Articles
DETECTION AND DIFFERENTIATION OF PLANT VIRUSES BY VARIOUS ELISA PROCEDURES
Article number
127_11
Pages
147 – 158
Language
Abstract
Variants of ELISA (figure 1) differ in their sensitivity and their ability to detect serologically related viruses.
The broadest range of serologically related viruses was detected by means of indirect ELISA on plates not precoated with antibodies.
Unfortunately, this technique can be used only with purified virus preparations, because the adsorption of virus particles onto the plates is greatly inhibited by crude plant sap.
Precoating of plates either with intact antibodies or their F(ab’)2 fragments greatly reduces the inhibitory action of crude plant sap, but also leads to a narrowing of the specificity in heterologous reactions.
With tymoviruses and tombusviruses this narrowing of the specificity was more pronounced when F(ab’)2 fragments rather than intact anti-bodies were used for coating the plates.
The highest degree of specificity in heterologous reactions was reached with the direct double-antibody sandwich method of ELISA. Indirect ELISA procedures on plates precoated either with intact antibodies or their F(ab’)2 fragments were usually about two twofold dilution steps more sensitive than the direct double-antibody sandwich method.
On precoated plates heterologous reactions of tymo-, tombus-, tobamo- and carlaviruses were always weaker than the homologous ones independent of the concentrations of the coating material and the detecting antibodies.
Under suitable conditions the additional working step usually necessary in indirect ELISA procedures could be avoided by using preincubated mixtures of enzyme-labelled anti-globulin antibodies and serum containing the detecting antibodies.
At high dilutions of antisera this short procedure was in homologous reactions even more sensitive than the regular two step procedure.
Attempts to differentiate very closely related isolates of either radish mosaic virus or Andean potato latent virus by using F(ab’)2 fragments either for coating the plates or after labelling with alkaline phosphatase for detecting the trapped virus particles failed.
The broadest range of serologically related viruses was detected by means of indirect ELISA on plates not precoated with antibodies.
Unfortunately, this technique can be used only with purified virus preparations, because the adsorption of virus particles onto the plates is greatly inhibited by crude plant sap.
Precoating of plates either with intact antibodies or their F(ab’)2 fragments greatly reduces the inhibitory action of crude plant sap, but also leads to a narrowing of the specificity in heterologous reactions.
With tymoviruses and tombusviruses this narrowing of the specificity was more pronounced when F(ab’)2 fragments rather than intact anti-bodies were used for coating the plates.
The highest degree of specificity in heterologous reactions was reached with the direct double-antibody sandwich method of ELISA. Indirect ELISA procedures on plates precoated either with intact antibodies or their F(ab’)2 fragments were usually about two twofold dilution steps more sensitive than the direct double-antibody sandwich method.
On precoated plates heterologous reactions of tymo-, tombus-, tobamo- and carlaviruses were always weaker than the homologous ones independent of the concentrations of the coating material and the detecting antibodies.
Under suitable conditions the additional working step usually necessary in indirect ELISA procedures could be avoided by using preincubated mixtures of enzyme-labelled anti-globulin antibodies and serum containing the detecting antibodies.
At high dilutions of antisera this short procedure was in homologous reactions even more sensitive than the regular two step procedure.
Attempts to differentiate very closely related isolates of either radish mosaic virus or Andean potato latent virus by using F(ab’)2 fragments either for coating the plates or after labelling with alkaline phosphatase for detecting the trapped virus particles failed.
Authors
R. Koenig, H.L. Paul
Keywords
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