Peroxidase label

Figure 2-10. Fully optimized ONIOM QM MM transition states for the enzymatic reaction in glutathione peroxidase. Labels (TS-II-III and TS-III-IV) correspond to labels in the potential energy diagram in Fig. 3-7. Numbers show important bond distances in A. (Adapted from Prabhakar et al. [28]. Reprinted with permission. Copyright 2006 American Chemical Society.)...

Taylor CR, Burns J. The demonstration of plasma cells and other immunoglobulin containing cells in formalin-fixed, paraffin-embedded tissues using peroxidase labelled antibody. J. Clin. Pathol. 1974 27 14-20. 

Figure 8.1 The results of IHC of two experiments using Dynabeads (Dynal, New York, NY) coated with biotinylated anti-mouse IgG (first experiment) and protein S-100 (second experiment), (a) Positive control showing red color (S-100) localized in the melanoma cells, (b) Strong positive red color circles all beads coated with biotinylated anti-mouse antibody after the heating AR treatment (first experiment), (c) Using the heating AR treatment, S-100-coated polymer beads show positive red color around the beads as circles (second experiment), (d) Negative control of the first experiment. No red color could be seen for polymer beads (arrows) that had been treated with exactly the same protocol as that of slide (b), but omitting the avidin-biotin-peroxidase (label). Bar = 50pm. Reproduced with permission from Shi et al., J. Histochem. Cytochem. 2005 53 1167-1170. See color insert.

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. 

Avrameas, S., and Ternynck, T. (1971) Peroxidase labelled antibody and Fab conjugates with enhanced intracellular penetration. Immunochemistry 8, 1175. 

Nakane, P.K. (1975) Recent progress in the peroxidase-labeled antibody method. Ann. N.Y. Acad. Sci. 254, 203. 

Nakane, P.K., and Kawaoi, A. (1974) Peroxidase-labeled antibody. A new method of conjugation. J. Histochem. Cytochem. 22, 1084-1091. 

Y. Xiao, H.X. Ju, and H.Y. Chen, Hydrogen peroxide sensor based on horseradish peroxidase-labeled Au colloids immobilized on gold electrode surface by cysteamine monolayer. Anal. Chim. Acta. 391,... 

For visualization of peroxidase label in tissue sections, Vector Laboratories offers the traditional substrates DAB and 3-amino-9-ethyl carbazole (AEC) as well as several proprietary substrates, producing colors as listed in Table 2.1. 

Immunohistochemical labeling for electron microscopy is based on the same principles as immunohistochemistry for light microscopy. The differences are that specimen sections must be much thinner (50 100 nm) and the label must be electron-dense. The first electron-dense labels used for immunolabeling at the electron microscope level were ferritin and peroxidase. Peroxidase label can be visualized using DAB reaction product which becomes electron-dense after osmi-cation. With the advent of colloidal gold particles as markers in immunocytochemical... 

Niewola et al. [183, 185] have described a rapid, convenient and accurate method, based upon an enzyme-based immunosorbent assay (ELISA) for the determination of Paraquat residues in soil. Polystyrene plates, coated with paraquat-keyhole limpet haemocyanin (KLH) conjugate, are incubated with the test samples and a known amount of monoclonal antibody. Residual antibody that has not reacted with free Paraquat in the sample combines with paraquat-KLH on the plate. The determination of the fixed antibody is achieved by the addition of peroxidase labelled rabbit antimouse immunoglobulin G followed by reaction with a chromogenic substrate. The enzyme activity of the solid phase is determined from the absorbance measurements, which are inversely proportional to the concentration of Paraquat. The method shows high specificity and correlates well with the traditional ion exchange-spectrophotometric method for the determination of Paraquat [178]. 

Then, the peroxidase-labeled streptavidin (Kirkegaard Perry) reagent is used in step 15. The labeled streptavidin is added at the appropriate dilution, which must be experimentally determined (see Note 2). It does not need to be prepared a minimum of 30 min in advance like the ABC reagent. 

