Lipase Characteristics Research Of P.fluorescens (Lip) And Preparation Of Hybridoma Cell Lines Secreting Monoclonal Antibodies Against The Lip

As we all know, under the influence of milking conditions, the raw milk is veryvulnerable to microbes after milking machines and other equipment processed. Inthe subsequent cooling process, the psychrophile that can withstand low temperaturewill survive, and become the predominant strains soon, for reproduction andmetabolism in milk. Although psychrotrophic bacteria can be killed during thesterilization process, but the heat resistance lipase its secretion can maintain thermalstability because of its special structure, and still remain active after pasteurizedeven UHT sterilization, thereby decomposing the fat in milk, causing spoilage ofmilk and dairy products.However, the traditional detection method ofpsychrotropHic bacteria takes a long time, and cannot reflect the case ofpsychrotropHic bacteria in raw milk quickly and accurately, thus cannot assesswhether the raw milk is qualified and suitable for processing, and then causing hugelosses in production. Therefore, developing a fast method of PsychropHile detectionis needed. Studies have shown that there is a significant existing relationshipbetween the number of psychrotropHic bacteria and the activity of heat-resistantlipase they secreted. So the psychrotropHic bacteria in raw milk can be predicted bymeasuring lipase. This study aimed to establish a hybridoma cell lines that secretedmonoclonal antibody anti-Pseudomonas fluorescens lipase, and lay the materialfoundation of the ELISA method for of rapid detection of Pseudomonas fluorescenslipase.In this study, put Pseudomonas fluorescens, which is the predominantpsychrotropHic strains in raw milk, as the antigen. First of all, a preliminarypurification by ultrafiltration method to lipase, which make the purity of lipasereach73.4%, meet the requirement concentration of the antigen. Then the influenceon lipase activity was examined at different temperatures reaction time, different pH,different Ca2+concentration, the results showed that the heat-resistant lipase in the50℃water bath for30min,63℃water bath for30min,72℃water bath for20min,90℃water bath for10min,121℃0.1Mpa temperature treatment, there is a certaindegree of survival activity, indicating that the heat resistance of lipase. Through theinfluence of pH on Lip, a conclusion can be drawn that optimum reaction pH forPseudomonas fluorescens lipase is pH8.0. In vitro of Pseudomonas fluorescens,there was not significantly affected by concentration of Ca2+on Pseudomonasfluorescens lipase activity.In this study,6weeks old Balb/c mice were immunized with antibody, which is mixed by Pseudomonas fluorescens lipase and QuickAntibody adjuvant, and thetiter of anti-lipase antiserum was tested by the double agar diffusion method and theindirect ELISA detection, and optimize reaction conditions of antiserum titerdetected by indirect ELISA. The results of research indicate that, the double agardiffusion method was chosen. After the third immunization, the mice have reached atiter of1:16. Then two more booster immunization was taken, and using a moreaccurate indirect ELISA method to detect the continues Antisera titer. At last theantisera titer has reached1:6400.The study also optimized experimental conditions of testing anti-Pseudomonasfluorescens lipase antiserum titer by indirect ELISA. The results show that: Thecoating solution was selected carbonate buffer of0.1mol/L pH9.6, the besttemperature and time of packet placement at4℃for10h; The best dilution ofcoating antigen and antiserum were: antigen diluted1:100; antiserum diluted1:1600;Best confinement: Select1%closed1h; Optimize the best dilution of enzymesecondary antibody is1:10000, the optimal reaction time was1h.Finally, adopt the method of indirect ELISA, controlled for monoclonalantibody of the commercialization inherent lipase, ant-lipoprotein lipase, in rawmilk. The results indicate that the antiserum of anti-Pseudomonas fluorescens lipaseobtained in this study can detect the Pseudomonas fluorescens lipase specifically,which shows specificity of anti-Pseudomonas fluorescens lipase antiserum. Theresults of this research have laid a certain foundation for the subsequentdetermination of Pseudomonas fluorescens lipase in raw milk by using monoclonalantibody.