Production of serological reagents and development of a test procedure for simultaneous detection and identification of tomato spotted wilt and impatiens necrotic spot tospoviruses

摘要

A rapid immunological procedure that employed antibody-adsorbed nitrocellulose membranes was developed for the detection and identification of tospoviruses. Viruses were prepared in a phosphate buffer after extraction from infected Nicotiana benthamiana. Tospovirus nucleocapsids were released from virions in the presence of 1% Nonidet P40 and purified by sucrose density gradient and caesium sulfate gradient centrifugations for immunization. Polyclonal antisera were produced in rabbits. Mouse hybridomas secreting monoclonal antibodies were prepared and antibodies were produced in ascitic fluids in tumour transplanted mice. Preparation of nitrocellulose membrane strips was accomplished by individual application of virus specific rabbit antisera on previously designated spots so that each was identified with its specificity. Following incubation in a blocking solution, the nitrocellulose membrane strips were air-dried or freeze-dried and stored at 4deg or -20℃. In tests, the nitrocellulose membrane strips were immersed for 15 min each in tissue extracts, a solution containing mixtures of virus-specific mouse monoclonal antibodies and, finally, alkaline phosphatase-labelled goat anti-mouse immunoglobulin antibody solution. Upon incubation in a substrate solution, the development of a purple colour on spots that targeted for specific viruses identified the virus in the infected tissue extract. The trapping antibody-adsorbed nitrocellulose membrane strips could be stored for at least 4 weeks without loss of potency for virus detection.