Name: elisa test types and signal detection
Uploaded: 2023-04-30T11:58:46.000Z
Duration: 1 min 33 s
Channel: JoVE

Enzyme-linked immunosorbent assay or ELISA is a labeled immunoassay with wide applications in detecting and quantifying analytes like antibodies, peptides, glycoproteins, hormones, and viruses.
The detection antibodies that bind to the antigen or target molecule are conjugated with specific enzymes which, upon substrate addition, produce measurable signals.
Depending on the capture and detection methods, there are four major types of ELISA.
Direct ELISA uses an enzyme-conjugated primary antibody for detecting the sample antigen coated on the plate.
While for indirect ELISA, the attached antigen captures the&#160; sample primary antibody, to which the conjugated secondary antibody binds.
Sandwich ELISA involves two antibodies, one coated on the plate to capture the sample antigen, while the other enzyme-conjugated antibody binds to the specific antigen-epitope forming the sandwich.
In competitive ELISA, the antigen and the primary antibody are pre-incubated and added to the plate that is coated with the same antigen.&#160;
The higher the sample antigen concentration, the more antigen-antibody complexes are formed, leaving fewer free antibodies to bind to the competing antigen from the plate,

enzyme-linked immunosorbent assay

Enzyme-Linked Immunosorbent Assay

In 1971, Peter Perlman and Eva Engvall developed an Enzyme-linked immunosorbent assay (ELISA or EIA). ELISA differs from western blot in that the assays are conducted in microtiter plates or in vivo rather than on an absorbent membrane.
There are many different types of ELISAs, but they all involve an antibody molecule whose constant region binds an enzyme, leaving the variable region free to bind its specific antigen.&#160; Enzyme-substrate reaction allows the antigen to be visualized or quantified. In ELISAs, the substrate for the enzyme is most often a chromogen, a colorless molecule that is converted into a colored end product. The most widely used enzymes are alkaline phosphatase and horseradish peroxidase, for which appropriate substrates are readily available. In some ELISAs, the substrate is a fluorogen, a nonfluorescent molecule that the enzyme converts into a fluorescent form. ELISAs that utilize a fluorogen are&#160; termed&#160; fluorescent enzyme immunoassays (FEIAs). Fluorescence can be detected by either a fluorescence microscope or a spectrophotometer.
There are several types of ELISA, based on differences in the format of detection and general workflow. In direct ELISA, antigens are immobilized in the well of a microtiter plate. An antibody that is specific for a particular antigen, and is conjugated to an enzyme, is added to each well. If the antigen is present, then the antibody will bind. After washing to remove any unbound antibodies, a chromogen is added.The presence of the enzyme converts the substrate into a colored end product.
Indirect ELISA is an extremely sensitive and flexible procedure. Here, in addition to a primary antibody, a secondary antibody is added for detection purposes. The secondary antibody&#160; quantifies how much antigen-specific antibody is present in the sample by the intensity of the color produced from the conjugated enzyme-chromogen reaction.
Sandwich ELISA is more specific and sensitive than direct and indirect ELISA. The goal is to use antibodies to precisely quantify the specific antigen present in a solution, such as the antigen from a pathogen, a serum protein, or hormone from blood or urine. The primary antibody captures the antigen and, following a wash, the polyclonal enzyme-conjugated secondary antibody is added. After a final wash, a chromogen is added, and the enzyme converts it into a colored end product. The amount of color produced (measured as absorbance) is directly proportional to the amount of enzyme, which in turn is directly proportional to the captured antigen. The complex workflow and several optimizations make this process error-prone.
For competitive ELISA, crude samples can be directly used. The experimental setup is highly flexible, wherein direct, indirect, or sandwich ELISAs can be adapted to a competitive format. The sample analyte concentration is determined by the signal interference. The &#34;competition&#34; comes from the fact that if a sample antigen is being tested, then the incubation of the sample with a primary antibody will result in lesser antibodies available to bind to the wells coated with the same antigen. Thus, the intensity of the signal produced in the well, due to the competitive binding, which is concentration dependent, becomes inversely correlated to the amount of sample antigen.
Depending on the purpose, the subtype of ELISA is chosen for a suitable outcome. For detecting large proteins comprising multiple epitopes, sandwich ELISA is most appropriate, whereas competitive ELISA is ideal for small protein&#160; detection. ELISA has significant applications in diagnostics, such as pregnancy testing, detecting food allergens, and identifying cancer biomarkers.

