| In recent years,with the gradual control of swine infectious diseases,mycoplasma become the focus of disease control as the representative of chronic respiratory infectious diseases.This study focuses on the diagnosis of Mycoplasma hyopneumoniae and Mycoplasma hyorhinis.(1)Mycoplasma hyopneumoniae(Mhp)is the pathogen of mycoplasmal pneumonia of swine(MPS),a chronic respiratory disease with high morbidity and low mortality.The disease can cause secondary infection.At present,the domestic widespread use the inactivated vaccine to prevention and control MPS.The antigen content is one of the keys to determine the quality and immunogenicity of the MPS vaccine.In this study,we will establish an in vitro potency test method for inactivating vaccines in order to become an alternative to animal testing for efficacy testing of MPS inactivated vaccines.In this study,a double antibody sandwich ELISA method was established based on P46 protein and the reaction conditions were optimized.The method was used to quantitatively detect the culture antigen and the finished vaccine,and compared with the traditional detection method,it was proved that the ELISA could be used as a reliable method for quantification of Mhp in vaccine production.(2)Mycoplasma hyorhinis(Mhr)is commonly found in pig’s nasal,tracheal and bronchial secretions,which can cause clinical symptoms such as multiple serositis,pneumonia,arthritis and otitis media.This disease can resulting in decreased pig growth performance and Mixed respiratory pathogens mixed infection,aggravated the condition.Mhr infection rate is high,adaptable,extremely difficult to evolvein in clinical pig farm.In recent years,it causes increasing clinical attention.At the same time,Mhr is related with a variety of human tumors,it belongs to zoonotic pathogens.But at present,Mhr’s diagnostic technique is not yet complete,and its infection mechanism research is still in its infancy.In this study,a monoclonal antibody against Mycoplasma hyopneumoniae was fully identified,which provided an important test tool for the pathogenesis and pathogen diagnosis of Mhr.This research was developed as follows:1.An double antibody sandwich ELISA method was established,using anti-P46 monoclonal antibody as capture antibody and anti-P46 protein polyclonal antibody as detect antibody.The optimal coating concentration of monoclonal antibody was 2.5 μg/mL.The optimal dilution of anti-P46 protein polyclonal antibody and HRP-conjugated IgG was 1:40 000 and 1:10 000,respectively.Furthermore,the coefficients of variation of inter-assay and intra-assay experiments were both below than 10%.The lowest detection quantity of M.hyopneumoiae was 9.754 ng/mL.No cross reaction with Mycoplasma hyorhinis,Mycoplasma hyosynoviae and Mycoplasma flocculare was detected.The established ELISA method was employed to detect artificially diluted samples.The concentration rate of the samples was calculated by directly detecting the samples(single point method)or by detecting a serial of dilutions of the samples(parallel line method),and the latter was more stable.The method used to detect the mycoplasma pneumoniae bacterial antigen content compared with CCU basically consistent.2.The contents of P46 antigen in MPS inactivated vaccine were determined by sandwich ELISA using low temperature freezing and thawing method,95%ethanol method,trichloromethane method,n-butanol method and acetone method.The results were compared,so as to screen out the method for determining the content of antigen in MPS inactivated vaccine,and compared with the rabbit immune antibody test method,explores the feasibility of this method as an alternative to vaccine efficacy test.The results showed that the acetone treatment method could reflect the antigen content of MPS inactivated vaccine.Compared with other methods,the method had high extraction efficiency and low consumption,and the interference of the demulsifier on the effective antigen was small,Which can accurately detect the difference of antigen content in the inactivated vaccine with different antigen contentafter inactivated vaccine.Using this method to deal with the finished vaccine,then use the sandwich ELISA to detection,which can accurately detect the antigen content difference in the different titer of the vaccine.3.BALB/c mice were immunized with whole cell protein of Mycoplasma hyorhinis,and then the monoclonal antibody(McAb)was prepared by hybridoma technique.The isotype of the McAb was identified,and then the titer and specificity were analyzed.The prepared McAb was used to detect Mycoplasma hyorhinis in the colony immunoboltting assay and indirect immunofluorescence assay.A hybridoma cell line was obtained,and was named Mhr-08.The McAb belonged to IgG1 isotype,and the light chains was κ type.The titer was detected to be 1:102 400 by ELISA.The result of Western-blot indicated that this McAb strongly reacted to a 43kDa protein of Mycoplasma hyorhinis,without cross-reaction with other swine mycoplasmas,Escherichia coli or KM2 medium.It was revealed that the McAb recognized a surface membrane protein and was successfully applied to detect Mycoplasma hyorhinis in colony immunoboltting assay.The mycoplasmas bound to the tracheal epithelial cells was successfully detected by using this McAb in indirect immunofluorescence assay.In conclusion,a McAb against Mycoplasma hyorhinis was successfully prepared,which provides as a useful tool for the diagnosis and pathogenesis researches.In summary,this study explored the method of determining the antigen content of Mhp NJ strain inactivated vaccine,and initially determined the detection method of antigen content in inactivated vaccine.But the method used to replace animal experiments to evaluate the efficacy of the vaccine need to be further explored and improved.At the same time,a monoclonal antibody against Mycoplasma hyopneumoniae was prepared,which provided a tool for pathogenicity and pathogen detection of Mycoplasma hyorhinis. |
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