Unlabeled antibody method

The sequential application of the primary antibody, the excess of the linking antibody and the anti-enzyme antiserum has the problem that the linking antibody will also adsorb non-specific Ig and that only a fraction of the complexes will be able to react with the enzyme in the last step (low detectability). 

Specific antibodies to the enzyme may be purified with immunosorbents (Section 7.1.8.2) and should, theoretically, give better results (Stemberger, 1969). However, this is not the case with POase. Only antibodies with low affinity will be obtained which will not retain efficiently the enzyme in the EIA. It has been estimated (Stemberger, 1979) that about 75% of the POase is lost from low-affinity antibodies during washing. Consequently, this method cannot be strongly recommended for POase. 

In contrast, this method is suitable for APase (Mason and Sammons, 1978). Antibodies to bovine intestinal APase (type VII from Sigma, or type I purified according to Section 10.2.3.3) were produced in the rabbit (Sections 5.3.3.1 and 5.3.4). The enzyme (2.5 mg) was coupled to CNBr-activated Sepharose (0.25 ml Section 7.1.8.2.2) and the specific anti-APase antibodies from 0.5 ml aliquots of rabbit antiserum (or Ig preparation) were retained on such minicolumns which were eluted subsequently with 100 mM glycine-HCl buffer, pH 2.5. The total yield over 7 adsorption/elution cycles is about 1-1.5 mg of antibody per ml of antiserum. In the test, APase and the specific anti-enzyme antibody could then be added simultaneously with the linking antibody (Mason and Sammons, 1978). The specificity of the purified antibody made it also possible to use a crude preparation of the enzyme without any decrease in detectability. Nevertheless, this method is quite involved for routine applications of EIA and is primarily used in EIH, such as in the simultaneous immunoenzymatic detection of two different antigens or epitopes (Section 17.3.2.3). 

Most of these problems can be circumvented with monoclonal antibodies. This very promising approach provides the means for  

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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. 

Sternberger LA, Joseph SA. The unlabeled antibody method Contrasting color staining of paired pituitary hormones without antibody removal. J Histochem Cytochem. 1979 27 1424. 

Sternberger LA, Sternberger NH (1986) The unlabeled antibody method comparison of peroxidase-antiperoxidase with avidin-biotin complex by a new method of quantification. J Histochem Cytochem 34 599-605... 

Correspondingly, two principal immunohistochemistry methods are employed direct and indirect. The direct method is a straightforward one-step process that creates a direct reaction between the antigen and the labeled antibody. The indirect method requires the use of two antibodies a primary unlabeled antibody and a secondary labeled antibody. 

Whereas multicolor immunoenzyme staining is applicable only for separately located antigens (see Chap. 7), multicolor fluorescence immmunostaining makes it possible to colocalize antigens not only in the same cell but also in the same cellular compartment. Simultaneous immunolocalization of antigens using fluorescent antibodies can be fulfilled both by the direct (see Sect. 4.1) and indirect (see Sect. 4.2) methods. With the direct method, primary antibodies are labeled with fluorescent dyes, while with the indirect method, primary antibodies are applied as unlabeled antibodies and the visualization is performed with secondary antibodies that are labeled with fluorescent dyes. 

The use of specific antibodies labeled with a fluorescent dye to localize substances in tissues was first devised by A. H. Coons and his associates. At first, the specific antibody itself was labeled and applied to the tissue section to identify the antigenic sites (direct method) (1). Later, the more sensitive and versatile indirect method (2) was introduced. The primary, unlabeled, antibody is applied to the tissue section, and the excess is washed off with buffer. A second, labeled antibody from another species, raised against the IgG of the animal donating the first antibody, is then applied. The primary antigenic site is thus revealed. A major advantage of the indirect method is the enhanced sensitivity. In addition, a labeled secondary antibody can be used to locate any number of primary antibodies raised in the same animal species without the necessity of labeling each primary antibody. 

Hsu, S. M., Cossman, J. C., and Jaffe, E. S. (1983) A comparison of ABC, unlabeled antibody and conjugated antibody methods with monoclonal and polyclonal antibodies—an examination of germinal center of tonsils. Am. J. Clin. Pathol. 80, 429 35. 

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... 

Multi-step technique (3) This is an indirect/direct method combining unlabeled primary antibodies with directly-conjugated antibodies. The method starts with staining the unlabeled antibody/antibodies with the appropriate detection system, but without performing the final enzymatic staining reaction. The tissue is blocked with normal serum from the host of the first primary antibody before the second, directly-labeled primary antibody is added. The staining ends with the two enzymatic reactions being performed sequentially. 

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. 

Heterogenous fluorescence immunoassays can be carried out with the aid of the same separation procedures used in radioimmunoassay. The more expedient homogenous fluorescence immunoassays require quenching, enhancement, polarization, or shifting of the fluorescence of the label upon binding of the labeled analyte to its antibody. Occasionally, a second antibody, directed at the anti analyte antibody, will be used in a double-antibody method to precipitate the bound labeled and unlabeled analyte or to alter the optical properties of the label in such a way as to make the analysis more sensitive. 

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