP1含量变化，并鉴别蜂王浆的真伪。

关键词：新鲜度；蜂王浆；王浆主蛋白1；ELISA；高度特异性

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用高度特异性王浆主蛋白1多克隆抗体ELISA法检测蜂王浆新鲜度

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