| Immunoassay | |
|---|---|
| Intervention | |
| MeSH | D007118 |
An immunoassay is a specific type of biochemical test that measures the presence or concentration of a substance (referred to as the "analyte") in solutions that frequently contain a complex mixture of substances. Analytes in biological liquids such as serum or urine are frequently assayed (i.e., measured) using immunoassay methods. In essence, the method depends upon the fact that the analyte in question is known to undergo a unique immune reaction with a second substance, which is used to determine the presence and amount of the analyte. This type of reaction involves the binding of one type of molecule, the antigen, with a second type, the antibody. Immunoassays can be carried out using either the antigen or the antibody in order to test for the other member of the antigen/antibody pair. In other words, the analyte may be either the antigen or the antibody.
For antigen analytes, an antibody that specifically binds to that antigen can frequently be prepared for use as an analytical reagent. When the analyte is a specific antibody its cognate antigen can be used as the analytical reagent. In either case the specificity of the assay depends on the degree to which the analytical reagent is able to bind to its specific binding partner to the exclusion of all other substances that might be present in the sample to be analyzed. In addition to the need for specificity, a binding partner must be selected that has a sufficiently high affinity for the analyte to permit an accurate measurement. The affinity requirements depend on the particular assay format that is used.
An analyte that has been identified using immunoassay methods is referred to in relevant literature as "immunoreactive." The following excerpt may help illustrate how these terms are used with each other: "By this immunoassay, the concentration and molecular size of immunoreactive gamma 2-MSH in plasma of healthy subjects were examined, and the results were compared with those by competitive enzyme immunoassay. Immunoreactive gamma 2-MSH measured by competitive enzyme immunoassay was a mixture of substances with high molecular weights (100-500 kDa), and its concentration was calculated to be 50-60 pmol/L using gamma 2-MSH (1-12) as standard."[1]
In addition to binding specificity, the other key feature of all immunoassays is a means to produce a measurable signal in response to a specific binding. Historically this was accomplished by measuring a change in some physical characteristic such as light scattering or changes in refractive index. With modern instrumentation such methods are again becoming increasingly popular. Nevertheless most immunoassays today depend on the use of an analytical reagent that is associated with a detectable label. A large variety of labels have been demonstrated including radioactive elements used in radioimmunoassays; enzymes; fluorescent, phosphorescent, and chemiluminescent dyes; latex and magnetic particles; dye crystalites, gold, silver, and selenium colloidal particles; metal chelates; coenzymes; electroactive groups; oligonucleotides, stable radicals,and others. Such labels serve for detection and quantitation of binding events either after separating free and bound labeled reagents or by designing the system in such a way that a binding event effects a change in the signal produced by the label. Immunoassays requiring a separation step, often called separation immunoassays or heterogeneous immunoassays, are popular because they are easy to design, but they frequently require multiple steps including careful washing of a surface onto which the labeled reagent has bound. Immunoassays in which the signal is affected by binding can often be run without a separation step. Such assays can frequently be carried out simply by mixing the reagents and sample and making a physical measurement. Such assays are called homogenous immunoassays or less frequently non-separation immunoassays.
Regardless of the method used, interpretation of the signal produced in an immunoassay requires reference to a calibrator that mimics the characteristics of the sample medium. For qualitative assays the calibrators may consist of a negative sample with no analyte and a positive sample having the lowest concentration of the analyte that is considered detectable. Quantitative assays require additional calibrators with known analyte concentrations. Comparison of the assay response of a real sample to the assay responses produced by the calibrators makes it possible to interpret the signal strength in terms of the presence or concentration of analyte in the sample.
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Classification of immunoassays depends on whether one (noncompetitive) or two (competitive) antigens are used in the test system:
The antigen in the unknown sample competes with labeled antigen to bind with antibodies. The amount of unbound, labeled antigen is then measured, which is directly proportional to the concentration of sample antigen. This is because the presence of more free, labeled antigens means there was little to no binding sites available on the antibodies as they were all saturated with the sample antigen, which means it's high in concentration.
The antigen in the unknown sample competes with labeled antigen to bind with antibodies. The amount of labeled antigen bound to the antibody site is then measured. In this method, the response will be inversely related to the concentration of antigen in the unknown. This is because the greater the response, the less antigen in the unknown was available to compete with the labeled antigen.
The unknown antigen in the sample binds with labeled antibodies. The unbound, labeled antibodies are washed away, and the bound, labeled is measured, which is directly proportional to the amount of unknown antigen.
The antigen in the unknown is bound to the antibody site, then labeled antibody is bound to the antigen. The amount of labeled antibody on the site is then measured. It will be directly proportional to the concentration of the antigen because labeled antibody will not bind if the antigen is not present in the unknown sample. This type is also known as sandwich assay as the antigen is "sandwiched" between two antibodies.
A typical immunoassay kit contains the following material and solutions:
This is a plastic plate that contains 96 wells pre-coated with antibodies directed specifically to the antigen of interest (i.e. capture antibody). As some of the wells must be devoted to blank, standard, and control solutions, depending on the manufacturer, the plate may allow up to 37 different samples tested in duplicate, or 21 in triplicate.
A blank solution is one that contains all the substances used in the assay except the antigen to be tested. It ensures that cross-reacting substances are eliminated so that signals and readings obtained accurately reflect the antigen measured.
ِA standard solution contains a known amount of the antigen of interest. Depending on the manufacturer, a series of standard solutions with different concentrations, or one with a diluent, may be provided. Standards are used to construct a standard curve to which readings from samples are compared. These different concentrations determine the range of the assay. Sometimes the standard solution is provided in a lyophilized (powder) form. In this case, reconstitution to a liquid form is required.
A control solution is one with a known amount of the antigen and will either give a positive signal (positive control) or a negative signal (negative control). Both types must be run with every assay as a quality control measure.
As the name implies, diluents are used for diluting specimens (when out of assay range), standards, and controls. An assay diluent may also be used. It is applied to the wells just before adding samples, and serves to eliminate interference or non-specific binding that may occur due to the specimen matrix.
Required for the sandwich assay, the capture antibody binds to the antigen of interest and generates a detectable signal for quantification. This antibody is usually polyclonal in nature, and is conjugated with an enzyme (most commonly, horseradish peroxidase), so that, upon substrate addition, a colored product is formed. The density of the color can then be measured spectrophotometrically as a function of antigen concentration.
This is a buffer that removes unbound antibodies floating in the well.
A specific substance for the conjugate enzyme, which, upon degradation, produces a colored product whose intensity could be measured.
In most test systems, the substrate is allowed to react with the enzyme for 20 minutes, before the reaction is terminated. This ensures a high degree of accuracy and sensitivity.
Owing to their high sensitivity (ability to detect very small concentrations) and specificity (minimal to no cross-reactivity), immunoassays have been employed in the measurement of blood leves of vitamins, hormones, and porphyrins.
Immunoassay is used in sports anti-doping laboratories to test athletes' blood samples for prohibited recombinant human growth hormone (rhGH, rGH, hGH, GH).[2]
"The Immunoassay Handbook", 3rd Edition, David Wild, Ed., Elsevier,2008
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