Immunoassay
As immunoassays ( engl. : Immunoassay ) are collectively a number of methods in the bioanalytics referred whose common fundamental principle of the detection, and thus the detection of an analyte in a liquid phase by the binding of an antigen to an antibody is. Depending on the configuration of the assay, both antigen and antibody can be the analyte to be detected. When performing immunassays, the high specificity and binding strength of the bond between antigens and antibodies is used. The immunodiffusion test can be described as a simple predecessor of specialized procedures .
general basics
Analyte
In immunassays, the analyte is in a liquid phase, usually dissolved in a buffer . It is also possible to use body fluids or other sample fluids, such as untreated water samples or liquid food or corresponding extracts . In addition, particulate structures with antigenic surface features, such as bacteria , viruses , cells or material particles, which are present in a suspension , can also be detected by immunassays.
Detection techniques
For detection and quantitative determination, the use of labeled reagents is necessary in immunassays . Different markings are possible depending on the assay format, application and desired sensitivity.
Marking by means of enzymes , which catalyze a chemical reaction , is widespread , in which either a specific color is created by a substrate ( chromogenic substrate) or light is emitted via chemiluminescence . Another optical method is marking with fluorescent dyes . Both the color intensity in the case of chromogenic substrates and the light intensity in the case of luminescent and fluorescent substrates or markings can be measured with appropriate devices. Assays based on enzyme labels are called enzyme-linked immunosorbent assays (ELISA). In radioimmunoassays (RIA), low-level radioactive substances are used for labeling, detection and quantification are carried out by measuring the radioactivity .
Further options for marking are the binding of the detection reagent to gold colloids or colored polymer particles. In both cases, if a sufficient amount is bound, visual recognition with the naked eye is possible. Marking with magnetic particles and thus the measurement of the magnetic field strength as a detection principle as well as electrochemical methods, for example based on impedance measurements , are used less frequently .
Fixed phase
While the analyte (the antigen) is in the sample liquid, its binding partner (the antibody) is usually bound (immobilized) to a solid phase. The selection of the solid phase depends on the intended use and, in part, on the selected detection method.
The most frequently used reaction vessels are so-called microtiter plates . These are plastic plates that contain a defined number of cavities (depressions, wells) with a certain volume in a certain grid . The frequently used plates in the so-called 96-well format, each with twelve cavities in eight rows, have a volume per cavity of around 350 microliters. Microtiter plates are typically used for ELISA format assays. Radioimmunoassays, on the other hand, are often carried out in individual plastic test tubes.
For simple assays, cuvettes are sometimes used, but membranes are often used as the solid phase. This format is known as an immunoblot or dot blot . The corresponding primary antibodies are also bound to membranes in the test strips known as lateral flow assays , which are used, for example, for pregnancy tests. The instrumental effort and, as a rule, also the time required for these variants are not as high as when carrying out a microtiter plate, but the sensitivity and the sample throughput are also lower.
quantification
If an immunassay is only used for yes / no detection of an analyte, it is called qualitative detection. In this case, no calibration using a calibration curve is necessary. Some assay formats are only suitable for such qualitative detection. A semi-quantitative detection enables a “more than” or “less than” statement by comparing it with one or more comparison samples with a known analyte concentration. For an exact concentration determination, however, a calibration curve is necessary, for which samples with known analyte concentrations are also measured. After creating a curve in which the measurement signals are plotted against the respective concentrations of the analyte in the comparison samples, the concentration of the analyte in the samples to be examined can be read from the curve or calculated using a mathematical function. In order to make the signals comparable with each other, a limit value (cut-off, CO) can be defined, which arithmetically results from a blank value and the negative control. The measured signals are then related to this cut-off as a quotient (S / C or S / CO) and expressed as a multiple of the limit value.
Assay principles
Competitive assay
A competitive assay is used to detect an antigen if either only a single specific antibody is available for it or if the antigen does not have sufficient binding sites for the unhindered binding of two antibodies. This assay variant is therefore also suitable for the detection of haptens .
In a competitive assay, an antibody specific for the antigen is bound (immobilized) to a solid phase. The sample solution containing the analyte and a second solution (tracer solution) with the antigen to be detected are added in a known concentration and marked with a detection system. During the reaction time (incubation time) the unlabeled antigen present in the sample solution in an unknown concentration and the marked antigen present in a known concentration compete for the binding sites of the immobilized antibody. The higher the concentration of the antigen from the sample, the less labeled antigen is bound to the antibody. The detection of the antigen and the measurement of its concentration are indirectly possible via the detection and, if necessary, a quantitative determination of the marking.
Non-competitive assay
For the detection of an antigen by means of a non-competitive assay, also known as a sandwich assay, two different antibodies are required that recognize the antigen and do not interfere with each other in their binding to the antigen. One advantage of the IRMA in comparison with the competitive format is the greater accuracy at low to very low concentrations of the substance to be determined (the antigen), a disadvantage in the false-low results at very high concentrations of the antigen (“high-dose hook -Effect) ".
In a sandwich assay, too, one of the two antibodies is initially bound (immobilized) to a solid phase. This is known as the primary antibody or also as a catcher. After adding the sample solution, the contained antigen binds to the primary antibody. The sample solution is then removed and the second antibody, known as the secondary antibody or detector, is added in dissolved form to the solid phase. The detector now also binds to the antigen bound by the primary antibody. For the also indirect detection and quantification, the secondary antibody is either labeled itself or it is detected using a labeled reagent.
Direct assay
A direct immunassay is used to detect antibodies. The corresponding antigen is bound to a solid phase. After adding the sample solution, the antibody it contains binds to the antigen. The sample solution is then removed and the bound antibody detected by a labeled reagent and, if necessary, quantified.
Applications
Immunassays are among the most widely used methods in bioanalytics, e.g. B. the ELISA , the Western Blot , the ELISPOT and the Indirect Immunoperoxidase Assay . Due to the biophysical and biochemical properties of the antigen-antibody binding, they offer a very high specificity and sensitivity with a comparatively moderate outlay in terms of equipment, costs and time. Another advantage over alternative methods is the ability to automate and construct high-throughput devices.
Immunoassays are used, for example, in laboratory medicine for the determination of a large number of parameters in various body fluids such as blood , serum or urine . They are used to predict, diagnose and monitor the progress of diseases as well as to detect toxins or to monitor drugs in the body. In microbiology , they can be used to identify pathogens.
In sports medicine , immunassays are used to detect certain doping substances . Other important areas of application are environmental, food and agricultural analysis. Here they are used, among other things, to detect environmental toxins, allergens in food or genetically modified organisms .
While most immunassays are carried out in specialized laboratories due to the equipment required for this, there are some formats for on-site execution. This can be, for example, in a doctor's practice or in an agricultural area in the open field. These tests are designed as test strips and are therefore easy to use. Some of these tests, such as pregnancy and drug tests , can also be done by laypeople.
literature
- David Wild (Ed.): The Immunoassay Handbook. Third edition. Elsevier Science Publishing Company, Amsterdam, Boston, Oxford 2005, ISBN 0-08-044526-8
- Werner Luttmann, Kai Bratke, Michael Küpper, Daniel Myrtek: The Experimenter: Immunology . Second edition. Spectrum Akademischer Verlag, Heidelberg 2006, ISBN 3-8274-1730-9 , pp. 103-131
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