Studies On The Method Of McAb-ELISA Detection Of Pebrine Of Bombyx Mori Eggs

Pebrine disease which is caused by Nosema bombycis is the very popular infectious diseases in silkworm and is very wide distributed.Nb is a eukaryotic obligate intracellular parasites.It caused the pebrine disease by parasitized in silkworm bodies and is a destructive disease because of vertical transmission.They cause severe losses to silk farms. Up to now, the detection of microsporidia relied mainly on microscopy mother moth in sericulture,in order to supply non-Nb silkworm eggs. The finished egg quarantine as a "correction check" approach has been wide concern in recent years,and used as the seized items standards by the Ministry of agriculture.The researchers also concerned about the Nb detection by use modern molecular biology and immunology techniques.The port system have the greater responsib- ility to import and export silkworm eggs,there are also certain risk.So, it is essential to study a immunology technology whitch could be used to import and export quarantine in the silkworm products.The polyclonal antibodies can identify more epitope surface proteins. However, compared with the monoclonal antibody,pAb have more cross-reaction and sensitivity is low.Our study was based on pAb can identify more epitope surface proteins, could capture spores more easy,and monoclonal antibody have more specific characteristics. Through direct marker enzyme in the monoclonal antibody,Intent to obtain a simple and useful antibody-sandwich ELISA detection method. and grinding method of processing Microsporidia Bao was significantly improved results The and antibody,Multi-epitope identification number.The purified rabbit polyclone antibody of Nosema bombycis was used to be coated on the 96-wellplate, used as capturing antibody,after test samples were added, then Horseradish peroxidase conjugated monoclonal antibodies was added as detecting antibody, and to establish a double-antibody sandwich ELISA assay for the detect,each of steps were optimized.The study shown that the coating contrast shows that the Nb treated by liquid nitrogen freezing and thawing nitrogen is more easy to coat than the normal Nb.Preparation of the monoclonal antibody verified by western blotting recognition of the spores is the surface antigen.The optimal concentration of recombinant polyclonal antibody for coating of plate was 10 ng/mL, the optimal coating condition for ELISA was 4℃overnight, the work concentration of HRP-labeled monoclonal antibody was 1:2000, the sample for detecting and HRP-labeled monoclonal antibody should be incubated at 37℃for 1.5 h and 1 h respectively, the substrate for ELISA was incubated at 37℃for 15 min before terminated with the stopping solution.The sensitivity was detected bu use the method condition have established, it was shown that the detected amounts of purified Nb was 3.125×105 spores/mL,and detected amounts of the simulated silkworm rggs infected with Nb was 5×106 spores/mL.The detected amounts need to elevate by further shudies.