Articles
PRODUCTION OF SEROLOGICAL REAGENTS AND DEVELOPMENT OF A TEST PROCEDURE FOR SIMULTANEOUS DETECTION AND IDENTIFICATION OF TOMATO SPOTTED WILT AND IMPATIENS NECROTIC SPOT TOSPOVIRUSES.
Article number
432_18
Pages
140 – 147
Language
Abstract
A rapid immunological procedure that employed antibody-adsorbed nitrocellulose membranes was developed for detection and identification of tospoviruses.
Viruses were prepared in a phosphate buffer by 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 cesium 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 tumor 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 an incubation in a blocking solution, the nitrocellulose membrane strips were air-dried or freeze-dried and stored at 4°C or -20°C. 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 purple color on spots that targeted for specific virus identified the virus in the extract of infected tissues.
The trapping antibody-adsorbed nitrocellulose membrane strips could be stored for at lest 4 weeks without loss of potency for detection of viruses.
Viruses were prepared in a phosphate buffer by 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 cesium 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 tumor 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 an incubation in a blocking solution, the nitrocellulose membrane strips were air-dried or freeze-dried and stored at 4°C or -20°C. 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 purple color on spots that targeted for specific virus identified the virus in the extract of infected tissues.
The trapping antibody-adsorbed nitrocellulose membrane strips could be stored for at lest 4 weeks without loss of potency for detection of viruses.
Authors
H.T. Hsu, X.P. Liu
Keywords
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