Potato Virus Y - Diagnostic Techniques For Detection of Potato Virus Y - ELISA

ELISA

In the past, crops were inspected visually to determine whether or not they were disease free. Visual inspection was also used as a basis for seed certification. Determination of viral status through visual inspection is incredibly difficult as the symptoms may be masked or the infection latent. As a result, post season tests and inspections were introduced. These tests involved the cultivation of previously harvested material in greenhouses. The resulting plants were inspected for a more accurate estimate of viral status. Although this method of screening did offer some degree of monitoring of viral presence it was subjective and highly ineffective. Enzyme-linked immunosorbent assay (ELISA) screening of crops and seed potatoes replaced visual inspection in the early 1970s. The use of ELISA offered routine diagnostic laboratories a quick, effective and sensitive method of screening for a wide range of potato plant viruses.

Detection of pathogens using ELISA relies on the interaction between the antigen and specific antibodies and has become a popular and cost-effective means of routine detection. In an ELISA the solid phase can be coated with the sample of interest containing the antigen. The efficiency to which the antigen binds to the solid phase is dependent on temperature, length of exposure as well as concentration. Solid phases used include nitrocellulose membranes, paper, glass, agarose and polystyrene or polyvinylchloride microtiter plates. Microtiter plates are the most widely used solid phase because they are easy to handle, allow for automation and for analysis using microtiter plate readers. A drawback of these plates is that they are highly absorptive and this increases the incidence of non-specific binding of components used in the ELISA. Non-specific binding to the plates is reduced through the use of buffers containing proteins such as casein and non-ionic detergents such as Tween 20. After coating, excess sample is removed and the plate typically treated with a 1% casein containing solution. Subsequent to this the solid phase is treated with antibodies raised against the antigen of interest. After each incubation step the plate is washed with Tween 20 containing PBS. These washing steps are aimed to wash away any non-specifically bound components. Nonspecifically bound components are less strongly bound than the specific bound ones. Detection is achieved either through the addition of an enzyme-coupled antibody or the addition and detection of a biotinylated antibody. In a system using an enzyme-coupled antibody the subsequent addition of an appropriate substrate results in the formation of a colour proportional to the amount of antigen. Alternatively the plate can be coated with antibody followed by incubation with the sample that is to be detected. This, in turn, can be detected as described above and is then referred to as the double antibody sandwich (DAS) ELISA. Both of these systems, however, have a disadvantage in that coupling of the enzyme to the antibody may result in steric hindrance which in turn may result in a loss in function of the antibody and/or the enzyme. This may be overcome through the use of a biotin-avidin or biotin-streptavidin bridge. In this type of system biotin is coupled to the antibody. The biotin molecule has no influence on the working of the antibodies and is easily detectedusing avidin or streptavidin conjugated to a suitable enzyme. Streptavidin has an extremely high affinity for biotin which results in even a higher degree of specificity than a system in which the enzyme is coupled directly the antigen. To establish whether or not the antigen is present, a substrate specific for the enzyme used is added. The enzyme then converts the substrate to a coloured product and the colour intensity can be correlated to the amount of antibodies bound and thus the amount of antigen present. A DAS-ELISA has the advantage that it can increase the specificity of the ELISA and reduce the occurrence of non-specific binding. As a result the DAS-ELISA principle is commonly employed in ELISA’s for the detection of plant pathogens in plant sap without prior purification of the pathogen.

The ELISA is considered to be a safe, inexpensive and rapid method for detection of plant viruses. The inexpensive nature and relative simplicity thereof allows for it to be used as a workhorse within the agricultural sector and is used to screen thousands of samples per year. Unfortunately ELISAs are not completely failsafe. Virus levels within potato tubers, which are screened by ELISA for use as seed potatoes, are normally low while the tubers are dormant. ELISA detection of viruses in these potatoes is difficult and absorbance values may fall below the set cut-off value. For this reason, seed tuber screening is performed on sprouting rather than dormant tubers. Although this results in more reliable readings than direct tuber testing, it does delay the certification of seed potatoes. Another disadvantage of an immuno-based detection method is that changes at the gene level may have an influence on the immunogenicity of the antigen to be detected. In terms of potato plant viruses, mutations within the CP gene may cause the CP to undergo conformational changes rendering antibodies produced against the previously present virus less effective.