Antibody labeling particulates

Fig. 31 (A) Principle of a sandwich immunoassay using FDA particulate labels. The analyte is first immobilized by the capture antibody preadsorbed on the solid phase (a) and then exposed to antibody-coated microparticle labels (b). Every microparticle contains 108 FDA molecules. High signal amplification is achieved after solubilisation, release, and conversion of the precursor FDA into fluorescein molecules by the addition of DMSO and NaOH (c). (B) Calibration curves of IgG-FDA microcrystal labels with increasing surface coverage of detector antibody (a-d) compared with direct FITC-labeled detector antibody (e). The fluorescence signals increase with increasing IgG concentration. FDA microcrystals with a high IgG surface coverage (c,d) perform better than those with lower surface coverage (a,b). (Reprinted with permission from [189]. Copyright 2002 American Chemical Society)...

Antibody molecules cannot be seen with the light microscope unless they are labeled. A variety of labels have been used including fluorescent compounds and their active enzymes, all with the property of inducing the formation of a colored reaction product from a suitable substrate system to allow visualization. Some of these systems can be employed in electron microscopy by rendering the reaction product electron dense through appropriate treatment. Alternatively, particulate labels such as gold, ferritin, or viral particles can be used (Leong 1993). 

Particulate solid phases have originally been used for the separation of radiolabeled antibody-antigen complex from free labeled antibody in immunometric assays (Wide and Porath, 1966 Woodhead et al., 1974). 

The rapid expansion of lectin-based applications for the detection and quantification of glycoconjugates has been led by the development of commercially available, purified and chemically derivatized lectins, and in some cases, anti-lectin antibodies. Over 50 purified plant lectins are sold commercially by a number of producers and vendors, with this number growing annually. Equally important is the ease by which investigators can obtain lectins labeled with various fluorescent dyes, haptenic moieties, biotin, and radioactive atoms, as well as conjugated to enzymes and solid-phase supports. These derivatized lectins are useful for either direct or indirect detection and quantification techniques, or for the physical separation of particulate-bound or soluble glycoconjugates. Table 4 lists many of the commercially available lectin reagents and sources. 

All antibody solutions should be clear and free of particles or other precipitated material essential to eliminating background labeling in immunocytochemistry. IgG purification removes any particulate material from the whole serum, supernatant, or ascites fluid that could cause background. 

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. 

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... 

Lanthanide chelate-dyed polystyrene particles containing carboxylic acid groups have been covalently coated with antibodies, streptavidin [95], and nucleic acids [96] for different biomolecular-binding assays. Particulate labels can be coated with antibodies and used as such, but the obvious steric and kinetic problems associated with the large molecular size can be particularly avoided by indirect detection of the bound antibodies using, e.g., biotin-streptavidin interaction [97,... 

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