Skip to 0 minutes and 12 seconds Hello. Today, we’re in the life sciences laboratories at Lancaster University to see some of the techniques we can use to determine if somebody is suffering from or has recently suffered from a virus. Student Katharina Hartman and researcher Dr Lisa Bishop will be joining me to do some of the demonstrations. The first technique we’re going to look at is western blotting, which was developed by W N Burnette in Seattle in the 1980s, based on earlier work by Ed Southern. Western is a triple play on words referencing Southern, the first initial of WN Burnette’s name, and also the geographical location of the city of Seattle. So, Katharina, what does western blotting involve?
Skip to 0 minutes and 51 seconds In western blotting, we use a thin gel, which we place in a tank of buffer solution. Then we put the serum samples from the patients into the top of the gel and apply an electric current. The sample then flows into the gel. And the protein from the serum are separated according to their size. Larger ones run slower, and smaller ones, faster. Katharina is now loading the gel.
Skip to 1 minute and 25 seconds So, the gel has been running for a while now. And the blue dyes nearly reached the bottom. So what happens now then, Katharina? Once the gel has run to the bottom, we’re ready to remove it from the tank. It’s then laid flat, and a nylon membrane is placed on top of it. So the proteins are drawn out of the gel and onto to the membrane. Is that why we say it’s blotting? Yes. And the proteins are then fixed in place and available to interact with the antibodies. If we have an antibody to a certain protein, we are able to determine if that protein was in the serum sample that we ran on the gel.
Skip to 1 minute and 56 seconds So Katharina, how do we know that the antibody has detected the protein? Well, the antibodies are themselves proteins and can interact with other antibodies known as secondary antibodies. So that’s quite complicated. We have an antibody that binds to a protein. And then we have another protein, another antibody, that binds to that antibody. Exactly. And that secondary antibody has a chemical group attached to it that we can develop to show some colour, like a photograph in the old days before digital camera. Ah, yes, I remember the old days before digital cameras even if Katharina doesn’t. Katharina is now performing the reaction that develops the colour in the membrane.
Skip to 2 minutes and 41 seconds So Katharina, we are now looking at the results of the western. So, what do the results show us? So, we can see here that these lines are negative. There’s nothing in them. But these other lines here have bands in them. Right. So that means that the patients corresponding to the lines that have bands in them do have viral proteins in their blood. But the others don’t? Yes. And we also expect that these bands here to be a certain size, corresponding to the size of the viral proteins we’re looking for. But if the bands are the wrong size, we wouldn’t assume that we had a positive result. But we would need to do further testing.
Skip to 3 minutes and 15 seconds So, we’ve just seen western blotting, which directly detects the presence of protein in the serum sample. So, a positive western blot result would indicate that the patient in question was either suffering from the virus or incubating the virus at the time the sample was taken. So, Lisa, we’re going to look at a different technique now called ELISA. What does ELISA stand for? ELISA stands for “enzyme linked immunosorbent assay”. It’s different from western blot, because in the ELISA, we’re looking for antibodies to the virus, rather than the viral protein itself. Right. So it’s actually the response to the virus rather than the presence of the virus that’s been detected. That’s exactly right.
Skip to 3 minutes and 55 seconds So the ELISA will give us information as to whether the patient has seen the virus in the past. And of course, by the time we do the ELISA, the patient may have recovered from the virus. And so western, which is the other technique we’re looking at today, would actually be negative. But the virus would tell us about the past experience of the patient. Yes. Exactly. So, no gel is needed, then, for the ELISA technique, Lisa? No. The first thing we do is incubate the serum from the patient with a viral protein, an antigen. This protein acts as an immune challenge to the antibodies in the patient’s serum sample.
Skip to 4 minutes and 32 seconds If the patient has antibodies to that virus, they’ll bind to the challenge proteins. Lisa is now setting up the ELISA. Secondary antibodies are used again. But because there is no gel, unlike the western, we have to read the output of the color development reaction using a spectrophotometer.
Skip to 5 minutes and 18 seconds So, now we’re reading the results of the ELISA plate that was set up earlier on. So what do the results tell us then, Lisa? Well, the colour and the number correspond to the optical density of the sample. So where the number and colour are strongest and highest, it’s an indication that the patient has a lot of antibody to that particular viral protein. Right. So the patients that have got higher numbers– that appear in here– are the ones that have been exposed to that particular virus at sometime in the past. And they now have antibodies to that virus.
Skip to 5 minutes and 56 seconds Whereas the patients here, that obviously have much lower numbers– in fact, in some cases quite close to zero– these are probably patients that have no antibody, which would mean that they’re patients that haven’t been exposed to that virus. That’s exactly right. Right.
Western Blotting and ELISA (Enzyme-Linked ImmunoSorbent Assay)
This video shows two techniques that are very commonly used in virology.
The first is Western Blotting, which detects viral antigens (proteins usually on the surface of viruses) using antibodies against those proteins. A positive Western Blot indicates the presence of viral antigen - which very often means live virus - in our patient. That patient may have an on-going viral infection.
ELISA (Enzyme-Linked ImmunoSorbent Assay) is a related technique, but instead of using antibodies to detect virus antigen, it uses virus antigen to detect antibody. A positive ELISA indicates the presence of antibody to a virus in our patient. That patient may have had a viral infection to which their immune system has responded. Often, this will mean they have no live virus, and will have recovered. Since antibodies can persist for a while after a virus infection has occurred, ELISA can detect infections that have occurred a while ago.
This might be a bit confusing at first, since these two techniques are to a certain extent mirror images of each other. The key thing to remember is that an antigen is a virus protein that presents a target for the immune system. The thing that the immune system launches against that antigen is an antibody. Antibodies attach themselves to antigens to destroy them, and that natural process is exploited in these two techniques.
Purified antibodies against certain viral antigens are used in the Western Blot. When the patient sample is run out on the gel, the purified antibody binds to it and (after the secondary antibody is added, which is an antibody-to-the-antibody) signals to us that the viral antigen is there.
Purified antigens from certain viruses are used in the ELISA. When the patient sample is added to the well, the antigen in the well binds the antibodies in the sample. Again a secondary antibody is used to amplify the signal. This time the signal indicates that the patient has antibodies against the virus.
So if the patient has antigen (Western Blot) it probably means that the infection is still active. And if the patient has antibody (ELISA) it probably means the patient has had the virus at some fairly recent time, but may not longer have an active infection.
- Western Blots ask “who is ill?”
- ELISAs ask “who has been ill?”
Links to some further reading can be found below.
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