Horseradish peroxidase. See

Appropriate antispecies second antibodies linked to horseradish peroxidase (see Note 2). 

FIGURE 9.13 Proposed reaction mechanism for H202 activation in horseradish peroxidase. (See the color version of this figure in Color Plates section.)... 

The working principle of the ELISA described here involves the use of LHRH peptide-coated wells to capture any anti-LHRH antibodies present in the sera of vaccinated mice. Captured antibodies are detected through the use of a horseradish peroxidase-conjugated antibody that is specific for mouse immunoglobulins and a substrate is then added to induce a color change. In this case, the substrate utilized is 2,2 -azino-bis 3-ethylbenzthiazoline-sulfonic acid (ABTS Sigma-Aldrich, USA), which is converted to a green soluble end product by horseradish peroxidase (see Note 7). The level of substrate-induced... 

Western blotting is similar to IFE, except that the separated proteins are blotted onto an overlaying strip of nitrocellulose or a nylon membrane by diffusion or electroblotting. The strip or membrane is then reacted with an antibody raised against the protein of interest and labeled with a radioisotope (e.g., T) or enzyme (e.g., horseradish peroxidase). (See Chapter 5 for further details and applications of this technique.)... 

Iron(III) porphyrins with imidazole anion(s) as ligands have been synthesized in non-aqueous media"" , and are potential models for heme-containing enzymatic species. Metalloporphyrin vr-cation radicals generated electrochemically (but never from iron porphyrins) are models for compound I or horseradish peroxidase " (see 14.8.5.3.2). 

Figure 15.9 The results of capture ELISA on native RNase A and formalin-treated RNase A. Right panel, native RNase A (curve 1) and unfractionated formalin-treated RNase A (curve 2). Left panel, individual fractions of formalin-treated RNase A monomer (curve 3), dimmer (curve 4), trimer (curve 5), tetramer (curve 6), and a mixture of oligomers with >5 cross-linked proteins (curve 7). The ELISA plate wells were coated with monoclonal antibody against bovine pancreatic RNase A (1 pg/mL) overnight at 4°C and then blocked with bovine serum albumin. The wells were incubated for lh at 37°C in the presence of various concentrations of antigen in lOOpL of PBS. After washing, each plate well received a 1 4000 dilution of horseradish peroxidase conjugated rabbit polyclonal anti-RNase A antibody followed by incubation at ambient temperature for lh. After washing, detection was achieved using a mixture of 2,2,-azino-di-(3-ethylbenzthiazoline-6-sulphonate) and hydrogen peroxide. Absorbance was monitored at 405 nm. See Rait etal.11 for details.

The major enzymes used in ELISA technology include horseradish peroxidase (HRP), alkaline phosphatase (AP), (3-galactosidase (P-gal), and glucose oxidase (GO). See Chapter 26 for a detailed description of enzyme properties and activities. HRP is by far the most popular enzyme used in antibody-enzyme conjugates. One survey of enzyme use stated that HRP is incorporated in about 80 percent of all antibody conjugates, most of them utilized in diagnostic assay systems. 

Enzyme labels are usually coupled to secondary antibodies or to (strept)avidin. The latter is used for detection of biotinylated primary or secondary antibodies in ABC methods (see Sect. 6.2.1). Enzyme labels routinely used in immunohisto-chemistry are horseradish peroxidase (HRP) and calf intestinal alkaline phosphatase (AP). Glucose oxidase from Aspergillus niger and E. coli (3-galactosidase are only rarely applied. 

The technique described here is for use with monoclonal primary antibodies of mouse origin, but can easily be adapted for use with polyclonal antibodies from other species (i.e., rabbit). This method uses a secondary biotin-labeled antibody and a detection system that employs a biotin-avidin horseradish peroxidase complex linker step, the so-called ABC (avidin-biotin complex) detection system (5) (see Chapter 25). In this detection system, avidin acts as a bridge between the biotinylated secondary antibody and a biotin-labeled peroxidase enzyme. The anchored enzyme, in the presence of H2O2 can then convert the substrate, diaminobenzidine, to a brown or black reaction product that is easily identifiable in the tissue section. 

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. 

The two most common enzyme labels for secondary antibodies are alkaline phosphatase (AP) used in conjunction with the substrate p-nitrophenyl phosphate, which results in a yellow reaction product, and horseradish peroxidase (HRP) used in conjunction with the substrate ABTS and H2O2, which results in blue-green reaction product see Chapter 23). 

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