Labels for Antibodies

Keywords Immunohistochemistry Antibody labeling Fluorescence microscopy Fluorescent immunocytochemistry Fluorescent immunohistochemistry Indirect immunocytochemistry Immunostaining 

Immunocytochemistry, DOI 10.1007/978-l-4419-1304-3 6, Springer Science+Business Media, LLC 2010 

Labels are molecules that can be attached to antibodies and will localize the antibodies in cells and tissues. For bright field microscopy, enzymes are the best compounds to label antibodies. Enzymes allow amplification wifh development of reaction products for labeling. A second approach for bright field microscopy is the use of particulates, such as enhanced gold or silver. The third approach is the popular fluorescent labels. In this chapter, the different types of labels are examined. In subsequent chapters, the methods of applying labeled antibodies will be examined. 

Different types of labels or tags attached to antibodies can be divided into three types  

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Horseradish peroxidase (HRP EC 1.11.1.7) is the most widely used enzyme label for antibodies. It is relatively small (44 kDa), stable, and has a broad specificity that allows is to be measured by absorption, fluorescence, and luminescence. Several of its products are intensely colored, which makes the enzyme convenient to use for immunocytochemistry and immunoblotting applications. 

Nitrenes, the nitrogen analogs of carbenes have been introduced as a reactive group in photoaffinity labels for antibodies by Fleet et al. (1969). Nitrenes tend to be more selective in their reactions and possibly will provide fewer products than carbenes (but see also 6.2.3). The types of reaction that they can undergo (eq. 6.2, Knowles 1972) include insertions into C-H bonds to yield secondary amines, cycloadditions to double bonds to form cyclic 3-member imines and... 

A third class of labels for antibodies that can be used for bright filed microscopic immunocytochemistry is particulate labels. Antibodies can be labeled with gold... 

Designing an experiment with a single 1° antibody involves many of the topics presented in the previous chapters, including reagents, types of label for antibodies, and the methods for applying antibodies. This chapter presents a step-by-step approach to designing an immunocytochemistry experiment and depends on information in the Immunocytochemistry Experimental Design Chart completed in Chapter 9. 

Fluorescent molecules (fluorophore) - molecules able to absorb one wavelength of light and emit a higher wavelength of light used as labels for antibodies in immunocytochemistry. 

Pt- and Pd-coproporphyrin complexes may also be used as labels for antibodies to develop phosphorescence immunoassays. Also, RTP provides a sensitive tool for diagnosis of vascular disorders and image oxygen distribution in cancerous tissues (porphyrins are known to concentrate in tumors). Many other potential uses of RTP in biological systems include the study of processes such as rotational motion, distances, excited state reactions. RTP measurements have an important role in the study of biological systems and areas for future development are apparent. 

Figure 15.22 T-cell receptor stucture shown as a ribbon diagram. The anbgen-binding site is formed by CDR loops (labeled 1 to 3) from the Va and Vp domain, as for antibodies.

Sandwich-type sensors are applicable for measuring large antigens that are capable of binding two different antibodies. Such sensors utilize an antibody that binds the analyte-antigen, which then binds an enzyme-labeled second antibody. After removal of the nonspecifically adsorbed label, the probe is placed into the substrate-containing solution, and the extent of the enzymatic reaction is monitored... 

The first application of immunologically based technology to pesticides was not reported until 1970, when Centeno and Johnson developed antibodies that selectively bound malathion. A few years later, radioimmunoassays were developed for aldrin and dieldrin and for parathion. In 1972, Engvall and Perlman introduced the use of enzymes as labels for immunoassay and launched the term enzyme-linked... 

For indirect immunoassay methods, the antigen (analyte) is bound to support materials and excess binding sites are blocked. Analyte and primary antibody are then added simultaneously, followed by the addition of enzyme-labeled secondary antibody and color reagent. The bound analyte (coating antigen) and free analyte (in... 

Figure 1 Schematic sequence of the direct and indirect competitive ELISA. The principle difference is that for direct competitive immunoassay, the well is coated with primary antibody directly, and for indirect competitive immunoassay, the well is coated with antigen. Primary antibody (Y), blocking protein (Y), analyte (T), analyte-tracer ( ), enzyme labeled secondary antibody ), color development ( J)...

Tc(V) gluconate and glucoheptonate are often used when the reaction should be carried out in a neutral aqueous- or mixed aqueous/organic medium, and a rapid exchange reaction and high radiochemical purity of the product is required. In radiopharmaceutical preparations, Tc(V) tartrate is also quite often used. For labelling modified antibodies Tc(V) tricine has recently been particularly recommended [33],... 

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