Development And Application Of Rapid Detection Strip For Antigen Of Avian Reticuloendotheliosis Virus

Avian Reticuloendotheliosis(RE),caused by the Avian Reticuloendotheliosis virus(REV)of type C retrovirus,is a general term of a series of symptoms,including short stature syndrome,acute reticular cell tumor and chronic tissue tumor.The pathogen of this disease is the third one that has the ability to cause poultry toumor.At present,although the methods used in REV detection are highly sensitive and accurate,the long time and professional equipment and workers were needed.To get a simpler and more convenient method to detect REV,we have designed a colloidal gold immunochromatographic test strip based on two monoclonal antibodies,REVp30-2C8 and REVp30-5G6,which were against the main immunogenic protein p30 of REV.Within only 5-15min,it can test RE pathogen with as much rapidness,convenience,accuracy,perfect specificity and sensitivity as ELISA kit.Consequently,this test strip can be used to test anal swab and throat swab.It is much more convenient than traditional detection method,and professional tools are not needed.In a word,with its low cost and good promotion value,this colloidal immunochromatographic test strip will play a significant role in the control of RE.1.Optimal Conditions for Colloidal Bold Binding with Two Monoclonal Antibodies.The method,designed by Frens to control the particle size of colloidal gold by controlling reduction dose,was used to prepare three sizes of colloidal gold particles with different radius of 15nm,30nm,40nm respectively.Their application was evaluated by visual observation,transmission electron microscopy and colloidal gold test.The hybridoma cells of the two monoclonal antibodies against REVp30 protein kept in our laboratory were recovered to prepare ascites through balb/C mice,of which the mAb,REVp30-2C8 and REVp30-5G6,were purified by Protein G-Sepharose 4B column.After optimal condition such as bingding pH and bingding protein amount,the size colloidal gold particles combined with these two mAbs,we identify them used electron microscopy.As a result,30nm colloidal gold particle has been selected as the binding particle with both antibodies2.Development and Optimization of Rapid Test Strip for REV AntigenThe colloidal gold immunochromatographic strips have been developed and optimized for clinical application.Firstly,the test strip has been designed,using saturated ammonium sulfate precipitation method to purify rabbit anti-mouse IgG polyclonal antibody as C-line antibody;secondly,the optimal amount of colloidal gold-antibody complex in the colloidal gold test strip,gold standard pad treatment,and T line C line treatment have been tested.Thirdly,the sensitivity,specificity,repeatability and keeping time of colloidal gold strip have been studied.Finally,the test strip,with REVp30-5G6 as the gold binding antibody and REVp30-2C8 as the T-line antibody,has been selected as the final product.The minimum detection of which is 195TCID50 of REV,which was sensitive as much as ELISA kit.The final product has no cross reaction with ALV,CAV,REV,IBDV and MDV,displaying excellent sensitivity and repeatability between and within batches.The strip is not resistant to heat,so it needs to be stored at 4? low humidity environment.In this environment,the strip can be maintained for a minimum of 12 months,and the sensitivity,specificity and other properties of the strip kept no change.3.Preliminary Application of the Colloidal Gold Immunochromatographic Test StripREV infection in SPF chickens were challenged under laboratory conditions.ELISA,CGIA,PCR and ELISA were used respectively to detect the anal swabs and throat swabs collected from the chickens.Compared results each other,we could make an evaluation for the application effect of the test strip.We found that the colloidal test strip developed in this project can detect REV antigen sensitively,conveniently and accurately,converting results into images visible to the naked eye.When detecting,no professional equipment is needed,so the application prospect of this assay is broad and bright.With low cost and remarkable economic benefits,the test strip can be widely used for rapid detection of REV at primary-level veterinary departments,poultry breeding companies,vaccine manufacturers and import-export commodity inspection departments.