Articles
ADVANCES IN THE PURIFICATION OF FILAMENTOUS VIRUSES FROM GARLIC AND IN ANTISERA PRODUCTION
Article number
433_71
Pages
623 – 630
Language
Abstract
The disease called "mosaico del ajo" is caused by a group of filamentous viruses (potyvirus and carlavirus) which are very difficult to isolate and purify.
Those difficulties are related with the presence of mucilage in garlic and the tendency of the viruses to aggregate and form insoluble complexes.
Initial attempts of purification were done with protocols that used PEG, Triton X 100, and many cycles of high and low centrifugation involving 4 to 5 days of work.
Unfortunately, final viral suspensions were associated with normal plant proteins and the antiserum raised had unspecific reactions on serological tests.
After several modifications in the purification protocol, the best results were obtained with the following steps: Homogenization of fresh tissue with borate buffer 0.5 M, pH 8.1 and organic solvents, and latter clarification.
The supernatant was layered on 20% sucrose borate buffer 0.05 M pH 8.1 + urea 0.5 M. The pellets were resuspended and layered on a CICs gradient 0–40 % (using borate buffer 0.05 M pH 8.1 + urea 0.5 M + 20% sucrose). Viral fractions were collected, concentrated and layered on a second gradient (sucrose 10–40%). Viral fraction was concentrated and resuspended.
Five purifications were spectrophotometrically scanned and gave an A258–260 peak and a A 245–247 minimum value.
The relation AMax / AMin was 1.09–1.11 and A 260 / A 280 was 1.25–1.35. Negative stain electron microscopy observation showed viral particles without other type of structures that would suggest presence of contaminants.
An antiserum was raised using Californian female rabbits.
Four intramuscular injections of viral suspensions at ten-fifteen days intervals and a last intravein injection were applied.
The collected antiserum had a titer of 1/6400 by microprecipitation and it did not show reaction against healthy plants.
The antiserum gave a titer of 1 / 512000 by PTA-ELISA and 1/8 x 106 by NC-ELISA. Using DAS-ELISA it was possible to detect virus concentration down to 16 ng/ml.
Those difficulties are related with the presence of mucilage in garlic and the tendency of the viruses to aggregate and form insoluble complexes.
Initial attempts of purification were done with protocols that used PEG, Triton X 100, and many cycles of high and low centrifugation involving 4 to 5 days of work.
Unfortunately, final viral suspensions were associated with normal plant proteins and the antiserum raised had unspecific reactions on serological tests.
After several modifications in the purification protocol, the best results were obtained with the following steps: Homogenization of fresh tissue with borate buffer 0.5 M, pH 8.1 and organic solvents, and latter clarification.
The supernatant was layered on 20% sucrose borate buffer 0.05 M pH 8.1 + urea 0.5 M. The pellets were resuspended and layered on a CICs gradient 0–40 % (using borate buffer 0.05 M pH 8.1 + urea 0.5 M + 20% sucrose). Viral fractions were collected, concentrated and layered on a second gradient (sucrose 10–40%). Viral fraction was concentrated and resuspended.
Five purifications were spectrophotometrically scanned and gave an A258–260 peak and a A 245–247 minimum value.
The relation AMax / AMin was 1.09–1.11 and A 260 / A 280 was 1.25–1.35. Negative stain electron microscopy observation showed viral particles without other type of structures that would suggest presence of contaminants.
An antiserum was raised using Californian female rabbits.
Four intramuscular injections of viral suspensions at ten-fifteen days intervals and a last intravein injection were applied.
The collected antiserum had a titer of 1/6400 by microprecipitation and it did not show reaction against healthy plants.
The antiserum gave a titer of 1 / 512000 by PTA-ELISA and 1/8 x 106 by NC-ELISA. Using DAS-ELISA it was possible to detect virus concentration down to 16 ng/ml.
Publication
Authors
M. Helguera, Pablo A. Lunello, C. Nome, Vilma C. Conci
Keywords
garlic (Allium sativum L.), filamentous viruses, purification, antiserum
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