Colloidal gold antibody labeling

It has been reported that some commercial preparations of colloidal gold-antibody complexes may contain free active antibody. Such free antibody will compete with antibody-colloidal gold particles for antigen binding sites and may reduce labeling intensity. The presence of free protein may be identified using a simple test procedure (20)... 

Colloidal gold used as a label is in sizes from 5 to 50 nm. Colloidal gold is easy to identify on sections in the electron microscope because it is uniform in size, uniformly dense, and spherical (Fig. 15.3b). Most commonly, colloidal gold is labeled with protein A that binds the Fc receptor of the primary antibody without the need for a secondary antibody (Ghitescu et al., 1991). 

Antibody O Analyte Colloidal gold particle Labeled antibody... 

One particularly novel carrier was reported to consist of 50-70 nm colloidal gold particles of the type often used in cytochemical labeling techniques for microscopy (Pow and Crook, 1993) (Chapter 24). Adsorption of peptide antigens onto gold and subsequent injection of the complex into rabbits in an adjuvant mixture resulted in rapid production of antibody of extremely high titer. The resultant antibodies could be used in immunocytochemistry at dilutions from l-in-250,000 down to l-in-1,000,000, which is orders-of-magnitude beyond the dilutions typically used with lower-titer antibodies. 

Colloidal gold-labeled (strept)avidin can be used as highly sensitive detection reagents for microscopy techniques (Cubie and Norval, 1989) (Chapter 24). Finally, cytotoxic substances coupled to (strept)avidin can be used to direct cell-killing activity toward a tumor-cell-bound, biotinylated monoclonal antibody (or other targeting molecule) for cancer therapy (Hashimoto et al, 1984) (Chapter 21). 

Jemmerson, R., and Agre, M. (1987) Monoclonal antibodies to different epitopes on a cellsurface enzyme, human placental alkaline phosphatase, effect different patterns of labeling with protein A-colloidal gold. J. Histochem. Cytochem. 35, 1277-1284. 

Labeling antibodies with colloidal gold was developed for electron microscopy, but the procedure can be used for light microscopy as well. 

Visualizing more than one epitope on one section can be accomplished by different fluorescence labeling or different sizes of colloidal gold coupled to primary or secondary antibodies. Primary antibodies from different species and adequate secondary antibodies labeled differently can be used. In case of primary antibodies from the same species, the hapten technique can be applied. A hapten is a small molecule that can be bound to antibodies dinitrophenol and arsinilate are typically used as haptens. Again, adequate secondary antibodies labeled differently can be used (14,17,32). A collection of protocols for multiple immu-nolabeling has been described by Beesley (37). 

Brown-rot fungus (isolate MAD-698) was grown on sterile 12 mm glass slips placed on the surface of 2% (w/v) malt agar removed when about 50% covered were localized using colloidal gold labeled monoclonal antibodies to the B-l, 4-xylanase fraction of P. placenta-, enzymes were localized on the hyphal surface... 

Romano EL, Romano M. Staphyloccal protein A bound to colloidal gold a useful reagent to label antigen-antibody sites for electron microscopy. Immunochemistry 1977 14 711-715. 

The devices usually contain antibody immobilized on a membrane surface above a filter and absorbent pad (Figure 7.16). The sample is placed in the device and passes through the membrane into the absorbent pad. Where analyte is present in the sample it is sequestered by the antibody on the membrane and is then visualized by addition of a labelled second antibody. Labelled second antibody not bound to analyte also passes into the absorbent pad, sometimes with the aid of a wash solution. The labelled molecule may be an enzyme, which would then require addition of a suitable substrate, or a label such as colloidal gold, which has the virtue of being visible without the aid of a second reagent. 

A report of a particle-based FPIA<56) details improvement in the limit of detection by two orders of magnitude over a conventional FPIA format for rabbit IgG, to 10 ° M. Goat anti-rabbit capture antibody (binder) is immobilized on polystyrene microspheres or colloidal gold particles, and the sample competes with a fluorescein-labeled rabbit IgG probe. The observed increase in sensitivity is apparently due to the large increase in effective mass of the binding complex. The polarization range is rather limited, though, reportedly due to low concentrations of immobilized antibodies. 

Apply goat antimouse or antirabbit secondary antibody labeled either with colloidal gold or with horseradish peroxidase for 30 min (see Notes 5 and 11). Wash for 2 X 2 min in TBS containing 0.1% Tween-20. 

Apply appropriate, labeled secondary antibodies together (e.g., goat antimouse and antirabbit immnnoglobnlins), labeled with either colloidal gold, horseradish peroxidase, or alkaline phosphatase, respectively. Alternatively, use secondary antibodies labeled with separate fluorescent dyes. Incubate paraffin sections for 1-2 h and frozen sections for less than 30 min. 

Bienz, K, Egger, D, and Pasamontes, L (1986) Electron microscopic lmmuno-cytochemistry. silver enhancement of colloidal gold marker allows double labeling with the same primary antibody J Histochem Cytochem 34,1337-1342. 

The sections are treated with 10% BSA in PBS (pH 7.2) for 4 hr to block nonspecific labeling. Incubation is carried out overnight at 4°C in the primary antibody, appropriately diluted in PBS. This is followed by washing three times for 5 min each in PBS and incubation for Dhr at 22°C in colloidal gold (15 nm)-conjugated secondary antibody, appropriately diluted in PBS containing 3% BSA. The sections are poststained with 5% uranyl... 

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