Enzyme-linked immunosorbent assay (ELISA)
Basic
The purpose of an ELISA is to determine if a particular protein is present in a sample and if so, how much. There are two main variations on this method: you can determine how much antibody is in a sample, or you can determine how much protein is bound by an antibody. The distinction is whether you are trying to quantify an antibody or some other protein. In this example, we will use an ELISA to determine how much of a particular antibody is present in an individuals blood.
ELISAs are performed in 96-well plates which permits high throughput results. The bottom of each well is coated with a protein to which will bind the antibody you want to measure. Whole blood is allowed to clot and the cells are centrifuged out to obtain the clear serum with antibodies (called primary antibodies). The serum is incubated in a well, and each well contains a different serum . A positive control serum and a negative control serum would be included among the 96 samples being tested.
Principle
ELISA involves detection of an "analyte" in a liquid sample by a method that continues to use liquid reagents during the "analysis" which consists of a controlled sequence of biochemical reactions that will generate a signal which can be easily measured quantified and interpreted as a measure of the amount of analyte in the sample.
As a heterogenous assay, ELISA separates some component of the analytical reaction mixture by adsorbing certain components onto a solid phase which is physically immobilized.
In ELISA a liquid sample is added onto a stationary solid phase
with special binding properties and is followed by multiple liquid reagents that are sequentially added, incubated and washed followed by some optical change (e.g. color development by the product of an enzymatic reaction) in the final liquid in the well from which the quantity of the analyte is measured. The qualitative "reading" usually based on detection of intensity of transmitted light by spectrophotometric which involves quantitation of transmission of some specific type of light through the liquid (as well as the transparent bottom of the well in the multi-well plate format). The sensitivity of detection depends on amplification of the signal during the analytic reactions. Since enzyme reactions are very well known amplification processes, the signal is generated by enzymes which are linked to the detection reagents in fixed proportions to allow accurate quantification - thus the name "Enzyme linked".
The analyte is also called the ligand because it will specifically bind or ligate to a detection reagent and thus ELISA falls under the bigger category of Ligand Binding Assays. The ligand-specific binding reagent is "immobilized" i.e. usually coated and dried onto the transparent bottom and sometimes also side wall of a well (the stationary "solid phase'/"solid substrate" here as opposed to solid microparticle/beads that can be washed away), which is usually constructed as a multi-well plate known as the "Elisa Plate". Conventionally, like other forms of immunoassays the specificity of Antigen-Antibody type reaction is used because it is easy to raise an antibody specifically against an antigen in bulk as a reagent. Alternatively if the analyte itself is an antibody its target antigen can be used as the binding reagent.
The purpose of an ELISA is to determine if a particular protein is present in a sample and if so, how much. There are two main variations on this method: you can determine how much antibody is in a sample, or you can determine how much protein is bound by an antibody. The distinction is whether you are trying to quantify an antibody or some other protein. In this example, we will use an ELISA to determine how much of a particular antibody is present in an individuals blood.
ELISAs are performed in 96-well plates which permits high throughput results. The bottom of each well is coated with a protein to which will bind the antibody you want to measure. Whole blood is allowed to clot and the cells are centrifuged out to obtain the clear serum with antibodies (called primary antibodies). The serum is incubated in a well, and each well contains a different serum . A positive control serum and a negative control serum would be included among the 96 samples being tested.
Principle
ELISA involves detection of an "analyte" in a liquid sample by a method that continues to use liquid reagents during the "analysis" which consists of a controlled sequence of biochemical reactions that will generate a signal which can be easily measured quantified and interpreted as a measure of the amount of analyte in the sample.
As a heterogenous assay, ELISA separates some component of the analytical reaction mixture by adsorbing certain components onto a solid phase which is physically immobilized.
In ELISA a liquid sample is added onto a stationary solid phase
with special binding properties and is followed by multiple liquid reagents that are sequentially added, incubated and washed followed by some optical change (e.g. color development by the product of an enzymatic reaction) in the final liquid in the well from which the quantity of the analyte is measured. The qualitative "reading" usually based on detection of intensity of transmitted light by spectrophotometric which involves quantitation of transmission of some specific type of light through the liquid (as well as the transparent bottom of the well in the multi-well plate format). The sensitivity of detection depends on amplification of the signal during the analytic reactions. Since enzyme reactions are very well known amplification processes, the signal is generated by enzymes which are linked to the detection reagents in fixed proportions to allow accurate quantification - thus the name "Enzyme linked".
The analyte is also called the ligand because it will specifically bind or ligate to a detection reagent and thus ELISA falls under the bigger category of Ligand Binding Assays. The ligand-specific binding reagent is "immobilized" i.e. usually coated and dried onto the transparent bottom and sometimes also side wall of a well (the stationary "solid phase'/"solid substrate" here as opposed to solid microparticle/beads that can be washed away), which is usually constructed as a multi-well plate known as the "Elisa Plate". Conventionally, like other forms of immunoassays the specificity of Antigen-Antibody type reaction is used because it is easy to raise an antibody specifically against an antigen in bulk as a reagent. Alternatively if the analyte itself is an antibody its target antigen can be used as the binding reagent.