Enzyme-linked immunosorbent assay (ELISA)is a laboratory technique that sets out to detect the presence and quantity of a specific antigen or antibody in a sample. This methodology is widely used in biomedical research, clinical diagnosis, and various industries, including food and beverage, agriculture, and environmental monitoring. Its sensitivity and specificity are second to none, making it a force to reckon with in the scientific world.
ELISA is built upon the principle of binding an antigen or antibody to a solid surface, typically a microplate well, and then detecting the bound molecule using an enzyme-linked detection system. The detection system utilizes an enzyme that catalyzes a reaction with a substrate to produce a measurable signal, such as a color change or fluorescence.
ELISA comes in several types, each with its own advantages and limitations, including direct, indirect, sandwich, and competitive ELISA. However, most ELISA assays typically involve the same basic steps, including coating, blocking, incubation, washing, detection, and signal generation, which all work in unison to produce results that are truly repeatable.
Coating involves incubating the surface of solid, such as a microwell plate, with the a solution containing the antigen or antibody of interest. After this, the surface is typically blocked to prevent non-specific binding of other proteins or molecules to the surface using a blocking solution, such as bovine serum albumin (BSA).
The sample of interest, such as a serum or a tissue homogenate, is then added to the coated and blocked surface and incubated for a specific period of time to allow the antigen or antibody to bind to the surface. Afterward, the surface is washed several times with a washing buffer to remove any unbound molecules.
The detection step involves the addition of a detection reagent, such as an enzyme-linked secondary antibody, which binds specifically to the antigen or antibody of interest that is bound to the surface. Following an incubation period, the surface is washed again to remove any unbound detection reagent.
Finally, the addition of a substrate that reacts with the enzyme linked to the detection reagent produces a measurable signal. This can be a color change or fluorescence, depending on the type of substrate used. The signal can then be measured using a spectrophotometer or a fluorescence reader.
In recent years, ELISA assay technology has been supplemented by several new technologies, such as multiplex analysis and high-throughput sequencing, which in turn allow ELISA to simultaneously detect and quantify multiple biomolecules in a single sample. Due to its sensitivity, specificity, and versatility, ELISA has become an important tool widely used in biomedical research and clinical diagnosis.
Although ELISA is a highly sensitive and specific technique that can detect very low levels of antigen or antibody in a sample, it does have limitations, including the possibility of false positives and false negatives, the need for careful optimization and validation of the assay, and limited dynamic detection range. Nevertheless, ELISA has become an important detection method in various industries such as biomedical research, clinical diagnosis, food and beverage, agriculture and environmental monitoring due to its simplicity, convenience and speed.