| Myxobolus honghuensis invariably caused an army of death and lesion of silver crucian carp which bring huge economic losses. So it is particularly crucial for the early detection of this parasitic disease,but at present, there is no early detection method of myxobolus honghuensis. One kind of myxobolus which named honghuensis has been accurately identified. Chemical methods were used to purify the parasite that avoided the immunological properties of the parasite been destroyed. Of these the soluble and insoluble components of the parasite were prepared and the immunogenicity of myxobolus honghuensis was researched by immunology, then established the corresponding ELISA method which based on the antigen characteristic that was used to detected the diseased fish.One species of myxobolus honghuensis was identified by morphological observation and bioinformatics analysis. The polar organic solvent was used to separate and purify the myxobolus honghuensis that guaranteed the soluble and insoluble antigen of the parasite which extracted by repeated freezing and thawing with liquid nitrogen and ultrasonic fragmentation were pure. Polyclonal antibody was produced in mice and guinea pig by two kinds of antigens immunization, The immunological properties of polyclonal antibody were analyzed by enzyme-linked immunosorbent assay(ELISA), SDS-PAGE protein electrophoresis and indirect immunofluorescence test(IFAT). ELISA result demonstrated that antiserum titers of mice and guinea pig which injected by soluble components were all above 1:20000,injected by insoluble components were all less than 1:50. SDS-PAGE manifested that the soluble protein was mainly distributed in 10 kDa ~ 200 kDa and there is no obvious distribution of insoluble components. Fluorescence localization with soluble components of the polyclonal antibody displayed that most of myxobolus honghuensis antigen was mainly located in the sporoplasm and few part located in spore shell valve, fluorescence localization with insoluble components of the polyclonal antibody was weak, in addition there is a faint cross reaction existed between Myxobolus and Thelohanellus.We establish a method of double antibody sandwich ELISA which detect the myxobolus honghuensis that use the purified polyclonal antibody as the detection reagent. Guinea pig and mouse polyclonal antibody optimal working concentration which determined by phalanx titration method was respectively 3.09μg/ml(1: 200) and 10.3μg/ml(1: 800). The working conditions of sealing liquid and antibody labeled with HRP enzyme was respectively 90 min in 37℃ sealed with 5% skim milk and 1:2000 affected in 37℃. The specific experiment indicated that: there is poor cross reaction with Koi herpes virus, Vivtorinox Aeromonas, Ciliates, Dactylogyrus. But there was a feeble cross reaction between the Thelohanellus wuhanensis and Myxobolus ampullicapsulatus. The repeated experiments demonstrated that the method proved a good repeatability, that because the batch and inter batch coefficient of variation was respectively less than 8% and 9%.Preliminary clinical application shown that the positive coincidence rate reached at 80%, the negative coincidence rate reached at 100%, while using PCR method and double antibody sandwich ELISA method to detect gill tissue lixivium from 90 tail silver crucian carp in the age of 10 weeks were respectively sampled from Shuangyang reservoir in Jilin province, a reservoir in Jilin City, a reservoir of Meihekou. Consequently the method of double antibody sandwich ELISA was proved to be applicable which not only provides a more simple, rapid, sensitive and widely used method for the detection of myxobolus honghuensis, but also provides a basis for the further establishment of a faster, more intuitive and convenient method. |
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