Study On Preparation Method Of Monoclonal Antibody To Listeria Monocytogenes

Listeria monocytogenes( LM for short)belonging to Listeria genus, is a kind of food-borne pathogenic bacteria which can cause human and animal diseases. Hence finding an easy and sensitive approach to detect Listeria monocytogenes content in foods is critical for supervising the Listeria monocytogenes-caused food-poisoning. To upgrade the method of testing Listeria monocytogenes, Monoclonal Antibody to Listeria monocytogenes are prepared, and during this process, this paper also makes an analysis on how to screen and select antibodies in a optimal way .This research uses the inactivate monocaryotic proliferation Lisztthe bacillus as the antigen(1×109/ml), with the purified fusion protein Listeria monocytogenes fully emulsified with equal volume of FCA , to firstly immunize the female BALB/c mice through the abdomen. The same immunization is undertaken every two weeks since then. When the immunizing work is done for five times, mice serum titer achieves above10, 000. Then taking its spleen, and with the SP2/0 myeloma cell, we carry on the cytomixis through the conventional approach to prepare the hybridoma cell. The proportion of the spleen cell and the SP2/0 myeloma cell is 5︰1, then screen and colonize them.This experiment is aimed at studying how to test antibodies. And by comparing testing methods of the indirect ELISA, the sandwich ELISA, the DAS-ELISA, DOT-ELISA and the slide agglutination test,we can safely reach the conclusion that the indirect ELISA and the DAS-ELISA are the appropriate methods for testing antibodies.Through a series of exploring on the action conditions of ELISA, the following findings could be presented. The coating condition includes, the inactivated Listeria monocytogenes solution ( 1×108/ml);rabbit serum coated with the ratio of 1︰10; 4℃overnight. The sealing solution is 2% bovine serum albumin;sealing temperature is 37℃;the antibody duration lasts for 1h;the HRP-goat-anti-mouse IgG's concentration is 1︰3000,the HRP-goat-anti-rabbit IgG's concentration is 1︰100;the function time of HRP-conjugated antibody is 40min;the function time of substrate solution is 15min.Based on the preparation of specific monoclonal antibody to Listeria monocytogenes, this experiment initially identifies the indirect ELISA and the DAS-ELISA as the appropriate methods for testing antibodies. We should firstly select the holes at the bottom of which hybridoma cell group grow. Then, we use the indirect ELISA method for screening. The screening approach should include the fowling procedure: coating the inactivated Listeria monocytogenes solution, sealing 2% bovine serum albumin;blending 50μl supernatant fluid with 50μl PBS solution evenly; sealing temperature 37℃for one hour ; the function time of the 1︰ 3000–diluted HRP-goat-anti-mouse IgG is 40min(temperatur 37℃), the function time of substrate solution is 15min. When the color reaction happens, cease the reaction and get the reading number. Then, we colon the cells initially identifies as positive, and use the sandwich ELISA method to test their specificity, so as to make sure that antibodies secreted by the positive hybridoma hole are antibodies to Listeria monocytogenes. To be specific, rabbit serum is coated with the ratio of 1︰10 ; sealing solution is 2% bovine serum albumin; then we add the inactivated Listeria monocytogenes solution;the temperature is 37℃, and for an hour. Following this, we again use 2% bovine serum albumin as the sealing solution, and mix 50μl supernatant fluid with 50μl PBS solution evenly; the action temperature is 37℃,and for one hour;the function time of the 1︰3000–diluted HRP-goat-anti-mouse IgG is 40min(temperatur 37℃), the function time of substrate solution is 15min. When the color reaction happens, cease the reaction and get the reading number.The indirect ELISA method is used while screening and selecting positive holes. This method is characterized by better sensitivity, stability simplicity, and low-cost as well as instant access. After identifying the positive holes, the sandwich ELISA method is used to make sure whether they are antibodies to Listeria monocytogenes.