Immune labeling
The immunolabeling (also immune or antibody staining called) is a biochemical method for the detection of a molecule by binding of an immunoconjugate .
properties
Immune labeling is a method of labeling molecules and uses antibodies or fragments labeled with a detection system. An immune label uses biotin , radioisotopes , reporter enzymes , oligonucleotides or fluorophores as a signal molecule (reporter) for detection. The detection is based on the affinity of antibodies for a certain tissue property ( epitope ) as an antigen-antibody reaction . Ideally, there is a specific and strong bond between the antibody and the epitope. The antibody is coupled to a detection system that makes its presence in the preparation visible. By means of various detection systems, even small amounts of epitopes can be amplified. The goal is to detect a signal at the location of the epitope (and only there) with sufficient strength.
The antibody that is directed against the epitope to be found is called the primary antibody. The antibody should be characterized by high specificity and affinity and should not show any cross-reactions with similar epitopes. The individual components of the detection system are added to the preparation in a multi-step process. This is why immunohistochemistry (IHC) is relatively tedious and error-prone. The result can also be influenced by the type of fixation, fixation duration, embedding methods , pretreatment methods ( antigen retrieval ) of the preparations, etc. A standardization of the test execution should therefore be aimed for. The antigen-antibody reaction depends on temperature, concentration, incubation time, agitation and the optimal reaction environment (pH value, salt concentrations). TBS-T buffer is mostly used as a buffer for binding antibodies or immunoconjugates .
In order to keep these variables as constant as possible and to be able to process the large sample volume in the laboratory, IHC machines of various designs were introduced. The assessment of immunohistochemistry is always carried out in the morphological context. Unspecific reactions and general background colorations (endogenous peroxidase, endogenous biotin) can prove to be a hindrance. If only a weak signal can be detected, it can be amplified by various methods (e.g. TSA) (signal amplification).
Direct method
The antigen (= protein) to be examined is brought together under defined conditions with a specific antibody that is directly coupled (conjugated) to an enzyme or fluorophore such as fluorescein , rhodamine or Texas Red . The antibody (and thus the enzyme) binds to the antigen, unbound antibody is rinsed off. In a further step, the enzyme is offered a substrate which reacts with the enzyme to form a dye. This dye forms where the immunochemical reaction took place and is visible. Simply put: antigen + antibody with enzyme + substrate / chromogen → color
In the case of fluorochrome-labeled antibodies, detection takes place directly in the fluorescence microscope. Direct immunofluorescence (DIF) is also well suited for multiple representations of different antigens in one preparation, in this case antibodies of different specificities are conjugated with fluorochromes of different emission wavelengths. The DIF is the oldest immunohistochemical technique and was first used in the 1950s.
Indirect method
In this method, also known as indirect immunofluorescence (IIF), a specific antibody (primary antibody) is applied to the tissue / cells to be examined in the first step. In a second step, an antibody is applied that is directed against the first antibody. It is the so-called secondary antibody, which is coupled with an enzyme and triggers the development of color with an enzyme-substrate reaction. Again a visible dye is created.
Simple: antigen + primary antibody + secondary antibody with enzyme + substrate / chromogen → color
The indirect method is available as a two-step method and a three-step method. In the three-step method, an additional antibody coupled with an enzyme (= tertiary antibody) is added. This binds to the secondary antibody. This step serves to amplify the signal and is useful if a small amount of epitope is to be displayed.
The indirect technique is also used for the detection of already bound endogenous antibodies, for example autoantibodies such as anti-neutrophil cytoplasmic antibodies (ANCA); or the patient serum to be examined contains these autoantibodies and is applied to test tissue. If the result is positive, the secondary antibody (here typically coupled with a fluorochrome) finds its binding partner.
PAP and APAAP method
These methods get their name from the peroxidase-anti-peroxidase ( peroxidase-anti-peroxidase method ) or alkaline phosphatase-anti-alkaline phosphatase complex, which is applied here after the secondary AK. The complex consists of three molecules of enzyme and two antibodies (from the same species as primary AK) that are directed against the enzyme. The secondary AK acts as a bridge between the primary AK and the PAP complex. This method showed increased sensitivity and lower background staining than the previous methods and was introduced as a “kit” for routine use in laboratories in the 1980s.
Labeled (strept) avidin-biotin method (LSAB)
Nowadays this staining method is the most widely used. The principle is based on the high affinity of streptavidin ( Streptomyces avidinii ) and avidin (egg white) for biotin . Streptavidin and avidin each have four binding sites for biotin.
The order of the reagents: unconjugated primary antibody + biotin-labeled (= biotinylated) secondary antibody + avidin-biotin-enzyme conjugate + substrate / chromogen → color.
Polymer methods
Here the primary AK (direct M.) or the secondary AK (indirect M.) is equipped with one or more polymer molecules (dextran or polypeptide). These polymers are marked with as much enzyme as possible, which in turn causes substrate and chromogen conversion. This results in an increased staining at the site of the antigen. The advantage is that there is no need to use biotin, which could cause background staining as "endogenous biotin". The method is usually more sensitive and faster than LSAB. The disadvantage is the size of the molecules that have to be brought into the tissue and which can lead to steric hindrances at the binding site.
