4 Immunology/Antibody Based Techniques

Transcript

Hello welcome to the fourth talk of this week, that is Immunology and antibody based techniques. In the previous talk, we have talked about the use of NGS platforms and plant disease detection and diagnosis. And here we will be talking about at different method that is Immunology or antibody based detection techniques. So Serological assays for plant pathogens, such as viruses, cannot be cultivated ad hoc and hence serological assays were developed to detect them. So definitely we need to have different technologies for those particular microbes that causes plant diseases like viruses which are not culturable and that is why serological methods is also helpful or useful to detect certain pathogens of that kind. More than a thousand other pathogens, like bacteria and fungi, can now be detected using polyclonal as well as monoclonal antisera and techniques such as ELISA western blots, immunostrip assays, dot-blot immune binding assays, as well as serologically specific electron microscopy (SSEM). So these are all techniques that is serology based, and these techniques are deployed for identification of various pathogenic virus, bacteria and fungi.

Among them, ELISA was first employed, 1970s and till now it is the most popular method used for high throughput detection of certain plant viruses. The sensitivity of ELISA varies depending on the organism, the sample freshness and a titre, for instance, a bacteria can be detected at 100cfu/mL-1 .Antibodies need to be stored at lower than -20o C, and it should not be defrosted several times because the antibody lose its properties if it is defrosted several times. So this is kind of a drawback of this particular technique. So ELISA in full, it is known as (enzyme linked immunosorbent assay) So ELISA is a molecular method for identification of disease based on antibodies and change in the assay. In this method, the target epitopes- that is the antigens of the pathogen such as from viruses, bacteria and fungi- are made specifically bind with antibodies conjugated to an enzyme. So, this is the major principle behind ELISA technique where the epitope or the antigen is made to bind with antibodies, which is linked with an enzyme.And then, when the substrate is added this enzyme can change color of the solution, but normally, it turns into yellow color The detection can be visualized based on color changes, resulting from the interaction between the substrate and immobilized enzyme. So here this is an example of PCR plate , where you can see that the yellow color wells, giving the positive result for the particular pathogen and the more darker the refers to the more number antigens present in the sample. So the lighter color wells indicate lesser amount of antigens presence in the well. The principle how ELISA works, based on this – There are four different types of ELISA that are designed that is direct ELISA, indirect ELISA, sandwich ELISA as well as competitive ELISA. All this four ELISA techniques are used based on their necessity and based on their applications. So let us talk about Direct ALISA – Where a target protein or a target antibody is immobilized on the surface of microplate wells. Here, the target proteins are immobilized in the microplate wells and it is incubated with an enzyme- labeled antibody, is the enzyme labeled antibody when it is incubated together and then, when the substrate is added, then the color of the solution turns yellow. So this is the process, we call it as direct ELISA and yellow color development indicates positive outcome of the interaction. Next is indirect ELISA – Where the target protein is immobilized on the surface of micro plate wells and incubated with an antibody to the target protein. We call it as primary antibody. Then if the secondary antibody is added against the primary antibody and after washing the activity of the microplate well bond enzyme is measured.

So here what happens? The protein is immobilized in the microplate well. Then, first primary antibodies are added which is not enzyme linked and followed by in the next step. Secondly, antibody is added, which is linked with an enzyme and then when substrate is added then this enzyme linked antibody with substrate interacts and the color of the solution turns yellow. So this gives again a positive indication of presence of a particular pathogen that is present in the same sample. Although indirect ELISA requires more steps, then Direct ELISA labeled secondary antibodies are commercially available, eliminating the need to level primary antibody each and every time.

So it saves certain amount of cost as primary antibody are not labeled with enzyme and rather the function of enzyme is achieved through use of commercially available secondary antibody. Next, is Sandwich ELISA – The antibody to a target protein is, immobilized on the surface, microplate wells and incubated first with the target protein and then with another target protein specific antibody, which is labeled with an enzyme. After washing, the activity of the microplate well-bound enzyme is measured. So in sandwich ELISA first of all, the antibody is immobilized in the mictoplate well, in contrast to the antigen then antigen is added, at the second stage where primary antibody will then bind with the antigen followed by addition of secondary antibody that is linked with enzyme and this will bind with the antigen and then when substrate is added the color of the solution turns to yellow.

