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中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志
中国生物工程杂志  2023, Vol. 43 Issue (1): 42-49    DOI: 10.13523/j.cb.2207033
技术与方法     
免疫检测信号放大抗体偶联物的制备及应用*
沈妍,姚玢妍,杨若楠,康文斌,林海英**()
福州大学生物科学与工程学院 药物生物技术与工程研究所 福州 350108
Preparation and Application of Immunoassay Signal Amplification Antibody Conjugates
SHEN Yan,YAO Bin-yan,YANG Ruo-nan,KANG Wen-bin,LIN Hai-ying**()
Medicine Biotechnology and Engineering Research Institute, College of Biological Science and Engineering, Fuzhou University, Fuzhou 350108, China
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摘要：

目的: 以聚赖氨酸(polylysine,PL)为骨架提高辣根过氧化物酶(horseradish peroxidase,HRP)与羊抗兔IgG的连接数量,比较几种化学偶联剂的偶联效果,通过免疫检测技术对其灵敏度进行检测和比较。方法: 对HRP与PL、聚合物HRP-PL与N-琥珀酰-S-乙酰乙酸(N-succinimidyl-S-acetylthioacetate,SATA)和2-亚氨基硫烷(Traut’s)两种试剂、羊抗兔IgG与琥珀酰亚胺基4-(N-马来酰亚胺甲基)环己烷-1-羧酸酯[succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate,Sulfo-SMCC]、活化后羊抗兔IgG及HRP-PL进行摩尔比的优化;对偶联物IgG-PL-HRP及商品化二抗分别进行斑点免疫印迹、ELISA和免疫组化,并计算偶联物IgG-PL-HRP的检测放大倍数。结果: 当PL与HRP摩尔比为1∶5,HRP-PL与Traut’s摩尔比为1∶15,羊抗兔IgG与Sulfo-SMCC摩尔比为1∶30,羊抗兔IgG与HRP-PL摩尔比为1∶10时,反应效率较高;商品化二抗及偶联物IgG-PL-HRP在斑点免疫印迹实验中的最低检测限分别为2.5 μg和312.5 ng,最大稀释倍数分别为50和100倍;在ELISA实验中的最大稀释倍数分别为5 000和20 000倍;在免疫组化实验中偶联物IgG-PL-HRP的检测特异性及强度均大于商品化二抗。结论: 成功合成抗体偶联物IgG-PL-HRP,且免疫检测信号放大倍数为商品化二抗的3~7倍,这对后续免疫诊断及生物学的研究具有重要意义。
关键词： 辣根过氧化物酶;  聚赖氨酸;  抗体偶联物;  信号放大    
Abstract:

Objective: Chemical coupling agent and polylysine (PL) were used to conjugate goat anti-rabbit IgG and horseradish peroxidase (HRP) to increase the number of HRP connections on antibodies to amplify the sensitivity of immune detection, and the sensitivity was detected and compared through immunodetection applications, which is of great significance for subsequent immune diagnosis and biological research. Methods: Tylthioacetate (SATA) and Traut’s were optimized for the polymer HRP-PL, the feed ratio of Succinimidyl 4-[N-maleimidomethyl]cyclohexane-1-carboxylate (Sulfo-SMCC) was optimized with goat anti-rabbit IgG and HRP-PL, and the reaction ratio of the two was optimized. The conjugated IgG-PL-HRP and commercial secondary antibody were detected by immunoblot, ELISA and immunohistochemistry, respectively, and the amplification of conjugated IgG-PL-HRP was calculated. Results: When the molar ratio of PL to HRP was 1∶5, the molar ratio of HRP-PL to Traut’s was 1∶15, the molar ratio of goat anti-rabbit IgG to Sulfo-SMCC was 1∶30, the molar ratio of goat anti-rabbit IgG to HRP-PL was 1∶10, the reaction efficiency was high. In the immune spot test, the minimum detection limits of commercial secondary antibody and conjugate IgG-PL-HRP were 2.5 μg and 312.5 ng, respectively, and the maximum dilution times were 50 and 100 times, respectively. In ELISA experiment, the maximum dilution times were 5 000 and 20 000, times respectively. In the immunohistochemical experiment, the detection specificity and intensity of conjugated IgG-PL-HRP were higher than those of commercial secondary antibody. Conclusion: The primary amplification conjugate IgG-PL-HRP was successfully synthesized and its immune detection signal amplification was about 3 ~ 7 times that of the commercial secondary antibody. It is of great significance to the following immunodiagnosis and biological research.
Key words: Horseradish peroxidase    Polylysine    Antibody conjugate    Signal amplification
收稿日期: 2022-07-18 出版日期: 2023-02-14
ZTFLH:  R392  
基金资助: *福建省自然科学基金(2020J01490)
通讯作者: **林海英 电子信箱:haiylin@163.com   
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沈妍
姚玢妍
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康文斌
林海英

引用本文:

沈妍, 姚玢妍, 杨若楠, 康文斌, 林海英. 免疫检测信号放大抗体偶联物的制备及应用*[J]. 中国生物工程杂志, 2023, 43(1): 42-49.

SHEN Yan, YAO Bin-yan, YANG Ruo-nan, KANG Wen-bin, LIN Hai-ying. Preparation and Application of Immunoassay Signal Amplification Antibody Conjugates. China Biotechnology, 2023, 43(1): 42-49.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.2207033    或    https://manu60.magtech.com.cn/biotech/CN/Y2023/V43/I1/42

图1  免疫检测信号放大原理
图2  HRP-PL的制备和纯化
图3  反应后HRP-PL的巯基含量及酶活
图4  羊抗兔IgG与不同比例Sulfo-SMCC反应后活性测定
图5  偶联物IgG-PL-HRP凝胶层析纯化紫外分析
图6  偶联物IgG-PL-HRP在斑点免疫印迹实验中的灵敏度分析
图7  商品化二抗和抗体偶联物IgG-PL-HRP不同稀释倍数的ELISA灵敏度分析
图8  偶联物IgG-PL-HRP在免疫组化实验中的灵敏度分析
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