Purification Of Feline Calicivirus(FCV) And The Establishment Of An Indirect ELISA For The Detection Of Anti-FCV Antibodies

Feline calicivirus(FCV)belongs to the Caliciviridae family,Vesivirus genus.And its genome consists of a single-stranded positive sense RNA.FCV is a small non-enveloped virus and its capsid protein subunit is arranged regularly appearing icosahedral symmetry.FCV is a highly infectious upper respiratory pathogen of feline causing oral and upper respiratory tract disease which is called infectious feline rhinitis-conjunctivitis Clinical signs of the disease include rhinitis,conjunctivitis,ulceration of the palate or tongue and bronchial pneumonia or interstitial pneumonia in severe acute case of FCV infection.Other clinical syndromes include chronic stomatitis and a limping syndrome.Vaccination has been available for its prevention and has been relatively successful in controlling disease.Detection of FCV mainly depends on virus isolation and RT-PCR.Virus neutralization test(VNT)and indirect ELISA are commonly employed for detection of titers of antibodies against FCV in infected or vaccinated cats.VNT is time consuming and laborious While indirect ELISA is a rapid,simple and sensitive serological test for surveiling anti-FCV antibodies in cats,but it requires higher purity whole virus is coating antigen.In this study,FCV propogated in F81 ??culture were purified and an indirect ELISA was developed for detection of antibodies to FCV in the serum samples of cats using the concentrated FCV viral antigen as the coating antigenIn this study,firstly FCV particles were precipitated by saturated ammonium sulfate solution and by sucrose density gradient ultracentrifugation.The relatively pure concentrated FCV was obtained.Then concentrated FCV was identified by TEM observations of the virus form,protein concentration,viral titers by TCID50,molecular weight of protein by SDS-PAGE and immunity by Western Blot.The results showed that the spherical goblet virus particles could be observed under the electron microscope.The protein concentration of the purified FCV was 2.35 mg/mL.Compared with pre-purified FCV,the TCID50 was 10-8.5 which had increased by one titer.The purified FCV identified by SDS-PAGE had a single band at 60 kDa.And the purified FCV identified by Western Blot had a special reaction with feline anti-FCV positive serumUsing concentrated FCV as coating antigen and mouse anti-feline IgG which is labeled by horseradish peroxidase(HRP)as secondary antibody,an indirect ELISA for the detection of anti-FCV antibodies was established.Firstly,matrix titration was performed to determine the optimal concentration of antigen and serum(primary)antibody.Subsequently further optimization including the time of coating antigen,the effects of different blocking agents and blocking time,the dilution of the mouse-anti-cat IgG-HRP and the substrate acting time were also carried out and at last the final conditions of the indirect ELISA for detection of antibodies against FCV in blood samples were determined as follows:thus,antigen coating concentration was 4 ?g/mL and the optimal serum dilution was 1:80.The optimal dilution of goat-anti-cat IgG-HRP was 1:2000.Through the optimization of the reaction,ultimately determined antigen coating condition was at 4? overnight.Blocking buffer was 5%bovine serum albumin(BSA),blocking time was at 37 C for 1h.The best action time of serum and goat-anti-cat IgG-HRP were both at 37 C for 1h.The freshly prepared OPD substrate was incubated for 10 min at room temperature in the dark.2M sulfuric acid terminated the reaction and optical density(OD)492nm of per well was measured on ELISA reader immediately.The cut off value was 0.318.In Detecting the 30 FCV clinical serum samples by the established indirect ELISA and VNT,the result indicated that there was high correlation between indirect ELISA and VNT and the coincidence rate of the two methods can be reached 90%.Through the study,the established indirect ELISA had a good specificity,repeatability and sensitivity.The purified FCV antigen afforded the material foundation for the preparation of subsequent monoclonal antibodies and we also successfully established an indirect ELISA using the relatively pure concentrated FCV as coating antigen for the detection of anti-FCV antibodies.And this method also offers an effective tool for the diagnosis and epidemiological investigation of FCV.