Preparation Of Monoclonal Antibodies Against SLT-IIe A Subunit And Establishment Of Competitive ELISA Method

Porcine Edema disease (ED) is a common cause of illness and death loss in pigs during the first 2 weeks after weaning. The disease is an enterotoxaemia caused by Shiga-like toxin Escherichia coli (SLTEC) that colonize the small intestine and produce SLT-IIe. SLT-IIe is a Shiga-like toxin II variant which is responsible for edema disease of pigs. It is consisting of five B subunits responsible for binding to specific cell receptors and an A subunit containing the enzymatic activity. SLT-IIe, through the binding of the B subunit and the specific intestine epithelium cell receptor, mediates A subunit into the cell ribosome. With the catalytic reaction between the Al subunit and the large ribosomal subunit 28SrRNA, the AA-tRNA is prevented to bind with rRNA, which blocks the synthesis of protein within the cell, resulting in the cell death. Therefore, SLT-IIe is an important material foundation for the pathogenic bacteria, and is the ideal target antigen for the preparation of the specific antibody.Porcine Edema disease is the most widespread cause of death in weaned pigs and account for substantial economical losses in pig farming industry. However, commercial vaccines and effective drug therapies are still lacking so far. In the study, SLT-IIe A subunit and B subunit were respectively cloned and expressed by indirect ELISA with purified protein. But the routine indirect ELISA usually has enormous false positive results induced by the E. coli protein remnant which needs improving. In this study, monoclonal antibodies against SLT-IIe A subunit was prepared, and a competitive ELISA was established thereafter. The principal results are as the following.1. Preparation and identification of monoclonal antibodies against SLT-IIe A subunitUsed two purified Escherichia coli expressed recombinant SLT-IIeA proteins as antigen to immune Balb/c mice, then the spleen cells and myeloma cells SP2/0 were fused. The indirect ELISA method and western-blot assay were employed to screen target hybridoma cells. Next the positive cells were cloned by Limited dilution. At last, ten strains of Hybridoma cell which could stably secrete antibodies against SLT-IIe A subunit were obtained which were respectively marked as 3E12, 2G11, 5B12, 2E3, 4F4, 4G2, 1A10, 4A7, 4B12, 3H7. The titers of ascites were up to 100*2⁸ to 100*2¹⁴. All of the ten monoclonal antibodies had a high relative affinity of the tested by ELISA. The titer of ELISA was from 100*2² to 100*2⁵. It was evaluated by Western blot that all the monoclonal antibodies were proved to react with target proteins, and eight of them were found to react with nature SLT-IIe. Only 3H7 and 4G2 showed certain neutralization in the assay. 2. Establishment of the competitive ELISA and its prime applicationMonoclonal antibody 3H7 was purified by caprylic acid and ammonium sulfate. After purification, the monoclonal antibody 3H7 was labelled with HRP using improved sodium periodic acid mothed. The competitive ELISA method used to detect the antibody against SLT-IIe was established by using SLT-IIeA protein as antigen and the HRP-labelled monoclonal antibody 3H7 as detection antibody. Chess board test showed that the concentration of recombinant protein for coating ELISA plates was 0.32μg /ml and the working titer for HRP-labelled monoclonal antibody was 1:3200. Over 40% of inhibition rate was the positive judging standard. Clinical tests showed that the ELISA had the advantages of high specificity, reproducibility and stability.This study is trying to establish a competitive method which adapts to various hosts by generating the monoclonal antibody against SLT-IIe A subunit and marking it by HRP. Meanwhile, various factors influenced the detection were examined and the specialty sensitivity and reproducibility were optimized, which provided a base for the further application of the assay kit.