Preparation Of Listeriolysin O Monoclonal Antibodies And Preliminary Detection Of Listeria Monocytogenes

Listeria monocytogenes (LMO) was a foodborne pathogen which could infect human andanimal. The bacterium could cause severe syndromes, Such as encephalitis, meningoencephalitisand septicemia, the case fatality rate was very high. Listeriolysin O (LLO) which was secreted byLMO was the virulence factor that allowed the LMO to escape from the primary phagocyticvacuole. The BALB/c mice were immunized with purified recombinant fusion Listeriolysin O whichwas expressed by prokaryotic expression system pGEX-6P-hly /BL-21. The screen ofanti-Listeriolysin O McAbs was performed with natural Listeriolysin O cultured in BHI culture mediumby indirect ELISA. SP2/0 was fused with spleen cells from BALB/c mice immunize. The hybridomasupernatants were selected by indirect. ELISA. The positive cells were subcloned three times bylimiting dilution. Six strains hybridoma cells line steadily secreting McAbs of anti-Listeriolysin Owere obtained and were namedⅠF5,ⅡC4,ⅡE5,ⅢG7,ⅣB7,ⅣE9, respectively.The isotypes of McAbs secreted byⅠF5,ⅡC4,ⅣE9,ⅡE5 were IgG1. Others were IgM. AllMcAbs belonged toκchain. The chromosomal number of hybridoma cell was 85～103. Thechromosomal number of SP20 cell was 55～60. The antibody titers of two of the total strainshybridoma cells culture supemate were 1:512. The antibody titers of three of the total strainshybridoma cells culture supernate were 1:256 and the last one was 1:128. The titers of ascitesprepared by one strain cell was 1:10~5. The McAb had good specificity by Western blot assay.Blocking ELISA essay indicated that the McAb had excellent combinative capability with thenatural Listeriolysin O. and had no combinative capability with other erythrocytolysin. It is concludedthat the hybridoma secreting specific anti-Listeriolysin O McAb were constructed.The microreaction palte was invested with purified recombinant fusion Listeriolysin O. McAbsof ascites was used as diagnostic antibody that detected the culture supernate of LMO in BHIculture medium and TSB-YE culture medium on different time. A preliminary method wasestablished by ELISA to detect the LMO in the laboratory. 5 hours After the LMO be cultured inTSB-YE culture, Listeriolysin O can be detected by ELISA, and the number of bacterium in 1 mlculture fluid was 9.5×10~5. 5.5 hours After the LMO be cultured in BHI culture, Listeriolysin O canbe detected by ELISA, and the number of bacterium in 1 ml culture fluid was 2.7×10~6. In theexperiment, It was found that the number of LMO was more in BHI culture than in TSB-YE cultureon identical time and the quantity of LLO expressed was more in TSB-YE culture than in BHI culture.