Development Of Colloidal Gold-based Immunochromatographic Assay For Detection Of Listeria Monocytogenes

Listeria monocytogenes (LM) is one of the most virulent foodborne pathogens, which has been listed as one of four monitored foodborne pathogens by WHO. LM can infect both human and other animals, and cause listeriosis. Common clinical syndromes of lisiteriosis include mononucleosis, meningitis, septicaemia, abortion and stillbirths. LM menace seriously on human health and the development of economy, also cause huge economic losses. Thus it is important to develop a rapid, specific and sensitive method to screen LM in food samples. This research is aming to develop a specific and sensitive colloidal gold-based immunochromatographic test strip for detection of LM in food samples.In the present research, anti-LM polyclonal antibody was produced in Japanese rabbits using either 0.3%(v/v) formalin inactivated LM or LM cell debris as antigen. The titer of antiserum is higher than 30000, however, the antiserum reacts with Staphylococcus aureus, Enterobacter sakazakii and Shigella flexneri. Antibody was purified with both octanoic acid-ammonium sulfate precipitation and magnetic beads adsorption. Monoclonal antibody was obtained from Balb/c mice immunized with LM flagella. Six hybridoma cell lines were screened out by indirect ELISA reacting with LM. However, no stable hybridoma cell was obtained after limited dilution once.A rapid colloidal gold-based immunochromatographic test strip was developed for detection of LM using sandwich format with two different monoclonal antibodies. One of the monoclonal antibody against LM was conjugated with colloidal gold particles as reporter, and the other monoclonal antibody was immobilized on a porous nitrocellulose membrane to define the detection zone. The optimal concentrations of the monoclonal antibody conjugated with colloidal gold and the monoclonal antibody immobilized on membrane were determined as 20μg/mL and 2.0 mg/mL respectively. The sensitivity of the strip in LM pure culture was found to be 107 CFU/mL. No cross reactivity was observed with other bacteria (E. coli, S. aureus, E sakazakii, S. flexneri). Detection in LM-spiked food samples showed reproducible results after 48 h selective enrichment. Accelerated stability test at 37℃indicated that the test strips can be stored at room temperature for 15 months.A colloidal gold-based immunochromatographic test strip was successfully developed in the present research, advantages of this strip can be found in its user-friendly format, high specificity, long-term stability, easy handling, and can be used for onsite high through put screening.