Establishment And Preliminary Application Of Indirect Elisa For Mycoplasma Hyopneumoniae Serum Antibodies Detection

Mycoplasma hyopneumoniae (Mhp) is the causative agent of Swine Enzootic Pneumoniae, a disease which is characterized by highly infective, chronic, high morbidity and low self-mortality.Infected swine has a retarded growth and inefficient food conversion.Mhp destroy the respiratory tract mucosa-cilia barrier, so it can induce the infection of other pathogenic bacteria and increase the mortality. The control of M. hyopneumoniae infections critically requires suitable vaccines and diagnostic tools. Two commercial ELISAs are commonly used for monitoring the health status of pig herds, a monoclonal antibodybased blocking ELISA and an indirect ELISA (IDEXX HerdChek M. hyopneumoniae, IDEXX Laboratories).Comparison of ELISA results revealed a high specificity of both assays whereas the sensitivity is low and strongly depends on the vaccination status. It was reported that these two ELISA methods wrapped a single antigen of comparison. In this experiment, we expressed main antigenal proteins of Mycoplasma hyopneumoniae:P36, P46, P97R1, DnaK and synthesized specificity short peptide MHP366.Developed indrect ELISA detection method based on various protein antigens for Mhp antibody, in order to improve the sensitivity of the test method.1. Preparation a lot of standard positive serum by utilizing of the preservation of Js virulent strain in the lab, and prepared standard negatie serum. Collected more than300portion negative and positive serum from different areas and different background; this provided powerful references.to the establishment of antibody test methods to M. hyopneumoniae.2. Expressed a variety of immune protein of Mycoplasma hyopneumoniae:P36, P46, P97R1and DnaK in E.coli expression system and synthesized specificity short peptide MHP366wich can be used to differentiate infected from vaccinated animals.3. Established single protein indirect ELISA methods, and the methods are optimized, Based on the single protein indirect ELISA methods established a indirect ELISA method which coated a variety of immune protein of Mycoplasma hyopneumoniae and optimized.And the optimized program is as follows:P46protein was diluted to lOμg/ml, P36to0.5μg/ml, P97R1protein to5μg/ml, DnaK protein to5μg/ml, MHP366peptide0.1μg/ml, the above protein and peptide were mixed in a same ratio.The mixing protein was coated overnight at4℃after incubation for1h at37℃on96-well microtiter plates. And the plates were subsequently blocked1h with10%horse serum.Then incubated for60min with sera diluted to1:40by PBS containing0.05%Tween-20(PBST), the working concentration of HRP-goat anti-swine IgG was1:10000, the chromogenic time was incubated at37℃for15min.4. The specific peptide MHP366can identification of antibodies of natural infection pigs, and can detect infected piglets from immuned pigs; it is very important for the diagnosis and control of Enzootic pneumonia (EP). Further research need to do for differentiating infected from vaccinated animals.5. The indirect ELISA method established in this study is coated with multiple immune antigenic protein of M. hyopneumoniae, compared with a single protein it can improve the sensitivity of the test method, purified protein guarantee the specificity of detection method, so this method can more accurately monitoring the infection status and animal immune status.