猪源ST11型艰难梭菌TcdB毒素受体结合域双抗体夹心ELISA的建立

Development of a double-antibody sandwich ELISA targeting the receptor binding domain of TcdB toxin of ST11 type Clostridium difficile of porcine origin

摘要:

艰难梭菌是一种重要的人兽共患肠道病原菌，广泛存在于人和多种动物体内。ST11型艰难梭菌是国际上流行最广泛、危害最大的亚型之一。我国作为养猪业大国，猪源艰难梭菌的检测方法欠缺，给猪场艰难梭菌的防控留下隐患。本研究旨在建立一种特异、敏感的双抗体夹心ELISA，为猪源ST11型艰难梭菌流行病学调查提供血清学检测方法。首先采用原核表达并纯化出97 kDa的受体结合域(receptor binding domain, RBD) 蛋白，并利用杂交瘤技术成功筛选出能稳定分泌针对RBD蛋白的单克隆抗体杂交瘤细胞AE2D3，经检测其抗体亚型为IgG2b (κ)。其次以针对RBD蛋白的单克隆抗体为检测抗体、兔多克隆抗体为捕获抗体，运用棋盘法确定了捕获抗体和检测抗体的配对浓度、抗原包被条件、封闭条件、检测抗体和待检样品的孵育条件、羊抗小鼠IgG/HRP和TMB显色液反应条件。经测定，此方法的临界值OD₄₅₀为0.152，与13株非ST11型的艰难梭菌无交叉反应，对RBD蛋白的最低检测浓度为8.83 ng/mL。这一特异、敏感、可用于兽医临床检测猪源ST11型艰难梭菌的双抗体夹心ELISA，为养猪业ST11型艰难梭菌流行病学调查提供了可靠的血清学检测方法。

关键词:艰难梭菌;ST11型;RBD蛋白;单克隆抗体;双抗体夹心ELISA

Abstract:

Clostridium difficile is an important zoonotic intestinal pathogen, which is widely present in humans and a variety of animals. The ST11 type C. difficile is one of the most widespread and harmful subtypes in the world. As a large country in pig farming, China lacks efficient methods for detecting C. difficile of porcine origin, leaving hidden dangers for the prevention and control of C. difficile. The aim of this study was to develop a specific and sensitive double-antibody sandwich ELISA for the epidemiological investigation of ST11 type C. difficile of porcine origin. Firstly, a 97 kDa receptor binding domain (RBD) was expressed in a prokaryotic host and purified. A hybridoma cell line AE2D3 capable of stably secreting monoclonal antibody targeting the RBD was screened, and the antibody subtype was determined to be IgG2b (κ). Secondly, a double antibody sandwich ELISA method was developed, where the monoclonal antibody targeting the RBD was used as a detection antibody, and the rabbit polyclonal antibody was used as a capture antibody. The chessboard method was used to determine the matching concentration of the capture antibody and the detection antibody, the antigen coating conditions, the blocking conditions, the incubation conditions for detection antibody and samples to be tested, as well as the reaction conditions of HRP-conjugated and reaction conditions of TMB chromogenic solution. The negative cutoff OD₄₅₀ was 0.152, and no cross-reaction with 13 strains of non-ST11 type C. difficile was found. The minimum detection concentration of RBD was 8.83 ng/mL. This specific and sensitive double-antibody sandwich ELISA provides a reliable serological detection method for epidemiological investigation of the ST11 type C. difficile in pig industry.

Key words:Clostridium difficile;ST11 type;RBD protein;monoclonal antibody;double-antibody sandwich ELISA