||单核细胞增生性李斯特菌(Listeria monocytogenes,LM)简称单增李斯特菌,是李斯特菌属的代表种,是一种能够引起人畜共患病的食源性致病菌.建立快速、灵敏的检测食物中单增李斯特菌的方法对于控制单增李斯特菌引起的食物中毒具有重要意义.为了改进该菌的检测方法,我们制备了抗单增李斯特菌的单克隆抗体,并在抗体制备过程中,分析选择了筛选抗体的最好方法.本研究首次免疫雌性BALB/c小鼠,采用热灭活后的单核细胞增生性李斯特杆菌全菌为抗原(1*109个/ml),与等体积弗氏完全佐剂充分乳化后,经腹腔免疫,以后每隔2周免疫一次,经过五次免疫后,待小鼠血清效价达到1︰100000以上,在加强免疫后取其脾脏与SP2/0骨髓瘤细胞按常规方法进行细胞融合,制备杂交瘤细胞,脾细胞与SP2/0骨髓瘤细胞的比例为5︰1,再进行筛选和克隆化.本试验对抗体检测方法进行了研究,通过对间接ELISA,夹心ELISA,双夹心ELISA,DOT-ELISA,简易ELISA,血凝板凝集等检测方法的比较,确定了细胞上清液中抗体的检测方法为间接ELISA法和双夹心ELISA法.本研究通过对ELISA反应条件的系列摸索,确定了以下工作条件:包被条件为:经热灭活的单增李斯特菌液1*108个/ml,兔血清以1︰10倍稀释度包被,4℃,过夜;封闭条件为:2%牛血清白蛋白溶液37℃作用1h;待测抗体作用条件为37℃,1h;HRP-羊抗鼠IgG的工作浓度为1︰3000倍,HRP-羊抗兔IgG的工作浓度为1︰100倍,条件为37℃,40min;底物液作用条件为37℃,15min.本研究在制备单增李斯特菌的特异性单克隆抗体的基础上初步建立了检测细胞上清液中抗体的间接ELISA法和双夹心ELISA法,首先选出杂交瘤细胞团长至孔底1/10面积的孔,用间接ELISA法进行初筛,筛选方法为:包被热灭活的单增李斯特菌液,2%牛血清白蛋白溶液封闭,取50μl细胞上清液与50μl PBS溶液混匀,37℃作用1h,1︰3000倍稀释的HRP-羊抗鼠IgG,37℃作用40min,与底物液在37℃条件下显色15min,终止反应,读数.对初步鉴定为阳性的细胞克隆孔,再进行双夹心ELISA法检测特异性,以确定阳性孔杂交瘤细胞所分泌的抗体是抗单增李斯特菌的抗体.具体方法为:以1︰10倍稀释度包被兔血清,2%牛血清白蛋白溶液封闭,加入热灭活的单增李斯特菌液,37℃作用1h,再以2%牛血清白蛋白溶液封闭,取50μl细胞上清液与50μl PBS溶液混匀,37℃作用1h,1︰3000倍稀释的HRP-羊抗鼠IgG,37℃作用40min,与底物液在37℃条件下作用15min显色,终止反应,读数.以挑选出分泌抗单增李斯特菌的抗体的杂交瘤细胞孔.本研究设计在筛选细胞阳性孔的过程中,选用间接ELISA法,此方法灵敏度高,稳定性强,操作相对简便,费用低,取材方便.当间接法测到稳定阳性孔时,再进行双夹心ELISA法,确定此阳性孔为分泌抗单增李斯特菌的抗体的杂交瘤细胞团.
||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.