• Posted on November 13, 2012 6:05 pm
    By Chris
    96 well ELISA test plate

    As I missed histology yesterday I rescheduled to take my credit test today, whilst I didn’t originally feel that I had done well, I got a C in the written part and a B in the practical part. I was pretty pleased with this as it means I’ve only got an extra little bit of revision to do on certain areas for my final at the end of semester.

    Immunology today was then on the ELISA test, which stands for the Enzyme-Linked Immunosorbent assay which is one of the most common diagnostic and analytic tools available. In fact most of you reading this would have seen ELISA tests for sale in the shops in the form of pregnancy tests. Within the laboratory ELISA is usually carried out using wells in specially designed microtiter plates, and within LABS where multiple samples are processed each hour automatic reading machines are also used.

    96 well ELISA test plateThe basis of an ELISA test is the binding of antigen the sample solution with molecules within the test solution to cause a change of colour. The test solution is usually coated onto the test plate in a dry form before the test, although it can also be used in solution form. Each test solution is specific to a defined disease or antibody and so there needs to be some knowledge of what is being tested for before a test can be carried out.

    Just to clarify for my new readers, an antigen is a substance that when it enters the body it causes the immune system to respond. And antibodies are markers produced by the body that attach to antigens and act like tags to identify the foriegn substance so it can be destroyed by the immune system. For the purposes of ELISA this is then linked to another enzyme which causes a visible change in colour (or flourescence).

    There are different methods that are used for this binding which are…

    • Direct ELISA
    • Indirect ELISA
    • Competitive ELISA
    • Sandwich ELISA

    Direct ELISA

    Is the original and most simple form of ELISA testing, the antigen is added to the test plate and then a protein is added to block all the other binding sites apart from the one being tested. The test solution is prepared seperately with the antibody being bound to the enzyme (forming a labelled antibody) before it is then added to the test plate. A positive result is a change in colour.

    Indirect ELISA

    This type of ELISA test is different to the direct ELISA as it is done in two stages. The antigen is first added to the test plate, and the blocking protein applied. A primary antibody is added to bind the the antigen, and then a secondary labelled antibody is added which binds the primary antibody. This method is less expensive and quicker as the labelled antibody no longer needs to be specific to the antigen being tested. A positive result is a change in colour

    Competitive ELISA

    This one is like its name and causes competition for the binding of antibodies. The sample solution with antigens is incubated with a primary unlabled antibody which binds to the antigen. This is then added to a antigen coated test plate where any unbound antibody will bind to the antigen coating the plate (if there is any unbound off course as it has already been incubated with the antigen). This is where the competition is as the more antigen in the test sample, the less free antibodies able to bind to the test well antigen. The excess sample is then washed off the plate and a secondary labelled antigen applied which binds the primary antibody, obviously if there was a lot of antigen in the sample there would have been no antibody to bind to the test well so with a positive results there will be no (or very little) change in colour.

    Sandwich ELISA

    No i’m not talking about the latest Subway sandwich here… This method is less common and requires 2 binding sites on each antigen as they are bound by two different antibodies (hence the sandwich). The first antibody that is coated onto the test plate is usually a polyclonal antibody which means it can recognise many different antigens and binds as much as possible from the test solution when applied. The secondary labelled antibody is usually monoclonal meaning that it binds to specific antigens. This test is more accuate as it tests for two different binding regions on the antibody, in addition to having the ability to use it with complex samples where multiple antigens may reside.

    Having such a powerful diagnostic tool available is great for medicine, the biggest drawback is that each test is relatively expensive and generally specific to a single antigen. The microtiter plates in each kit can only be used once for example, however in addition as with the pregnancy test kits for humans, rapid test kits have been developed for single use in veterinary practice for specific diseases.

    I am way over my word count so am going to leave it there for today 🙂

    Posted in categories: Vet School Diary
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