Fig. 3. Diagram illustrating the amplified biotin substrate method. The biotinyi tyramide compound TSA forms biotin when oxidized by peroxidase. The deposition of biotin in the vicinity of the original ABC reagent allows for further amplification by the addition of peroxidase-labeled avidin or more ABC, followed by substrate (A, immunoglobulin , biotin A, avidin , peroxidase).

Elias, J., Margiotta, M., and Gabore, D. (1989) Sensitivity and detection efficiency of the peroxidase antiperoxidase (PAP), avidin-biotin complex (ABC), and the peroxidase-labeled avidin-biotin (LAB) methods. Am. J. Clin. Pathol. 92,62. 

Nakane, P. K. (1968) Simultaneous localization of multiple tissue antigens using the peroxidase-labeled antibody method a study on pituitary glands of the rat. J. Histochem. Cytochem. 16, 557-560. 

Detection reagent (substrate) for enzyme-labeled antibody 2,2-azino-di(3-ethyl-benzthiazohne sulfonate-6) (ABTS) 0.3 g/L in 0.15 M sodium citrate with 0.1% H2O2 for the detection of horseradish peroxidase-labeled secondary antibody or p-nitrophenyl phosphate (pNPP) 1 g/L in 1M diethanolamine in water for the detection of an alkaline phosphatase-labeled antibody (Kirkegaard and Perry Laboratories). 

Secondary antibodies are enzyme-linked antibodies directed against the primary antibody host animal s immunoglobulin (usually an IgG). If the primary antibody is mouse anticytokeratin the secondary antibody would be horseradish peroxidase-labeled or alkaline phosphatase-labeled antimouse IgG. 

EuLISA based on primary and Eu-labeled secondary antibody Based on primary and peroxidase-labeled secondary antibody EuLISA based on primary and Eu-labeled secondary antibody Peroxidase-coupled assay... 

Figure 6. Ilyphae from N-limited nutrient agar grown cultures of C. versicolor showing (a) a thick walled skeletal hypha and (b) a thin walled generative hypha labelled for lignin-peroxidase. Label is seen throughout the hypha.1 walls with little labelling in the cytoplasm. Magnification x 15,700.

Figure 8. Higher magnification of generative hyphal walls from N-limited nutrient agar grown cultures labeled for lignin-peroxidase. Label on the hyphal tip where a thickening of the wall and mucilage form a cap at the tip. Magnification x 44,000.

Ol. Olsson, T., Kostulas, V., and Link, H., Improved detection of oligoclonal IgG in cerebrospinal fluid by isoelectric focusing in agarose, double-antibody peroxidase labeling, and avidin-biotin amplification. Clin. Chem. 30/7, 1246-1249 (1984). 

Bacon, J.R. and Rhodes, M.J.C., Development of a competition assay for the evaluation of binding of human parotid salivary proteins to dietary complex phenols and tannins using a peroxidase-labeled tannin, J. Agric. Food Chem., 46, 5083, 1998. 

Sialic acids may also be specifically stained by using peroxidase-labelled Limulus polyphemus agglutinin in combination with 3,3 -diaminobiphenyl or Aleian Blue, and the procedure was tested with various, mammalian tissues by light-microscopy.199... 

Kricka, L. J., Stott, R A W., and Thorpe, G. H. G (1991) Enhanced chemiluminescent detection of horseradish peroxidase labels in ligand binder assays, in Luminescence Techniques in Chemical and Biochemical Analysis (Bayens, W R. G., De Kekeleire, D, and Korkidis, K, eds.), Dekker, New York, pp 599-635... 

Whitehead, T. P, Thorpe, G. H G, Carter, T. J. N, Groucutt, C., and Kricka, L J. (1983) Enhanced luminescence procedure for sensitive determination of peroxidase-labeled conjugates in immunoassay. Nature 305, 158,159. 

Wash and incubate the blots in peroxidase-labeled antispecies antibody at room temperature, as indicated m Table 1. 

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