Preparation And Preliminary Application Of Monoclonal Antibodies Against Hematodinium Sp. In Portunus Trituberculatus.

Hematodinium sp. is one of the main pathogens which lead to marine crustaceans parasitic diseases and it is the major pathogen that causes the death of economically important crustaceans such as Portunus trituberculatus in recent years. Its host is with wide distribution, a wide range of popularity and has extremely high mortality rate and it results in significant economic losses to the large-scale farming of coastal crab and mud crab in Zhejiang. Microscopic examination found that there was sharp decline in the number of blood lymph cells and blood lymph was mostly replaced by parasites with similar size and shape of blood cells. As there were few parasites in latency and the early infection period, and there were few differences between the morphological features of parasites and those of crab blood cells in some life stages, it is difficult to accurately distinguish them under a microscope and cause a lot of inconvenience .to the parasite clinical diagnosis.The diagnosis of parasitic infections generally relies on conventional etiology and epidemiology diagnosis. Etiology check is time-consuming and it is sometimes difficult to diagnose some pathogens. The sensitivity of etiology in low infection pathogen detection is not good and it does not apply to large-scale clinical inspection. Molecular diagnostic techniques or the immunological detection of the parasite greatly improve the sensitivity and specificity, especially the application of some new immunological methods such as in vitro culturing, antigen purification, monoclonal antibody technology, and other markers in the parasitic research, which create favorable conditions for the immunodiagnostic of parasitic diseases. The aim of this study is to build related immune markers through monoclonal antibody research of Hematodinium sp. and enable to locate the parasites and carry out quantitative detection of pathogens. This can resolve the difficulties in early diagnosis of parasitic disease problems; at the same time, through the use of this technology, large-scale survey of clinical samples can be carried out to explore the life history characteristics and transmission of the pathogen.There are two parts in the paper. The first part of the paper is about the development and specific identification of monoclonal antibodies of Hematodinium sp. The experiment used Hematodinium sp. from Portunus trituberculatus with "milk sickness" and Scylla serrata with "yellow water" disease, which were selected from Zhejiang coastal crab artificial breeding farms. Indirect Enzyme-Linked Immunosorbent Assay (ELISA) was used to screen hybridoma cells and limited dilution method was performed to sub clone the positive clones. After three cycles of subcloning, three McAbs against Hematodinium sp. were selected and designated as 2B₂, 3G₄ and 4G₇ respectively. The three McAbs in culture liquid were proved to have high ELISA titers and inducing ascites by using this. The ELISA titer in culture liquid and ascites were 5.12×10^-4 and 8.00×10^-4 in the indirect ELISA, respectively. By using the immunoglobulin subtypes kit, three McAbs were identified to be IgG.The identification of the monoclonal antibody specificity via indirect fluorescence-antibody examination showed that Hematodinium sp. was specific labeled with bright yellow green under UV, while the normal haemocytes remained dark. Through monoclonal and polyclonal antibodies, double antibody-based sandwich enzyme-linked immunosorbent assay detecting Hematodinium sp. was built. The positive compatibility rate of the double sandwich ELISA was 100%. Results showed that the monoclonal antibody characetered by high valence and high specificities can be used to diagnose Hematodinium sp. in Portunus trituberculatus in an early stage.The second part is about the early application of monoclonal antibody. a double antibody-sandwich ELISA has been developed to detect Hematodinium sp. The micrplates were coated with monoclonal antibody at concentration of 10μg/ml and working concentration of the polyclonal antibody purified from polyclonal antiserum was 14.3μg/mL, the sample dilution concentration was1:800 and the enzyme-conjugated antibody was 1:20000 for this double antibody sandwich ELISA assay. By antibody-sandwich ELISA, the positive rates of the Hematodinium sp. was more than 97.6 and the sensitivity of this method to detect the antigens of Hematodinium sp. was 100pg/mL. These data indicated that a sensitive, specific and reproducible sandwich ELISA for detection of Hematodinium sp.was developed.It provided a useful tool for diagnosis of Hematodinium sp. infection.