Double immune labeling
A double immunolabeling or double immunostaining referred to the visualization of two epitopes on a test sample by use of two antibodies or immunoconjugates . Reporter enzymes , radioactive isotopes (short-lived α- or β-emitters), colloids of noble metals or fluorophores are usually coupled directly to the antibodies as immunoconjugates via heterobifunctional crosslinkers .
The sample can e.g. B. a Western blot , cells for a flow cytometry , a transmission electron microscope preparation or a thin section stained immunohistochemically or by immunofluorescence . The double marking is also used in the course of a virological diagnosis .
Direct coloring
For double immunostaining, the sample is first incubated with the two antibodies directed against two different epitopes . If the antibodies against these epitopes are each coupled with a reporter enzyme, one speaks of direct labeling. In the case of indirect labeling, the antibodies directed against the epitopes sought are referred to as primary antibodies, as immunoconjugates are then used.
Indirect staining
In the case of indirect labeling via primary antibodies and immunoconjugate, an additional washing step and an immunoconjugate incubation are necessary. For this purpose, the signal is amplified through the use of an immunoconjugate that is directed against several epitopes on the F (c) fragment of the antibody, whereby many reporter molecules are grouped on the primary antibody. In addition, the immunoconjugates can be used modularly against all primary antibodies of a type, which avoids coupling of each primary antibody to a reporter molecule and thus reduces costs. If two different indirect stains (with separate primary antibodies and immunoconjugates with reporter enzymes) are used at the same time, two primary antibodies from two different types are used to differentiate the two signals , in which the immunoconjugates do not cross-react. Different reporter enzymes are mostly used. A serial double immunostaining can also be carried out exclusively with peroxidase conjugates, in which, after an initial staining, the peroxidase is inactivated with dilute sodium azide solutions and then the unbound sodium azide is washed out and stained again with a peroxidase conjugate.
Reporter enzymes
As reporter enzymes z. B. the horseradish peroxidase (with TMB , DAB , ABTS , AEC or - in the form of chemiluminescence - with luminol ) and the alkaline phosphatase (with BCIP and NBT or with Naphtol-AS-MX-phosphate and Fast Red TR) are used. The different precipitating color reactions are carried out in series.
Fluorophores
In the case of fluorescent multiple markings, the fluorophores are selected so that the ranges of the excitation and emission wavelengths are as far apart as possible. This reduces excessive exposure of the fluorescence to other color channels of the fluorescence detector and the associated fluorescence compensation, which leads to a loss of signal strength and thus increases the detection limit .
Reporting systems
The various dyes differ in their color and solubility, among other things. Soluble dyes are preferably used in an ELISA , while in immunohistochemistry and Western blot mostly precipitating dyes are used.
Peroxidase (POD, Horseradish peroxidase HRP)
The peroxidase (usually the horseradish peroxidase ) is hydrogen peroxide offered as a substrate. The released protons oxidize the previously almost colorless chromogen to its colored end product with the formation of water.
- ABTS
- AEC ( 3-amino-9-ethylcarbazole ) forms a rose-red end product
- CN ( 4-chloro-1-naphthol ) reacts to form a blue dye
- DAB ( 3,3′-diaminobenzidine ) forms a brown end product
- Luminol and other dioxetanes react to form chemiluminescence with the following autoradiography
- TMB ( tetramethylbenzidine ) forms a blue end product, which forms a stable yellow color complex when the reaction with sulfuric acid is stopped
Alkaline Phosphatase (AP)
The alkaline phosphatase available organic phosphate compounds as a substrate. The AP splits off phosphate and the released compound reacts to a colored end product.
- 5-Bromo-4-chloro-3-indoxyl phosphate (BCIP) is converted into a violet to blue dye in combination with nitro blue tetrazolium chloride (NBT): the tetrazolium salt oxidizes indoxyl to blue indigo dye. The tetrazolium salt is reduced to the blue formazan dye .
- Naphthol-AS-MX-Phosphat forms a red dye: Naphthol AS reacts with Fast Red TR to form an insoluble azo dye .
- Neufuchsin produces a pink-red reaction product
Radioisotopes
Radioisotopes are coupled to antibodies in the course of a molecular marking and, after protein purification, are registered by autoradiography or a scintillation counter .
- 131 iodine
- 32 phosphorus
- 3 tritium
See also
literature
- Friedrich Lottspeich , Haralabos Zorbas: Bioanalytics . Spektrum Akademischer Verlag, Heidelberg 1998, ISBN 3-8274-0041-4 .
- Hubert Rehm , Thomas Letzel: The Experimenter: Protein Biochemistry / Proteomics. 6th edition. Spectrum Akademischer Verlag, Heidelberg 2009, ISBN 978-3-8274-2312-2 .
Individual evidence
- ↑ R. Lequin: Enzyme immunoassay (EIA) / enzyme-linked immunosorbent assay (ELISA) . In: Clin. Chem. Band 51 , no. 12 , 2005, p. 2415-2418 , doi : 10.1373 / clinchem . 2005.051532 , PMID 16179424 .