Preparation Of Polyclonal Antibody Against Listeria Monocytogenes And The Study Of Coupling The CdS Quantum Dots

Listeria monocytogenes has been known to be a human pathogen witch can causes a serious health threat to immunocompromised individuals,sach as meningitis,hematosepsis,abortion and so on.Due to an associated high fatality rate,this organism can be classified becoming an emerging problem in public hygiene.General detect assay is veracity,but it is time-consuming;molecular biological assay is speediness, delicacy specificity,but it is expensive and need high technic level;Vitek Automated System（VITEK）,vitek immune diagnostic assay system（VIDAS） is celerity,exact,but expensive.ELISA assay of immunological apply abroadly in the detection of pathogens for it is simple,convenient,speediness and delicacy.Since 20th century,radio-immumity, immunoenzyme,immunofluorescence test,chemical lumind immunoassay and electricity chemical lumind immunoassay had been set up,the reaction between the antigen and antibody can be localizated in a microcosmic field,and then detection of the microbe can reache a new level.In this reserch we coupling the Listeria monocytogenes monoclonal antibody with the CdS quantum dots,using its fluorescence,the new analysis method was estabilshed.This article by high titer polyclonal antibodies against Listeria monocytogenes were produced from rabbits immunized with the hest kill cells and 3%formalin kill cells immunogen.The result show that the rising trend of the antibodies to the immunity after 50d.Through the indirect enzyme-linked immunosorbent assay（ELISA）,the final titer of antibodies were 1:12800,getting high purity IgG by Caprylic acid-Ammonium sulfate assay pufificate antibodies.Conditions were optimized for using these preparations for an inderecte ELISA a. The result was shown that the optimal concentration of antigen was 10⁸cfu/mL;the dilution of antibody was 1:800;the dilutoin of the HRP-labeled goat anti-rabbit was 1: 2000;the confining liquid was 5%defatted milk powder,37℃,2h;serum sample for detecting and HRP-labeled goat anti-rabbit should be incubated at 37℃for 2h respectively,the substrate for inderecte ELISA was incubated at room temperature for 5min before reading the OD₄₉₂.The specific crossing test indicated that the Listeria monocytogenes monoclonal antibody had good specificity for the detection.The cross-reaction only happened with some specieses of the listeria.It was confirmed that there was no cross-reaction between Escherichia coli,,Salmonella.spp,Bacillus subtilis,Vibrio Parahemolyticus,Proteus mirabilis,Staphylococcus aureus and some other genus. Using the indirecte ELISA technique,a minimum detectable level of 10⁷cfu/mL Listeria monocytogenes in buffer was achieved.The CdS quantum dots was preparated by the CdCl₂ and the Na₂S solution.By optimizing the reaction time,the ratio of[Cd²⁺]to[S^2-],the volume of stabilizing angent（TGA）,the pH of the system and the reaction temperature to find a best way for making the CdS quantum dots.Conditions were optimized for the CdS quantum dots indicated that the optimal reaction time was 3h;the ratio of[Cd²⁺]to[S^2-]was 2:1;the volume of TGA was 50μL;the pH of the system was 8 and the reaction temperature was 30℃.The EDC·HCl and the NHS were used for coupling the Listeria monocytogenes monoclonal antibody IgG with the CdS quantum dots.After coupling the Listeria monocytogenes monoclonal antibody IgG with the CdS quantum dots,the fluorescence intensity had been reinforcing.The Listeria monocytogenes could be detected by the serum agglutination test and direct immunofluorescence test.This research establish a specific,sensitive,stabile and repeatable method which offers a new analysis method for the detection.