So, it indicates again positive off the particular pathogens present, and this is what we call it as Sandwich ELISA. Since the antigen is present in between two antibodies, so this particular type of ELISA method is known as sandwich ELISA. The immobilized antibody, that is orange in color and the enzyme labeled antibody which is green in color, must recognize different epitopes of the target protein. So both the antibody’s that is the yellow color antibody as well as the green color antibody the both recognizes different epitopes of the target protein, and that is a important criteria for designing of for use of antibodies for sandwich ELISA. Compared to direct ELISA the sandwich ELISA the (combining antibodies to two different epitopes on the same target protein) has a higher specificity and Sandwich ELISA useful for applications that require high accuracy. So this is another method ELISA for those samples which requires high accuracy data generation

The last one is competitive, ELISA here antibodies specific for a target protein is immobilized on the surface of microplate wells and incubated with samples containing a target protein and is known amount of enzyme-labeled target putting.

After the reaction the activity of the microplate well-bound enzyme is measured. So this particular technique is used again, just like a Sandwich ELISA. First, the primary and the antibody is immobilized in the microplate well then addition of the enzyme linked protein that is added and when the substrate is added, then this enzyme linked antibody proteins will turn into yellow color gives a positive indication. So the competitiveness arises, depending on the amount of the actual antigen present in the sample. More the amount of antigen present in the sample, lesser the amount of enzyme-labeled antigen will be present, and that is why the color will be lighter in color in this case, as we have more amount of antigen, presence it will occupy more number of antibody on the less amount of enzyme linked antibody, will be occupied here, and that is why the color of the solution will be lighter yellow. Whereas, in case of lesser amount of antigens presence, more number of antibodies, will be occupied by enzyme linked antibodies, and the color of the solution will be brighter or darker yellow. So this shows the competitive ability, of the antigens, and that indirectly tells us the story whether the amount of antigen that is present in higher quantity or lower quantity in the sample. Next technique is Immunofluorescence (IF) – So it is fluorescence microscopy-based optical technique, it is used for detection of pathogens infections in plant tissues. For this technique plant samples are fixed in to a microscopic slides in thin tissue sections, then detection is achieved through conjugating, a fluorescent dye to the specific antibody to visualize the distribution of the target molecule throughout the sample. So what it does is that, just like in other microscope we prepare a thin slide of the section of the target sample fixed in a microscope slide and then at fluorescence dye and then observed under fluorescence or confocal microscope. So the specific dye’s will go and bind to specific sites and once the dye binds to the target sites, then under fluorescence microscopy , then this fluorescence colour is very observed, and we can then confirm the presence of the particular pathogen or not. So, Immunofluorescence has been used to detect onion crop infection by the fungus, Botrytis cinerea.

The Immunofluorescence has also been combined with other techniques such as FISH for detection of crown rot pathogen Dickeya species in potatoes, which is an emerging disease in Europe. So it has a lot of significance whether Immunofluorescence has been used singly or in combination with the technology of FISH that is Fluorescence in situ hybridization. Even new and emerging pathogens pathogens can be detected in plant samples.

The other method is Flow Cytometry (FCM), so this is a laser-based optical technique, widely used for cell counting and sorting biomarker detection and protein engineering. But this technique can now also be used for detection of plant pathogens whether there is sufficient amount of a particular cell count is available based on it the detection of the pathogen can be achieved. So technically, samples having the pathogen cells is sent through a fluid along with the tube and then it is subject to a narrow tube and during they passes of the sample through the narrow tube the light source is allowed to visualize the samples and, depending on the cell nature, the light get scattered, some of the light get passed and some of the lights get steak scattered and this scattering pattern of the light is then recorded by different electronic devices and based on these electronic signal, since different pathogens has a different capacity to reflect the lights so, based on this technology, based on this electrical signal that is generated the pathogen is identified in the sample. So, this is again a very new technology and it is still not been very highly used in plant pathogen detection, but still it has a lot of scope for detection of plant pathogens, although it had been very frequently being used in determining the microbial pathogens that is causing food poisoning in certain food samples. So, with this we have come to an end of today’s talk that is antibody or immunological based technology for detection of plant pathogens where, we have talked about different techniques of ELISA then , Immunofluorescence and then Flow Cytometry. Next, time we’ll be talking about advanced DNA based point of care, diagnostic methods, for detection of plant pathogens.

Thank you very much.