Labeling immuno

ImmunO lSS iy. Chemiluminescence compounds (eg, acridinium esters and sulfonamides, isoluminol), luciferases (eg, firefly, marine bacterial, Benilla and Varela luciferase), photoproteins (eg, aequorin, Benilld), and components of bioluminescence reactions have been tested as replacements for radioactive labels in both competitive and sandwich-type immunoassays. Acridinium ester labels are used extensively in routine clinical immunoassay analysis designed to detect a wide range of hormones, cancer markers, specific antibodies, specific proteins, and therapeutic dmgs. An acridinium ester label produces a flash of light when it reacts with an alkaline solution of hydrogen peroxide. The detection limit for the label is 0.5 amol. 

Based on IgG-bearing beads, a chemiluminescent immuno-biochip has been also realized for the model detection of human IgG. Biotin-labeled antihuman IgG were used in a competitive assay, in conjunction with peroxidase labelled streptavidin59. In that case, the planar glassy carbon electrode served only as a support for the sensing layer since the light signal came from the biocatalytic activity of horseradish peroxidase. Free antigen could then be detected with a detection limit of 25 pg (108 molecules) and up to 15 ng. 

Thakur, M.L., and DeFulvio, J.D. (1991) Technetium-99m labeled monoclonal antibodies for immuno-scintigraphy. J. Immunol. Metb. 137, 217-224. 

Gonadotropes Gonadotropes Dual immuno-labeling Childs et al. 2001... 

Gonadotropes Gonadotropes Dual immuno-labeling Sanchez-Criado et al. 2005... 

Antibodies against sugars (carbohydrate residues) can be difficult to obtain and lectins are a solution to these problems. Lectins are naturally occurring plant and animal proteins or glycoproteins that selectively bind noncovalently to carbohydrate residues. Lectins can be labeled directly or secondary antibodies against lectins enables the use of other immuno techniques (30) including electron microscopy (31). 

Verkleij AJ, Leunissen JLM. Immuno-Gold Labeling in Cell Biology, CRC Press, Boca Raton, FL, 1989. 

Enzyme-linked immunosorbent assay (ELISA) is comparable to the immuno-radiometric assay except that an enzyme tag is attached to the antibody instead of a radioactive label. ELISAs have the advantage of nonradioactive materials and produce an end product that can be assessed with a spectrophotometer. The molecule of interest is bound to the enzyme-labeled antibody, and the excess antibody is removed for immunoradiometric assays. After excess antibody has been removed or the second antibody containing the enzyme has been added (two-site assay), the substrate and cofactors necessary are added in order to visualize and record enzyme activity. The level of molecule of interest present is directly related to the level of enzymatic activity. The sensitivity of the ELISAs can be enhanced by increasing the incubation time for producing substrate. 

Note Altematively, uranyl acetate staining may be carried out after immuno-labeling. 

Stirling, J. (1990) Immuno- and affinity probes for electron microscopy a review of labeling and preparation techniques. J. Histochem. Cytochem. 38, 145-157. 

In this method, the primary antibodies are combined into a single cocktail and applied simultaneously in a single-reaction step. The same is done for the labeled secondary antibodies. Antibodies of different animal species or immunoglobulin isotypes (noncrossreactive) or autologous antibodies that are directly coupled to different enzymes/fluorochromes are required. Immuno-fluorescent methods are particularly suited for this method. 

Goode, D. and Mangel, T. K. (1987) Backscattered electron imaging of immuno-gold labeled and silver-enhanced microtubules in cultured mammalian cells. 

Horisberger, M. (1989) Quantitative aspects of labeling colloidal gold with proteins, in Immuno-gold Labeling in Cell Biology (Verkleij, A. J. and Leunissen, J. L. M., eds.), CRC, Boca Raton, FL, pp. 49-60. 

Slot, J. W. and Geuze, H. J. (1984) Gold markers for single and double immuno-labeling of ultrathin cryosections, in Immunolabelling for Electron Microscopy (Polak, J. M. and Vamdell, I. M., eds.), Elsevier, New York, pp. 129-142. 

Additionally, two studies have measured colorectal epithelial cell proliferation and apoptosis in human non-neoplastic mucosa in combination with serum bile acid quantification. Ochsenkuhn et al have reported a positive correlation between serum DCA levels and proliferation measured by flow cytometric cell cycle analysis. However, a more recent study of colorectal adenoma patients failed to detect a correlation between serum DCA and immuno-histochemical Ki-67 antigen labelling. Instead, this latter study revealed a positive correlation between serum DCA and the degree of TUNEL-positive epithelial cell apoptosis. ... 

---