Preparation Of Anti-Shigella Polyclonal Antibodies, Monoclonal Antibody And Genetic Engineering Antibody And Preliminary Research On Immunochromatographic Test Strip

Shigella, known as Dysentery Bacilli, is one kind of seriously harmful foodborne pathogen and listed as the first place of infectious diarrhea pathogens in China. It mainly invades colon, causing inflammation, the formation of ulcers and frequent mucinous hemorrhagic diarrhea. It led to a high rate of mortality especially in infants and young children. Enhancing the surveillance of Shigella would be helpful for prevention and control of large-scale infection outbreaks of the pathogen. There are many detection methods for foodborne pathogens now, such as traditional biochemical culture, PCR and immunological methods. Immunological methods become more and more popular because of its simple, fast, low-cost and broad-spectrum characteristics. The study aims to prepare a variety of anti-Shigella antibodies for establishing a fast and simple colloidal gold immunoassay test strip method for detecting Shigella which is of great practical importance for clinical diagnosis of Shigella infection as well as the supervision of food hygiene.Shigella bacteria debris was used as immunogen for immunization of rabbit to prepare serum with titer as high as1:1280000. Specific anti-Shigella polyclonal antibodies (pAbs) were obtained by octylic acid-ammonium sulfate precipitation and bacterial reverse absorption to remove the cross-reactivity with Escherichia coli and Enterobacter sakazakii etc. Four hybridoma cell lines which could steadily secrete anti-Shigella monoclonal antibody (mAb) were screened out by the cell fusion technique. Thereof4G9mAb was purified by octylic acid-ammonium sulfate precipitation, and proved to be paired well with the former purified pAbs. The mAb was conjugated with colloidal gold particles, and the pAbs were dispensed at the test line, while the donkey anti-mouse IgG dispensed at the control line. Finally colloidal gold immunoassay test strip for detection of Shigella was well prepared. The sensitivity for detection of pure culture Shigella suspensions was105CFU/mL; while the sensitivity of Shigella mixed with108CFU/mL of Bifidobacterium longum and Enterobacter sakazakii in milk was106CFU/mL. VL and VH gene sequence were obtained from4G9hybridoma cell line, respectively belonging to κ light chain and y heavy chain. Due to the termination codon found in VL gene sequence, only the VH was constructed to vector pET-lic and pGEX-4T-1, then transferred to the E. coli expression system. The reaction of expressed products with Shigella was quite low by ELISA, and thus not suitable for the preparation of colloidal gold immunoassay test strip for the detection of Shigella.The results in this study will provide a valid research model for building up the rapid detection method for other foodborne pathogens.