Articles
PRODUCTION OF A POLICLONAL ANTISERUM AGAINST TWO TOSPOVIRUSES OF CHRYSANTHEMUM (DENDRANTHEMA GRANDIFLORA): TOMATO SPOTTED WILT VIRUS (TSWV) AND IMPATIENS NECROTIC SPOT VIRUS (INSV)
Article number
482_30
Pages
203 – 208
Language
Abstract
TSWV (Tomato Spotted Wilt Virus) and INSV (Impatiens Necrotic Spot Virus) have been reported in Colombia since 1989. The most important varieties of Chrysanthemum (Dendranthema grandiflora Tsvelev), such as Polaris, Yellow Polaris, Vero and Yellow Vero are particularly susceptible to those viruses.
Production can be affected up to 80% when infected with one or both of them.
The visual screening for virus-free mother blocks is impossible because infected plants appear symptomless under long artificial photoperiod conditions and during the first 7 weeks of the production phase.
The commercial ELISA kits available from 1990 until 1994 for the two common strains known (TSWV-l and TSWV -i) were not sensitive enough and their costs doubled for an indexing program.
Frequently, the routine tests gave rise to false negatives.
At the present times those strains are considered as two different viruses of the Tospovirus group.
We present the results of the production of a policlonal antiserum against isolates of these viruses from Colombian mums, and the adaptation of an indirect ELISA test.
The viruses were isolated and increased on young Nicotiana rustica plants.
Three purification methods were evaluated.
The absorbance spectrum of each purification step (pellets and supernatants) allowed us to choose the best purification system.
The antigens obtained were emulsified in Freund’s adjuvant oil and injected five times (one per week) into a nine months old rabbit.
Blood samples were taken regularly in order to detect antibodies using chloroplasts agglutination or agar double diffusion (Ouchterlony) tests.
The gamma-globulins were isolated and purified and indirect ELISA tests run in order to evaluate the specificity of the antiserum obtained.
As antisera obtained from partially purified virus suspensions contain host-specific antibodies, six inmuno-absorptions were done with virus free sap.
We conclude that it is possible to use policlonal antibodies to detect these two viruses with only two inmuno-absorptions of the antiserum produced.
Production can be affected up to 80% when infected with one or both of them.
The visual screening for virus-free mother blocks is impossible because infected plants appear symptomless under long artificial photoperiod conditions and during the first 7 weeks of the production phase.
The commercial ELISA kits available from 1990 until 1994 for the two common strains known (TSWV-l and TSWV -i) were not sensitive enough and their costs doubled for an indexing program.
Frequently, the routine tests gave rise to false negatives.
At the present times those strains are considered as two different viruses of the Tospovirus group.
We present the results of the production of a policlonal antiserum against isolates of these viruses from Colombian mums, and the adaptation of an indirect ELISA test.
The viruses were isolated and increased on young Nicotiana rustica plants.
Three purification methods were evaluated.
The absorbance spectrum of each purification step (pellets and supernatants) allowed us to choose the best purification system.
The antigens obtained were emulsified in Freund’s adjuvant oil and injected five times (one per week) into a nine months old rabbit.
Blood samples were taken regularly in order to detect antibodies using chloroplasts agglutination or agar double diffusion (Ouchterlony) tests.
The gamma-globulins were isolated and purified and indirect ELISA tests run in order to evaluate the specificity of the antiserum obtained.
As antisera obtained from partially purified virus suspensions contain host-specific antibodies, six inmuno-absorptions were done with virus free sap.
We conclude that it is possible to use policlonal antibodies to detect these two viruses with only two inmuno-absorptions of the antiserum produced.
Authors
S. Vásquez, A. Angarita
Keywords
Indirect ELISA test, inmuno-absorptions, host-specific antibodies, Nicotiana rustica
Online Articles (61)