Preparation Of Monoclonal Antibody Of Anti-LLO Fusion Protein And Establishment Of Indirect Sandwich ELISA Method For Detection Of Listeria Monocytogenes

Listeria monocytogenes (LM) is micro-organisms of the genus Listeria, pathogen of listeriosis of animals including human. LM is intracellular parasitic bacteria, infection by LM is mediated by a number of virulence factors, in which most important is Listeriolysin O(LLO).LLO is a 58.6ku～60 ku pore-forming toxin, encoding by hly gene, necessary for LM infection. LLO was only found in LM, thereby preparing its monoclonal antibody and establishing detection methods of LM based on anti-LLO monoclonal antibody, which will facilitate the further study of LLO.This research consisted of four parts. In the first part, LLO was expressed in E.coli. BL21, inclusion body including LLO protein was dissolved in 8 mol/L urea, purified by Ni-chelating affinity chromatography, refolded by gradient dialysis with urea in vitro.In the second part, Balb/c mice were immunized with refolding LLO-his, the splenocytes of immunized mice was fused with Sp2/0 cell, anti-LLO hybridoma cells were screened by indirect ELISA, finally anti-LLO monoclonal antibody was obtained. In the third part, New Zealand white rabbits were immunized by 200μg refolding LLO for rabbit against LLO polyclonal antibody. In the forth part, indirect sandwich ELISA detection methods of LM was primarily established, which high affinity monoclonal antibody was used as coating antibody, rabbit against LLO polyclonal antibody as testing antibody.(1)LLO was expressed in E.coli. BL21; E.coli. BL21 was ultrasonication for inclusion body including LLO protein, which was dissolved in 8 mol/L urea, purified by Ni-chelating affinity chromatography, refolded by gradient dialysis with urea in vitro.Refolding LLO was concentrated by PEG 20 000,finally refolding LLO was obtained.(2)Balb/c mice were immunized with refolding LLO-his, 20μg refolding LLO per rabbit.after two weeks, immunization was done with refolding LLO emulsified with Freund's incomplete adjuvant; totally four times, interval was two weeks; one week after each immunization, immunized mice serum was collected via caudal vein, antibody titer was tested by indirect ELISA. When anti-LLO serum dilution times was 1×10⁵, meanwhile OD₄₅₀ of anti-LLO serum was twice as much as control', OD₄₅₀≥1.0, three day before immunization, immunizing mice were immunized via caudal vein with antigen without adjuvant; the splenocytes of immunized mice were fused with Sp2/0 cell; anti-LLO hybridoma cells were screened by indirect ELISA; after three times sub-clone, 2 strain hybridoma secreting anti-LLO antibody was obtained named as 3F5-G3 and 2B4-C8 respectively. Ascites was induced with atoleine. Hybridoma and antibodies in ascites and cell supernatant were identified, the antibody titre of ascites was up to 1:1×10⁵(3F5-G3) and 1:1×10⁷(2B4-C8);the number of chromosome was 97±5(3F5-G3) and 93±8(2B4-C8);the affinity constant of McAbs was about 0.49μg/mL(3F5-G3) and 0.26μg/mL(2B4-C8); the isotype of both McAbs was IgG1 withκlight chain; the specific band was observed between 2 McAbs and LLO, no band between both and vector protein in Western blot assay.Therefore,the specific McAbs against LLO were prepared in the study.(3)New Zealand white rabbits were immunized with 200μg refolding LLO by two immunization methods respectively. One is classical methods, the first immunization was done with antigen emulsified with Freund's complete adjuvant, after two weeks, immunization was done with refolding LLO emulsified with Freund's incomplete adjuvant, interval was two weeks; the second method was a improved method based on classical method, the first immunization was same, the 4th day after first immunization, immunization was done with same method, the 30th day after the second immunization, the third immunization was done with antigen emulsified with Freund's incomplete adjuvant. After three times, anti-LLO polyclonal antibody was obtained. The result of two immunization methods was statistically analyzed, the results of both methods was highly significant difference, showing the second method was feasibility. (4)High affinity monoclonal antibody was used as coating antibody, rabbit against LLO polyclonal antibody as testing antibody, ELISA work conditions were optimized, finally indirect sandwich ELISA detection methods of LM was primarily established.