In 1971, Peter Perlman and Eva Engvall developed an Enzyme-linked immunosorbent assay (ELISA or EIA). ELISA differs from western blot in that the assays ...

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elisa test types and signal detection

ELISA is an antibody-based test used to detect and measure specific molecules in a sample. It was developed in 1971 by Peter Perlman and Eva Engvall. Unlike western blot, ELISA is done in microtiter plates or in vivo rather than on an absorbent membrane.

In ELISA, an antibody has an enzyme attached to its constant region. The variable region stays free to bind a matching antigen. After binding, an enzyme-substrate reaction makes the target visible or measurable. In many tests, the substrate is a chromogen, which is a colorless molecule that changes into a colored product. Common enzymes include alkaline phosphatase and horseradish peroxidase. Some ELISAs use a fluorogen instead. A fluorogen is a nonfluorescent molecule that becomes fluorescent after the enzyme acts on it. These tests are called fluorescent enzyme immunoassays, or FEIAs, and the signal can be read with a fluorescence microscope or a spectrophotometer.

ELISA can be run in several formats. In direct ELISA, antigens are fixed in a microtiter well, and an enzyme-linked antibody is added. If the antigen is present, the antibody binds. After washing away unbound antibody, a chromogen is added, and color forms. Indirect ELISA adds a secondary antibody for detection. This makes the method highly sensitive and flexible. The secondary antibody helps measure how much antigen-specific antibody is in the sample by the color intensity produced in the enzyme-chromogen reaction.

Sandwich ELISA is more specific and more sensitive than direct or indirect ELISA. It is used to measure a target antigen in a solution, such as a pathogen antigen, a serum protein, or a hormone from blood or urine. The primary antibody first captures the antigen. After washing, a polyclonal enzyme-linked secondary antibody is added. A final wash is followed by chromogen addition, and the enzyme makes a colored end product. The absorbance of that color is proportional to the amount of enzyme and, in turn, to the amount of captured antigen. This workflow can be complex and error-prone because it needs several optimization steps.

Competitive ELISA is another flexible format and can use crude samples directly. Direct, indirect, and sandwich ELISAs can all be adapted to this format. Here, the sample analyte is measured by signal interference. The competition happens because sample antigen and well-coated antigen compete for the same primary antibody. If more sample antigen is present, fewer antibodies remain available to bind the wells. As a result, the signal from the well is inversely related to the amount of sample antigen. The best ELISA type depends on the target. Sandwich ELISA works best for large proteins with multiple epitopes, while competitive ELISA is better for small protein detection. ELISA is widely used in diagnostics, including pregnancy testing, food allergen detection, and cancer biomarker identification.

ELISA Test Types and Signal Detection

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ELISA is an antibody-based test used to detect and measure specific molecules in a sample. It was developed in 1971 by Peter Perlman and Eva Engvall. Unlike western blot, ELISA is done in microtiter plates or in vivo rather than on an absorbent membrane. In ELISA, an antibody has an enzyme attached to its constant region. The variable region stays free to bind a matching antigen. After binding, an enzyme-substrate reaction makes the target visible or measurable. In many tests, the substrate is...

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Learn how ELISA uses enzymes, antibodies, and color or fluorescence signals to detect antigens in direct, indirect, sandwich, and competitive formats.

ELISA Test Types and Signal Detection | JoVE K12

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ELISA Test Types and Signal Detection

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