Immunolabeling

Immunolabeling is a biochemical method for the detection of a molecule by binding of the immunoconjugate.

• 2.1 Direct staining
• 2.2 Indirect staining
• 2.3 reporter enzymes
• 2.4 fluorophores

• 3.1 peroxidase (HRP, horseradish peroxidase HRP)
• 3.2 Alkaline Phosphatase (AP)
• 3.3 Radioisotopes

Properties

Immunolabeling is a method for molecular marker. An immune marker used to detect biotin, radioisotopes, reporter enzymes, oligonucleotides, or fluorophores. The detection is based on the affinity of antibodies to a particular tissue characterization ( epitope) as the antigen -antibody reaction. Ideally, there is a specific and strong binding between antibody and epitope. The antibody is coupled to a detection system, which makes its presence in the product visible. Using various detection systems small amounts of epitope can already be represented reinforced. The goal is ( only), to recognize a signal of sufficient strength at the location of the epitope.

The antibody is directed against the epitope to be found is referred to as primary antibodies. The antibody should be characterized by high specificity and affinity, and show no cross- reactions with similar epitopes. In a multi- step process, the individual components of the detection system are applied to the preparation. Therefore, the IHC is a relatively time-consuming and error-prone. The result is also influenced by firming, period of fixation, embedding methods, pretreatment methods ( antigen retrieval ) of the preparations, etc. A standardization of the test procedure should therefore be undertaken. The antigen -antibody reaction is dependent on temperature, concentration, incubation time, agitation and the optimal reaction environment ( pH, salt concentrations).

In order to keep these variables as constant as possible and process the large volume of sample in the laboratory, IHC - machines of various designs were introduced. Assessment of the Immunohistochemistry always occurs in the morphological context. One obstacle to non-specific reactions and general background staining (endogenous peroxidase, endogenous biotin ) can thereby prove. Is only a weak signal detected, it can be explained by different methods (eg TSA) increase (signal amplification ).

Direct method

The antigen to be assayed ( = protein) is brought together under defined conditions with a specific antibody coupled directly with an enzyme or fluorophore such as fluorescein, rhodamine or Texas Red ( conjugated) is. The antibody (and hence enzyme ) binds to the antigen, unbound antibody is rinsed off. The enzyme, a substrate is provided in a further step, to form a dye that reacts with the enzyme. This dye is formed where the immunochemical reaction has taken place and is visible. Simply put: antigen antibody with enzyme substrate / chromogen color =

For fluorochrome- labeled antibodies, the detection is performed directly in the fluorescence microscope. The direct immunofluorescence (DIF ) is also well suited to multiple views of different antigens in a preparation, antibodies of different specificity with fluorochromes of different emission wavelengths are conjugated. The DIF is the oldest immunohistochemical technique and was first applied in the 50s.

Indirect method

With this as an indirect immunofluorescence (IIF ) technique referred to in the first step, a specific antibody ( primary antibody ) to be examined on the tissue / cells is applied. In a second step, an antibody is applied, which is directed against the first antibody. It is the so-called secondary antibodies, which is here coupled to an enzyme, and the color development initiates with an enzyme -substrate reaction. Re- creates a visible dye.

Simple: antigen primary 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 is a further added with an enzyme -coupled antibody ( = tertiary antibody ). This binds to the secondary antibody. This step serves to signal amplification and is useful when a small amount is to be shown on epitope.

The indirect technique is also used for the detection of already bound endogenous antibodies, for example, autoantibodies such as anti- neutrophil cytoplasmic antibody (ANCA ); and containing the product to be examined patient serum autoantibodies and this is applied to the test fabric. With a positive result, the secondary antibody is (in this case typically coupled with a fluorochrome ) its binding partner.

PAP, APAAP - method

These methods have their name from the peroxidase - anti - peroxidase or alkaline phosphatase - anti - alkaline phosphatase complex here after the secondary Ab is applied. The complex consists of three molecules of enzyme and two antibodies ( from the same species as the primary Ab) directed against the enzyme. The secondary Ab acts as a bridge between primary antibodies and PAP complex. This method showed higher sensitivity and lower background staining than the previous methods and was introduced as a "kit" for routine use in the 80s in the labs.

Labelled ( strept) avidin -biotin method ( LSAB )

Today, this staining method is the most widely used. The principle is based on the high affinity of streptavidin ( Streptomyces avidinii ), and avidin ( hen egg white ) for biotin. Streptavidin and avidin have four binding sites for biotin.

The sequence of reagents: Unconjugated primary biotin-labeled ( biotinylated = ) secondary antibody avidin- biotin -enzyme conjugate substrate / chromogen → color.

