04 - Put the M into STEM: Quantitative Techniques for Biotechnology
4. The secondary antibody, which recognizes the primary antibody, is added to the wells. If the antibody-antigen complex has formed in the well, the secondary antibody remains in the well after washing. Before performing the experiment, the secondary antibody is covalently linked to an enzyme that allows us to detect the presence of the antibody-antigen complex. 5. The substrate is added to all the wells where it reacts with the enzyme. It either produces color (chromogenic detection) or light (fluorogenic detection) in wells where there is antigen-antibody complex. Since each enzyme can quickly break down many substrate molecules into product, we can get results in a few minutes. QUANTITATIVE AND QUALITATIVE ELISA The results we get from performing the ELISA can be qualitative or quantitative. The qualitative ELISA gives us a Yes or No answer: Stronger signal than standard is positive, lesser signal is negative. One example of a qualitative ELISA that you may be familiar with is a pregnancy test. This at-home ELISA tests urine for the presence of hormone HCG, which is an indicator of pregnancy. When taken at the right time, a pregnancy test will give you a yes or no answer. In the Qualitative ELISA, we compare results from unknown to results from samples of known concen- tration. A standard curve is created where each well has a known concentration of antigen. We then perform the ELISA on the test sample and see where the signal matches the standard. For example, the ELISA is often used to test for the presence of known allergens in food, like gluten. A quantitative ELISA would let us know the exact amount of gluten was present in the sample.
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