A common question when it comes to biochemistry is how an ELISA works. ELISA is an enzyme-based assay that uses an antibody to measure the amount of antigen bound to a given sample. Various enzyme-substrate reactions can produce an output that can be measured by a microplate reader. Depending on which enzyme is in action, the assay can be performed in either spectrophotometric or fluorescence mode.
ELISAs are widely used in medical research. The main advantage of ELISAs is their specificity, stability, and speed of analysis. ELISAs are popular for protein, peptide, and small molecule analysis. The disadvantages of polystyrene-based microplates are that the captured antibodies exhibit random orientation due to hydrophobic interactions. This reduces the avidity of antigen-capturing antibodies and limits the sensitivity of the assay.
Another ELISA method is sandwich ELISA, which uses two different sets of antibodies to detect secreted antigens. In this method, the antigens are bound to the plate's surface using a method known as passive adsorption. Since the antigens are not purified, cross-reactivity is a problem in sandwich ELISAs. However, sandwich ELISAs are useful in certain circumstances where high-level accuracy is desired.
A second ELISA method involves the use of secondary antibodies. The primary antibody in the indirect ELISA uses an enzyme to detect proteins. This is more expensive than the former, but it provides the highest specificity. The secondary antibody adds extra steps, increases the assay time, and increases the chance of error. In addition, polyclonal secondary antibodies require more optimization to find a suitable pair. If the benefits of this method outweigh the disadvantages, they are worth it.
Another method is the Lowry assay, which was developed in 1951 by Oliver H. Lowry. This method involves two chemical reactions: the reduction of copper ions in an alkaline solution and the formation of a complex with peptide bonds. A spectrophotometer is used to detect this reaction. The color change is proportional to the amount of protein in the sample. There are two major methods for performing this test: the Lowry assay and the Modified Lowry Protein Assay.
An ELISA is a popular method to test for a variety of proteins. The enzyme used in this method binds to a sample and is called an enzyme. Typically, a secondary antibody is attached to the substrate. This method is more rapid than the other two, but requires that the sample be thoroughly prepared before use. The antigen should be completely diluted to ensure accuracy. If there are too many antigens in a sample, the enzyme may not detect it. After detetion, there maybe some residual substances on the ELISA plate. In order to reduce the errors caused by the residues, an ELISA washer is needed. This medical device has been widely used in the cleaning of ELISA plates in hospitals, blood stations, health and epidemic prevention stations, reagent factories and research laboratories.
In a competitive ELISA, the antigen and antibody compete for limited binding sites. The sample antigen has a higher concentration than the reference antigen, and the antibody has a lower concentration. A competitive ELISA uses a single antibody and a low molecular weight antigen, whereas the other two types of ELISA use multiple reagents. The competition in the ELISA demonstrates a direct correlation between antigen concentration and color development.
Sandwich ELISAs use two different antibody pairs. The capture antibody binds to the antigen and the detection antibody binds to the second. The antigen is detected through direct or indirect ELISA methods, and the quantification occurs between the upper and lower layer of the antibodies. The sandwich ELISA has fewer steps and a more accurate result than direct ELISAs. It's also more sensitive and has a larger dynamic range.
Another popular method is the enzyme-linked immunosorbent assay. It is a plate-based, immunoassay that detects proteins and peptides. The target antigen must be immobilized on a solid surface and then reacted with an antibody attached to an enzyme. The substrate is then incubated with the enzyme and a measurable product is released. This method of detection is very specific and accurate, and it is used to detect antigens in biological samples.
PXDN is a commonly studied protein in melanoma. ELISA can detect the protein using both invasive and non-invasive melanoma cell lines. The ELISA uses a fusion protein, known as His6-SUMO-POX10, to measure PXDN activity. The ELISA is carried out on a Corning high-binding microplate that contains 50 mL of cell lysate per well. The samples are then incubated at 37 degC for 2 hours.