Production Of Antibodies Of High Specificity And Affinity For Constitutively And Stably Expressed Listeria Monocytogenes Surface Antigens

Objectives: Antibodies of high specificity and affinity for constitutively andstably expressed Listeria monocytogenes(L. monocytogenes)surface antigens areessential for the development and optimization of antibody-based detectionmethods.The study aims to clone and express protein InternalinA (InlA) fromL.monocytogenes and prepare the polyclonal antibody against InlA. At the same time,using three methods to prepare antigens for immunization of mice to developmonoclonal antibodies(MAbs) with high specificity and affinity for surface antigensof all major serotypes of L.monocytogenes cells.Methods: Use biological software to design the primers of inlA gene, amplifythe inlA gene from L.monocytogenes by PCR and construct the gene into prokaryoticexpression vector PET28a(+), then transform the gene into E.coli BL21and expressoptimally; purify the recombinant product InlA by nickel affinity chromatography,identify the recombinant protein by mass spectrometry analysis, and test theimmunogenicity by ELISA. The purified protein is used as antigen for immunizationof rabbit to prepare polyclonal antibody and the binding affinity between polyclonalantibody and L.monocytogenes is determined.At the same time, L. monocytogenessurface proteins、formalin-inactivated whole cells and heat-killed whole cells wereused as antigens to immunize Balb/c mice. The mouse which showed the highestserum reactivity with L.monocytogenes and the lowest cross-reactivity was selectedfor hybridoma production. The initial600hybridoma-containing wells were screenedusing a comparative, negative-screening ELISA format, where one set of wells wascoated with L. monocytogenes54002cells and a duplicate set of wells were coatedwith an equal mixture of L. welshimeri, L. grayi and L. innocua cells. Using ELISA toanalyse the cross-reactivity of anti-L. monocytogenes MAbs with othermicro-organisms. Analysed the reaction profiles of antibodies by Western blot assay.Identified the72kDa protein by immunoprecipitation-mass spectrometry (IP-MS).Produced and purified the recombinant MurA protein and Western blot analysis of therecombinant protein.Results: The recombinant InlA protein with relatively molecular mass of96kD was over-expressed in E. coli BL21(DE3). The InlA antiserum were obtained with a titer as high as1∶1×105.The antibodies had no cross reaction with otherpathogenic microorganisms. Immunofluorescent staining showed that it only binds tothe cell surface of L.monocytogenes but not L. welshimeri. At the same time, a panelof hybridomas was produced using extraction of L.monocytogenes54002surfaceprotein as the immunogen. Detected by ELISA, MAb1B10,2C10and1G3onlyreacted with killed L. monocytogenes without any cross reactions; MAb1C10and3G8highly reacted with L. monocytogenes, L.innocua and L.welshimeri in ELISA, butseldom reacted with non-Listeria spp. However, only MAb1B10was capable ofbinding to the cell-surface antigens of all major serotypes of live L. monocytogenes,as demonstrated by ELISA and immunogold transmission electron microscopy. Thereactive epitope was identified as MurA protein of72kDa by IP-MS and western blot.Recombinant MurA produced in Escherichia coli was confirmed to be the antigenrecognised by MAb1B10,2C10and1G3.Conclusion: The polyclonal antibodies which binds to the cell surface ofLM specifically are prepared successfully. MAb1B10which reacted with liveL.monocytogenes, formalin-inactivated and heat-killed L.monocytogenes wasprepared. The results indicate that MurA could be exploited as a high specificmarker for identifying pathogenic L. monocytogenes in suspect food samples.These results would provide basis for developing immunomagnetic beads whichcould separate L. monocytogenes efficiently and specifically.