The enzyme-linked immunosorbent assay (ELISA) is a technique for detecting and measuring target proteins in a sample. ELISA is one of many methods available for detection of proteins in biological samples. It uses antibodies that specifically bind to the target protein as an indicator for its presence. The antibody is attached to the surface of a plastic plate through its binding site for protein. The detection antibody specifically binds target protein on the coated plate, where it becomes immobilized by noncovalent bonds formed between specific amino acid residues in each antibody chain and complementary ones on antigen molecules (the target protein has two identical sites). After adding substrate, chromogenic color develops in wells of the plate; color intensity is proportional to amount of bound target protein: more bound target protein results in darker color intensity than less bound target protein does; thus standard curve can be constructed by measuring color intensity at different concentrations of standard solution containing known amounts of purified target proteins
Antibodies are proteins that recognize and bind to a specific target protein. These proteins, called antibodies, were discovered by the Austrian scientist Dr. Karl Landsteiner in 1899. Antibodies are used in ELISA to detect the presence of a target protein on an immunochemical substrate, such as a microtiter plate well.
Sandwich ELISA is an enzyme-linked immunosorbent assay that detects target protein using two antibodies. The sandwich method is based on the use of two antibodies, one with a high affinity for the target antigen and one with low affinity for it. The higher affinity antibody will bind to the antigen in solution, while the lower affinity antibody binds to that complex. Next, a third molecule (i.e., an enzyme) that can only react when both antibodies are present attaches itself to this complex and produces a color change visible in a plastic plate during incubation at room temperature.
The coating antibody is attached to the surface of a plastic plate through its binding site for protein. There are two types of coating antibodies: polyclonal and monoclonal. Polyclonal antibodies bind to multiple epitopes on the target protein, while monoclonal antibodies bind to a single epitope on the target protein.
After adding substrate, chromogenic color develops in the wells of the plate. The intensity of this color is proportional to the amount of bound target protein. To measure target protein concentration, a standard curve must be constructed by measuring color intensity at different concentrations of target protein standard.
The ELISA method can be used to detect a variety of proteins including hormones, cytokines and antibodies
The plate reader then measures the intensity of light emitted from each well, which corresponds to protein concentration in solution. This is done by subtracting the background (light emitted from wells that do not contain any target protein) from the total signal (light emitted from all wells), thereby allowing you to calculate how much target protein was present in each well.
To measure target protein concentration, a standard curve must be constructed by measuring color intensity at different concentrations of target protein standard. The standard curve is a plot of color intensity vs. protein concentration. You can then use this standard curve to find the concentration of protein in unknown samples.
The color intensity is measured with a spectrophotometer and is proportional to the amount of bound target protein that has been detected by your antibody (see figure).
When ELISA was first developed, it used radioactive materials as labels that emitted light upon exposure to X-ray film or photographic emulsion. The radioactive label could be detected by exposing the film or photographic emulsion to X-rays, which caused the label to emit visible light, thus producing a detectable signal. The use of radioactivity allowed for much easier detection of the target protein than was previously possible. After WWII and throughout the 1950s and 1960s, however, concerns about radiation safety led scientists away from using radioactivity in these types of experiments.
ELISA is one of many methods available for detection of proteins in biological samples. It is an immunoassay that uses a specific antibody (or group of antibodies) to bind the protein of interest and a reporter enzyme-labeled secondary antibody to detect it. ELISA's versatility makes it useful for detecting proteins in body fluids or tissues from humans or animals, as well as identifying bacteria, viruses, parasites and fungi based on their unique surface antigens.
ELISA is a powerful tool for the detection and quantification of target proteins in biological samples. It is a very sensitive technique, which means it can detect small amounts of protein in complex mixtures. This makes ELISA an ideal technique for detecting pathogenic bacteria or viruses in clinical specimens, like blood or urine.