| Duck Tembusu virus disease is an infectious disease caused by Duck Tembusu virus(DTMUV),which is characterized by a significant drop in egg production rate,delayed growth and development,and ovarian hemorrhage,causing significant losses to the breeding industry.The sooner the DTMUV is diagnosed,the smaller the loss for the farmer.Therefore,the establishment of a highly specific and sensitive early detection method of DTMUV is the key to preventing and controlling the disease.The NS1 protein is a highly conserved non-structural protein of DTMUV,consisting of 352 amino acid residues with a molecular weight of 40 k Da.Studies have reported that NS1 protein can be detected in the early stages of DTMUV infection,suggesting that it is related to early infection of the virus.In this study,monoclonal antibodies directed against the NS1 protein were used to establish a rapid colloidal gold test strip to provide technical support for the early detection of duck tambushu virus disease.First,select the monoclonal antibody against the Tambusu virus NS1 protein prepared by our laboratory,and detect its reactogenicity and specificity by Western blot and IFA.After determining the good reactogenicity and specificity,it is combined with the colloidal gold solution to form a gold-labeled antibody.For the coating antibody,the mouse anti-NS1 protein polyclonal antibody(detection line)and goat anti-mouse Ig G(quality control line)prepared in this laboratory were selected to explore the conditions for constructing the double antibody sandwich immunochromatographic test strip.The results showed that the optimal binding p H of the NS1 monoclonal antibody-colloidal gold complex solution was8.1,and the optimal concentration was 7.5 ?g/m L.According to the release rate and completeness of the antibody on the membrane,VL98 gold standard pad and Sartorius CN95 membrane were selected.The dilution ratio of NS1 polyclonal antibody is 1:5,the secondary antibody Ig G concentration is 1.0 mg/m L coated on the NC membrane,and the C/T line band is clearly visible.Secondly,the preliminary construction of the test strip was analyzed from four aspects of sensitivity,specificity,stability and repeatability,and the feasibility of the test strip developed in clinical application was explored.The results showed that the test strip can specifically detect Tambusu virus within 3?10 minutes,with a minimum detection dilution of 1:125,and no cross reaction with Newcastle disease virus,avian leukemia virus,duck plague virus,and reovirus.In this paper,a method for rapid detection of Tambusu virus colloidal gold immunochromatographic test strips is established to verify that the selected monoclonal antibody against Tambusu virus NS1 protein has good reactogenicity and specificity,and on this basis Duck Tambusu virus NS1 monoclonal antibody colloidal gold test strip has been developed.The test strip is easy to operate,has strong specificity,high sensitivity and good repeatability.It can be used for rapid clinical diagnosis of DTMUV to prevent and treat the disease Provided technical support. |
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