Cell-based ELISA is a method used to find specific proteins or antigens in cells. It is a variation of a technique called Enzyme-Linked Immunosorbent Assay (ELISA), which uses antibodies to measure the amount of a target protein or antigen in a sample. With cell-based ELISA, the assay is done on whole cells instead of just their components.
The process for cell-based ELISA has a few steps. First, the cells of interest are put onto a 96-well plate and allowed to stick overnight. Next, the cells are fixed with a chemical to keep the antigens from moving around. Then, the cells are made permeable with a detergent so that the antibodies can get inside. To make sure the antibodies only stick to the right places, the non-specific binding sites on the cell surface are blocked with a protein like bovine serum albumin (BSA) or fetal bovine serum (FBS). After blocking, specific antibodies that attach to the target antigen are added to the wells. These primary antibodies are usually made to target the antigen in question and will stick to it really well. After the primary antibodies bind to the target antigen, the wells are washed to remove any unattached antibodies. Then, secondary antibodies linked to an enzyme like horseradish peroxidase (HRP) or alkaline phosphatase (ALP) are added. These secondary antibodies will stick to the primary antibodies, creating a sandwich. Once the wells are washed again to remove any unattached secondary antibodies, a substance called substrate is added. This substance will change color if the enzyme that the secondary antibody is linked to is present. The color change is measured with a machine that tells researchers how much of the antigen is in the sample.
Cell-based ELISA is useful for finding many kinds of antigens, including those on the surface of cells, inside cells, and those that control gene activity. It is better than traditional ELISA because it can find proteins in their normal shape, which is important for studying how they work. It can also show where the proteins are in the cell, which can help researchers understand what they do. Another good thing about cell-based ELISA is that it can find more than one antigen at the same time. This is done by using primary antibodies from different species or attaching different enzymes to the antibodies.
Although cell-based ELISA is helpful, it takes a lot of time and needs special equipment and knowledge to use. Also, fixing and permeabilizing the cells can change the shape of the antigens and affect how well the primary antibodies stick to them.
Overall, cell-based ELISA is a powerful method for finding specific proteins or antigens in whole cells. It is better than traditional ELISA in some ways and can help researchers study how proteins work in different biological systems.