Иммуносараптама: Антиденелерді қолдана отырып биохимиялық тест

Biochemical test for a protein or other molecule using an antibody

An immunoassay (IA) is a biochemical test that measures the presence or concentration of a macromolecule or a small molecule in a solution through the use of an antibody (usually) or an antigen (sometimes). The molecule detected by the immunoassay is often referred to as an "analyte" and is in many cases a protein, although it may be other kinds of molecules, of different sizes and types, as long as the proper antibodies that have the required properties for the assay are developed. Analytes in biological liquids such as serum or urine are frequently measured using immunoassays for medical and research purposes. Immunoassays come in many different formats and variations. Immunoassays may be run in multiple steps with reagents being added and washed away or separated at different points in the assay. Multi step assays are often called separation immunoassays or heterogeneous immunoassays. Some immunoassays can be carried out simply by mixing the reagents and samples and making a physical measurement. Such assays are called homogeneous immunoassays, or less frequently non separation immunoassays. The use of a calibrator is often employed in immunoassays. Calibrators are solutions that are known to contain the analyte in question, and the concentration of that analyte is generally known. Comparison of an assay's response to a real sample against the assay's response produced by the calibrators makes it possible to interpret the signal strength in terms of the presence or concentration of analyte in the sample.

Immunoassays rely on the ability of an antibody to recognize and bind a specific macromolecule in what might be a complex mixture of macromolecules. In immunology the particular macromolecule bound by an antibody is referred to as an antigen and the area on an antigen to which the antibody binds is called an epitope. In some cases, an immunoassay may use an antigen to detect for the presence of antibodies, which recognize that antigen, in a solution. In other words, in some immunoassays, the analyte may be an antibody rather than an antigen. In addition to the binding of an antibody to its antigen, the other key feature of all immunoassays is a means to produce a measurable signal in response to the binding. Most, though not all, immunoassays involve chemically linking antibodies or antigens with some kind of detectable label. A large number of labels exist in modern immunoassays, and they allow for detection through different means. Many labels are detectable because they either emit radiation, produce a color change in a solution, fluoresce under light, or can be induced to emit light.

Immunoassays employ a variety of different labels to allow for detection of antibodies and antigens. Labels are typically chemically linked or conjugated to the desired antibody or antigen.

Possibly one of the most popular labels to use in immunoassays is enzymes. Immunoassays which employ enzymes are referred to as enzyme immunoassays (EIAs), of which enzyme linked immunosorbent assays (ELISAs) and enzyme multiplied immunoassay technique (EMIT) are the most common types. Enzymes used in ELISAs include horseradish peroxidase (HRP), alkaline phosphatase (AP) or glucose oxidase. These enzymes allow for detection often because they produce an observable color change in the presence of certain reagents. In some cases these enzymes are exposed to reagents which cause them to produce light or chemiluminescence. There are several types of ELISA: direct, indirect, sandwich, competitive.

Radioactive isotopes can be incorporated into immunoassay reagents to produce a radioimmunoassay (RIA). Radioactivity emitted by bound antibody antigen complexes can be easily detected using conventional methods. RIAs were some of the earliest immunoassays developed, but have fallen out of favor largely due to the difficulty and potential dangers presented by working with radioactivity.

A newer approach to immunoassays involves combining real time quantitative polymerase chain reaction (RT qPCR) and traditional immunoassay techniques. Called real time immunoquantitative PCR (iqPCR) the label used in these assays is a DNA probe.

Fluorogenic reporters like phycoerythrin are used in a number of modern immunoassays. Protein microarrays are a type of immunoassay that often employ fluorogenic reporters.

Some labels work via electrochemiluminescence (ECL), in which the label emits detectable light in response to electric current.

While some kind of label is generally employed in immunoassays, there are certain kinds of assays which do not rely on labels, but instead employ detection methods that do not require the modification or labeling the components of the assay. Surface plasmon resonance is an example of technique that can detect binding between an unlabeled antibody and antigens. Another demonstrated labeless immunoassay involves measuring the change in resistance on an electrode as antigens bind to it.

Immunoassays can be run in a number of different formats. Generally, an immunoassay will fall into one of several categories depending on how it is run.

In a competitive, homogeneous immunoassay, unlabelled analyte in a sample competes with labeled analyte to bind an antibody. The amount of labelled, unbound analyte is then measured. In theory, the more analyte in the sample, the more labelled analyte gets displaced and then measured; hence, the amount of labelled, unbound analyte is proportional to the amount of analyte in the sample. The fluorescence polarization immunoassay (FPIA) measures the fluorescence polarization signal after incubation, without separating bound and free labels. Free labeled analyte analog molecules are added to the sample, and their Brownian motion differs when bound to a large antibody (Ab) versus free in solution. The analyte competes for binding to the Ab, and if the labeled analyte binds to the Ab, a signal is produced. The signal intensity is inversely proportional to the analyte concentration. More specifically, they are qualitative tests that detect whether hCG is present, using a lateral flow setup. The COVID 19 rapid antigen test is also a qualitative, lateral flow test. Other clinical immunoassays are quantitative; they measure amounts. Immunoassays can measure levels of CK MB to assess heart disease, insulin to assess hypoglycemia, prostate specific antigen to detect prostate cancer, and some are also used for the detection and/or quantitative measurement of some pharmaceutical compounds (see Enzyme multiplied immunoassay technique for more details). Drug testing also starts with a quick qualtitative immunoassay.

Immunoassays are used in sports anti doping laboratories to test athletes' blood samples for prohibited recombinant human growth hormone (rhGH, rGH, hGH, GH).

The photoacoustic immunoassay measures low frequency acoustic signals generated by metal nanoparticle tags. Illuminated by a modulated light at a plasmon resonance wavelength, the nanoparticles generate strong acoustic signal, which can be measured using a microphone. The photoacoustic immunoassay can be applied to lateral flow tests, which use colloidal nanoparticles.