Development And Preliminary Application Of The Listeria Monocytogenes CELISA Kit Based On ActA Monoclonal Antibody

Listeria monocytogenes(Lm)is an important food-borne zoonotic pathogen which can cause listeriosis in humans and animals characterized by meningitis,sepsis,and abortion.Recently,the causative agent of listeriosis has been frequently reported and threatened animal husbandry together with public health.Therefore,the establishment of a sensitive,specific,rapid and simple diagnostic method for the epidemiological investigation of listeriosis will be helpful for the prevention and control of the disease.Lm is a gram-positvie intracellular parasite and its pathogenicity is mainly associated with virulence factors.Among them,the bacterial surface protein ActA plays a critical role in the intercellular movement and dissemination of the bacteria.In this study,a competitive ELISA method was established with ActA as target antigen and enzyme-labeled monoclonal antibody.The competitive ELISA kit was further developed to effectively and specifically detect Lm infection,thus afford new technology for rapid diagnosis listeriosis.1.Expression and immunogenicity analysis of ActAThe immunogenicity of the target antigen is the basis for establishing an ideal serological method.In order to evaluate the antigenicity of ActA,the recombinant strain BL21(DE3)(pET30a-actA)was used to induce the expression of recombinant rHis-ActA protein,which was identified by Western blotting.Following,the serum antibody level of ActA was dynamically determined using indirect ELISA.The results showed that the specific antibody against ActA was produced at 6 days post infection,and the antibody titer was the highest on the 10th day(1:6400).The anti-ActA antibody lasted for 22 days,indicating that ActA has good antigenicity.Additionally,ActA monoclonal antibody hybridoma cell 3G11 was prepared from ascites and labeled with purification.The amino acid sequences of three complementarity determining regions in light chains and heavy chains variable region of the monoclonal antibody were further determined.This study indicated that ActA is a promising candidate antigen for serological detection of Lm.2.Establishment of competitive ELISA based on ActA monoclonal antibodyTo establish the competitive ELISA detection method,the coating concentration of ActA and the working concentration of HRP-labeled ActA monoclonal antibody were explored.In this study,the optimal concentration of rHis-ActA was 2.0 μg/mL,and HRP-3G11 was 36.2 ng/mL by competitive ELISA square array.On this basis,the procedures of competitive ELISA were optimized by controlling the single variable method.The detection conditions and steps were as follows:the antigen was diluted to the working concentration with CBS buffer and coated at 4℃ for 16 h.Nextly,the blocking solution(PBS with 2%skim milk)was added to the well and incubated at 37℃ for 3 h.Finally,50 μL serum(1:10 dilution)and 50 μL enzymelabeled antibody(1:32000,36.2 ng/mL)were added to the well and incubated for 1 h,following,the colour reaction was performed at 37℃ for 3 min.The cut off value of competitive ELISA method was determined to be 27.5%with ROC curve.Meanwhile,the results of the sensitivity,specificity were 100%and 93.3%,respectively.Importantly,the method had no cross-reaction with the positive serum of other common pathogens,and the test results had excellent repeatability.In conclusion,the competitive ELISA method had good stability,specificity and sensitivity,which laied a foundation for the serological diagnosis of listeriosis.3.Development and preliminary application of ActA-cELISA kitBased on the established competitive ELISA method,the ELISA kit was further developed.This study determined the shelf life and conformity rate with commercial kits as well as the quality inspection of this kit,a preliminary application was identified.The results showed that the variation coefficients of intra-batch,inter-batch and different operators were all within 15%,indicating that the kit had stable performance and repeatability.The enzymelabeled antibody protectant could effectively improve the shelf life of this kit,thus the validity period could be ensured for more than 10 months at 4℃ as well as maintained good detection effect and quality inspection.The comparative analysis showed that the results of 47 clinical sheep serum samples detected by ActA-cELISA kit were 95.7%consisted with the results of Diatheva.Finally,928 sheep and 510 bovine serum samples were detected by this kit.The results showed that the average positive rates of sheep serum and bovine serum samples were 23.0%and 14.1%,respectively.In summary,the establishment of ActA monoclonal antibody competitive ELISA method and the developed kit could effectively detect listeriosis.In conclusion,we establisheed a sensitive and specific competitive ELISA method based on the ActA protein with good antigenicity.On this basis,the competitive ELISA kit was developed.This kit has favorable repeatability and stability,which can effectively detect Lm infection.The competitive ELISA kit helps to lay a solid foundation for the precise prevention and control of listeriosis.