Unlabeled antibody-enzyme

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... 

Unlabeled antibody-enzyme methods Covalent label-... 

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. 

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. 

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... 

Fig. 21. Principles of ELISA. A In a direct ELISA the unlabeled antigen is attached to the solid phase. Enzyme-conjugated antibody is then added, followed by the enzyme substrate solution and color is allowed to develop. B ELISA with unlabeled antibody attached to the solid support. A variable amount of antigen is then added. A secondary antibody labeled with enzyme, followed by substrate solution, is added to all wells. The amount of color produced is proportional to the amount of antigen present. C Sandwich ELISA assay with the antigen sandwiched between an immobilized, antigen-specific primary antibody and an antigen- or species-specific secondary antibody. An enzyme-labeled tertiary antibody increases the assay specificity and sensitivity...

Noncompetitive ELISA. The usual principle here is the sandwich technique, which requires the antigen to have at least two antibody binding sites (epitopes). Unlabelled antibody is first fixed to microtitre plates a food sample containing antigen (analyte) is then added and allowed to react with the fixed unlabelled antibody (Figure 8.3). Unadsorbed material is washed out and enzyme-labelled antibody then added which reacts with a second site on the bound antigen. Unadsorbed Ab-E is washed off and enzyme activity assayed activity is directly related to the concentration of antigen. 

A disadvantage of both competitive assays is that the enzyme conjugate solution has to be mixed with the sample solution. A sample solution, however, may contain inhibitory substances such as proteases that can alter the activity of the antibody and/or the enzyme-label used. Where enzyme-labeled antibodies are employed, such problems may be circumvented by using an unlabeled antibody in the competition phase, followed by incubation with an enzyme-labeled second antibody that is directed against the primary antibodies (109). 

Noncovalent binding of HRP to antibody, also known as unlabeled antibody binding, is described in great detail by Sternberger (1). Instead of the use of bifunctional reagents, IgG class antibodies to HRP are used to form a soluble semicyclic immune complex consisting of two antibody and three enzyme molecules. The molecular weight of the peroxidase antiperoxidase, PAP complex is 400 - 430 kDa. 

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. 

---