Preparation Of Anti-listeria Monocytogenes Monoclonal Antibody、Polyclonal Antibodies And Single-domain Heavy Chain Antibody

Listeria monocytogenes (LM) is a commonly foodborne pathogens, which caninfect humans and other animals through contaminated food. LM has a highlyadaptive to the environment, and can survive in some extreme conditions, such aslow temperature, low pH and high salt conditions. Meanwhile, LM can acrossmultiple body barriers, causing severe infections including meningitis, septicemiaand abortion, the infection mortality rate can achieve at20%to30%. In recent years,Listeriosis outbroken in the U.S. and Europe caused huge casualties and propertylosses. Therefore, it is very important and urgent to establish some efficient methodsfor rapid and accurate detection of LM.In the present study, the heat inactivation LM was used as antigen to generateLM specific monoclonal antibodies. Six of anti-LM monoclonal antibodies (mAbs),which were named as3F2-A8、8C8-B11、10A11-A4、10E7-H6、11B9-F4and12A6-H11, respectively, were obtained. The titers of mAbs were1：320000、1：640000、1：640000、1：640000、1：640000and1：25600. Dot-blot results showthat the six of antibodies can not only recognize listeria genus but also some otherbacteria in different level. The results of western-blot analysis show that antigenicsubstances recognized by3F2-A8,8C8-B11,10A11-A4,10E7-H6and11B9-F4areteichoic acid.To prepare the specific antibodies of LM, we developed pGEX-4t-1-actA,pET-22b-InlB and PET-22b-iap recombinant vectors, the ActA, InlB and P60proteins were expression. The purified InlB and P60proteins were used as antigen toinject BALB/c mice for generating specific anti-serum. The results of dot-blotanalysis show that anti-InlB polyclonal antibodies can specifically identify LM withnegligible cross-reaction with listeria genus. Anti-P60polyclonal antibodies canreact with LM ATCC13932, LM CMCC54001as well as other48wild LM.Moreover, the anti-P60polyclonal antibodies exhibit significant cross-reaction withListeria innocua NCTC11288, Listeria ivanovii ATCC19119and Listeria welshimeri ATCC35897. The above results indicate that InlB and P60proteins can be used asspecific antigen for preparing highly specific monoclonal antibodies of LM.Meanwhile, anti-LM specific single-domain heavy chain antibodies wereobtained by subtractive panning method. First, the whole cell of heat inactivated LMwas used as antigen for panning anti-LM specific antibodies from the alpacanon-immune phage displayed library. After four rounds of conventional panning andone round of subtractive panning, a total number of random384phage clones wereidentified by phage-ELISA. One hundred and twelve of positive clones were pickedout and then sequenced. Among them, the specificity of ten clones which have highaffinity to LM, were further analyzed by phage-ELISA, and two of anti-LM specificsingle-domain heavy chain antibodies were obtained. Moreover, the expressionvectors named pET-25b-L5-78and pET-25b-L5-79were constructed, and two ofanti-LM single-domain heavy chain antibodies were expressed. The results ofperformance validation show that two of antibodies can maintain high bioactivityand stability under extreme pH, high concentration of denaturing agent and hightemperature condition. Furthermore, a sandwich ELISA for LM was developedbased on mAbs-4A7and single-domain heavy chain antibodies (L5-79), the detectionlimit of LM is5×106CFU/mL.In summary, we have used three methods for the preparation of anti-LMantibodies, and have successfully obtained six kinds of mAbs, two kinds of pAbsand two kinds of single domain heavy chain antibodies. The resultant antibodies inthis study will provide a good basic for development of immunological method forrapid screening detection of LM.