Original unlabeled antibody-enzyme method

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 method is performed at room temperature with incubation periods of 1 h each, though longer periods are sometimes used for the primary antibody and shorter for the subsequent steps. After each incubation, 3 washings of 5 min with PBS should be carried 

Detectability with this technique can be increased through the use of a double bridge. After the anti-POase step, another incubation with bridging antibody can be introduced, followed by incubations with anti-POase and POase, at the same concentrations as above (Fig. 17.3). 

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. 

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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.

The choice for the multiple immunoenzymatic staining method is determined by several parameters (i) whether or not the same enzyme should be used for both staining procedures (ii) if unrelated enzymes are used, the selection of the second enzyme, in addition to the generally used POase (iii) the design of the two systems (direct conjugation, unlabeled method, bridge method, etc.) to avoid interference between them and, (iv) the species origin of the various antibodies. 

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