When you are undergoing an ELISA test, you may be asking yourself what the primary antibodies used in the test are. This article will provide you with some information on the primary antibody used in an ELISA test. It is also important to know that there are negative controls that you can use if the blocking solution is ineffective. These negative controls allow you to control for non-specific binding sites on the surface of the plate.
ELISA tests are highly sensitive and quantitative, and the amount of antigen absorbed by the enzyme is measured as absorbance. This color is directly related to the concentration of antigen present. Unlike other tests, ELISAs can detect antigen concentrations as low as a nanogram (10-9 g per mL).
ELISA tests are also often indirect. The most common type is called sandwich ELISA, which uses two specific antibodies called matched antibody pairs. First, a capture antibody is coated on a microplate. Next, the sample is added to the plate. The immobilized protein is bound to the capture antibody, which acts as a template for the conjugated-detection antibody. Once this complex forms, the enzyme then releases multiple signal molecules. This process is highly specific, and the amount of the analyte present in a sample is directly proportional to the number of signals generated by the sandwich ELISA.
Another type of ELISA uses antibodies, called secondary antibodies, that recognize the primary antibody. These antibodies are monoclonal or polyclonal, and can be monoclonal or a mixture of the two. The primary antibody used in an ELISA test is generally monoclonal, although polyclonal antibodies are increasingly used to improve sensitivity. If you are unsure which one to use, consider using a monoclonal antibody.
Competitive ELISA is another type of ELISA. It uses a competing antigen in addition to the target antigen. This means that the sample antigen and the primary antibody are competing for the same molecule. The lower the amount of antigen in the sample, the stronger the signal. The opposite happens in sandwich ELISA. The antibody binds to the antigen in the sample and the labeled antibody is able to detect it.
Indirect ELISA uses the same process, but involves a second amplification step. The first part of indirect ELISA uses an unconjugated primary detection antibody to detect a specific antigen. Once the primary antibody binds to the antigen, an enzyme linked to the secondary antibody catalyzes a chemiluminescent or colormetric reaction. The signal is then read with a plate reader.
Competition ELISA uses the same principle as a competitive ELISA, but in this case the antigen is a known quantity. The sample containing the unknown antigen is added to the plate and the detection antibody is applied using relevant substrates. In high concentrations of antigen, the signal output will decrease, whereas a low concentration will only show a slight reduction. Ultimately, the competition ELISA uses the same principle and is therefore the preferred method of detection for detecting antigen in complex mixtures.
ELISA assays are measurable. The sample is layered onto the plate. The first antibody binds to the antigen. The second antibody is then added to the plate. Once the primary antibody binds to the antigen, the secondary antibody binds to the second antibody, containing the enzyme. When the sample is exposed to the second antibody, the enzyme binds to the second antibody. The reaction produces a detectable signal, which is often a color change. When finished, the ELISA plate should be cleaned by ELISA washer.