Polymer methods

This is the latest development, which is now increasingly moving into the labs. Here the primary AK (direct middle) or the secondary AK (indirect M. ) equipped with one or more polymer molecules ( dextran or polypeptide). These polymers are labeled with enzyme as much as possible, which in turn causes substrate and Chromogenumsetzung. Thus one achieves a stronger staining at the antigen site. The advantage is having to use biotin, which could cause as " endogenous biotin " background staining. The method is usually more sensitive and faster than LSAB. The disadvantage lies in the molecular size, which must be brought into the tissue and can lead to steric hindrance at the binding site.

Double immunolabeling

A double immunolabeling or double immunostaining refers to the visualization of two epitopes on a test sample by using two antibodies or immunoconjugates. As immunoconjugates reporter enzymes, radioactive isotopes ( short-lived α - or β - emitters), colloids of precious metals or fluorophores are usually coupled directly via heterobifunctional crosslinker to the antibodies.

The sample may be, for example, a Western blot, flow cytometry, a cell for a transmission electron microscope or a specimen stained by immunohistochemistry or immunofluorescence thin section. The double labeling is also used as part of a virological diagnosis.

Direct staining

To the double immune staining, the sample is incubated with the two antibodies directed against two different epitopes first. If the antibodies against these epitopes coupled to a respective one reporter enzyme, one speaks of a direct labeling. For an indirect marker directed against this epitope antibodies are referred to as primary, since then immunoconjugates are used.

Indirect staining

For an indirect marker on primary and immunoconjugate an additional washing step and incubation immunoconjugate are ever necessary. This takes place, signal amplification by the use of an immunoconjugate that is directed against multiple epitopes on the F ( c ) fragments of the antibodies, causing many reporter molecules are grouped in the primary antibody. In addition, the immunoconjugates can be used for all modular primary antibodies of a type which avoids coupling of each primary antibody with a reporter molecule, and thus reduces costs. If two different indirect staining ( with separate primary antibodies and immunoconjugates with reporter enzymes ) used at the same time, two primary antibodies from two different species are used to distinguish the two signals, where the immunoconjugates not cross-react. It usually different reporter enzymes are used. Likewise, however, can only be done with peroxidase conjugates a serial double immunostaining also, in which, after a first staining, the peroxidase is inactivated with dilute Natriumazidlösungen and then the unbound sodium azide is washed out.

Reporter enzymes

As a reporter enzymes such as horseradish peroxidase ( TMB, DAB, ABTS, AEC or - in the form of a chemiluminescence - with luminol ) and alkaline phosphatase ( BCIP and NBT with or with naphthol AS- MX phosphate and Fast Red TR ) are used. The different precipitating color reactions are performed serially.

Fluorophores

Fluorescent markers in multiple fluorophores are chosen so that the portions of the excitation and emission wavelengths are as far as possible apart. Characterized excessive blooming of the fluorescence is decreased in other color channels of the fluorescence detector and the fluorescence compensation necessary, which leads to loss of the signal strength, thus raising the detection threshold.

Enzymes, substrates and chromogens ( commonly used )

The different dyes differ inter alia in color and solubility. Soluble dyes are preferably used in an ELISA, while precipitating most dyes used in immunohistochemistry and Western blot.

Peroxidase (HRP, horseradish peroxidase HRP)

The peroxidase (usually horseradish peroxidase ) is offered hydrogen peroxide as substrate. The released protons oxidize the previously almost colorless chromogen to his colored end product to form water.

• ABTS
• AEC (3- amino-9 -ethylcarbazole ) forms a rose red end product
• CN ( 4-chloro- 1-naphthol ) to form a blue dye
• DAB ( 3,3 '-diaminobenzidine ) is a brown end product
• Luminol and other chemiluminescent dioxetanes react to form the following autoradiography
• TMB ( tetramethylbenzidine ) forms a blue product which is used for stopping the reaction with sulfuric acid, a stable yellow color complex forms

Alkaline phosphatase (AP )

Alkaline phosphatase available organic phosphate compounds as a substrate. AP cleaves the phosphate released and the compound reacts to form a colored end product.

• 5-bromo -4-chloro -3- indoxyl phosphate ( BCIP ) is implemented in combination with nitro blue tetrazolium (NBT ) to a purple to blue dye: The tetrazolium salt oxidizing indoxyl to indigo blue dye. Here, the tetrazolium salt is reduced to blue formazan dye.
• Naphthol AS- MX phosphate forms a red dye: Naphthol AS reacts with FastRed TR to an insoluble azo dye.
• Neufuchsin delivers a pink reaction product

Radioisotopes

Radioisotopes are coupled in the course of a molecular marker of antibody and registered for a protein purification by autoradiography or by scintillation counter.