Unlabeled antibody-enzyme methods

Unlabeled antibody-enzyme methods Covalent label- 

Detection of Epstein Barr virus nuclear antigen (EBNA) in B lymphocytes with the indirect anticomplement method (a-d, 0- Cells arrested in metaphase (a,b) revealed that EBNA is linked to chromosomes. The indirect (anti-Ig) method (e) has higher background levels due to Ig or receptors on the membrane. In (f) antiserum was omitted. Dilutions used 1 600 (a, b, c), t 5000 (d), and 1 200 (e). From Kurstak et al., 1978 courtesy Journal of Medical Virology. 

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Stemberger, L. A (1970) The unlabelled antibody-enzyme method of histochemistry Preparation and properties of soluble anigen-antibody complex (horseradish peroxidase-antihorseradish peroxidase) and its use m identification of spirochetes. J Histochem Cytochem 18,315—333... 

Original mlabeled antibody-enzyme method The original unlabeled antibody-enzyme method obviated problems encountered in conjugation, but required purified anti-POase antibodies. Fig. 17.3a shows the four sequential steps in this technique. The anti-POase antibodies should be pure, to prevent the bridging antibody to capture Ig other than anti-POase antibody. Anti-POase antibodies can be purified with specific immunosorbents (Section 7.1.9.2), but mainly antibodies with lower affinities are recovered which are easily lost during washing (Section 8.5). 

The first two steps of the PAP method are similar to those of the original unlabeled antibody-enzyme method. In the following step, PAP (40 pg/ml in buffer containing 1% normal serum from the same species as the bridging antibody) is applied to the preparation, followed by the revelation of POase. Detectability can be increased by a double bridge procedure after the last incubation step, another incubation with bridging antibody (anti-Ig) and with soluble PAP is carried out at the same concentrations as above. 

Petrali P., Hinton M., Monarty C., and Sternberger A (1974) The unlabeled antibody enzyme method of immunocytochemistry. Quantitative comparison of sensitivities with and without peroxi-dase-antiperoxidase complex. /. Htstochem Cytochem 11, 782-801 Pickel V M, Joh T H, Field P M, Becker C G, and Reis D. J (1975a) Cellular localization of tyrosine hydroxylase by immunohisto-chemistry / Histochem Cytochem 13, 1-12 Pickel V, M., Joh T H, and Reis D J (1975b) Ultrastructural localization of tyrosine hydroxylase in noradrenergic neurons of brain Proc Natl. Acad. Set. USA 71, 659-663... 

Sternberger LA, Hardy PH Jr, Cuculis JJ, Meyer HG (1970) The unlabeled antibody enzyme method of immunohistochemistry. J Histochem Cytochem 18 315-333... 

Burns J. Background staining and sensitivity of the unlabeled antibody-enzyme (PAP) method Comparison with the peroxidase-labeled antibody sandwich method using formalin-fixed paraffin embedded material. Histochemistry. 1975 43 291. 

Unlabeled antibody methods are very simple and give excellent results when monoclonal antibodies are used, otherwise soluble enzyme-anti-enzyme antibody complexes are far superior. 

Two approaches are possible in such techniques (i) the sequential addition of the antibodies and enzyme (unlabeled antibody method) and, (ii) the preparation of enzyme-anti-enzyme complexes, prior to their application, followed by their linking to the primary antibodies (Section 11.3.2) the latter approach has become very convenient with the advent of monoclonal antibodies. 

Direct and indirect immunostaining methods The direct method is a one-step staining method, and involves a labeled antibody reacting directly with the antigen in tissue sections. In this method, the primary antibody can be labeled with a fluorophore or biotin. In indirect immunostaining, the bound unlabeled primary antibody (first layer) is visualized with a secondary antibody (second layer) bearing label, such as a fluorophore, biotin or an enzyme. 

Figure 7.15 Enzyme-multiplied immunoassay (EMIT). The three reactants, test (or standard) antigen, enzyme-labelled antigen and a limited amount of antibody are allowed to react and reach an equilibrium position. The unbound labelled antigen which remains is the only source of enzyme activity, the bound enzyme being inactivated. This free enzyme can be quantitated using a direct kinetic assay method and is proportional to the amount of unlabelled antigen originally present.

For ELISA, an enzyme is linked chemically to the antibody. The labeled antibody is allowed to bind to the unlabeled antigen, under conditions where nonspecific adsorption is blocked, and any unbound antibody and other proteins are washed away. Binding is detected by a reaction that converts a colorless substrate into a colored reaction product. The color change can be read directly in the reaction tray, making data collection very easy, and ELISA also avoids the hazards of radioactivity. This makes ELISA the preferred method for most direct-binding assays (Plested et al. 2003). 

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