Horseradish peroxidase chromogen

After incubation with primary and secondary antibody conjugates (see Basic Protocol or see Alternate Protocol), bound antigens are typically visualized with chromogenic substrates. The substrates 4CN, DAB/NiCl2, and TMB are commonly used with horseradish peroxidase (HRP)-based immunodetection procedures, whereas BCIP/NBT is recommended for alkaline phosphatase (AP)-based procedures (see Table B3.4.1). After incubation with primary and secondary antibodies, the membrane is placed in the appropriate substrate solution. Protein bands usually appear within a few minutes. 

Simultaneous detection of multiple antigens provides a spatial relationship between the antigens of interest and saves time, effort, and tissue specimens. In the standard immunoperoxidase technique, horseradish peroxidase is used to oxidize the colorless chromogen DAB into a brown end-product in the presence of hydrogen peroxide. When nickel chloride is included in the reaction mixture, the final reaction product is black. By... 

Clearly, a number of chromogenic systems are available and may be varied between laboratories. The horseradish peroxidase-DAB system is probably the most widely favored as the brown reaction product contrasts well against a wide range of counterstains and mountants. DAB is not only alcohol resistant but can be visualized in the electron microscope. Osmification can produce a more intense dark brown-black color and a similar effect is achieved by post-treatment with nickel sulphate or cobalt chloride but such enhancement is seldom necessary. The reaction product is relatively stable with fading occurring only after years in routine storage. 

Immunoreactive IL-1/ levels were analyzed by a mouse specific, sandwich ELISA. The immunoaffinity-purified polyclonal sheep anti-mouse IL-1 / coating antibody (S5/150799/JW, 1 pg/ml), the biotinylated, immunoaffinity-purified, polyclonal sheep anti-mouse IL-1 (3 detecting antibody (329/010800/SP, 1 500 dilution), as well as the recombinant mouse IL-1 / standard were kindly provided by Dr Stephen Poole (National Institute of Biological Standards and Controls (NIBSC), Hertfordshire, UK). Poly-horseradish peroxidase-conjugated streptavi-din (Sanquin, Amsterdam, The Netherlands) was used at 1 5000 dilution and the color was developed by using the chromogen o-phenylenediamine (Sigma). 

Immunoenzymatic staining methods utilize enzyme substrate reactions to convert colorless chromogens into colored end products. Of the enzymes used in these applications, only horseradish peroxidase and calf intestine alkaline phosphatase will be considered in some detail. Because of its low sensitivity, glucose oxidase (Aspergillus niger) is only rarely used today. 

Unquenched endogenous alkaline phosphatase activity may be seen in leucocytes, kidney, liver, bone, ovary bladder, salivary glands, placenta and gastro-intestinal tissue. Add levamisole to the alkaline phosphatase chromogen reagent or use another enzyme label such as horseradish peroxidase. Intestinal alkaline phosphatase is not quenched by the addition of levamisole. Pretreat the tissue with 0.03 N HCI. 115-121... 

Immunohistochemical staining can be direct or indirect. Direct immunohis-tochemical staining methods utilize only a primary antibody, which may be conjugated to horseradish peroxidase, biotin, alkaline phosphatase, or other chromogens. In the case of biotin-labeled primary antibodies, avidin or strep-tavidin linked to peroxidase binds to the biotin allowing detection of reactivity of the test antibody with the tissue. Indirect immunohistochemical staining methods utilize secondary, tertiary, or even quaternary antibodies, any of which may be linked either to biotin or enzyme (e.g., peroxidase). 

It should be noted that the relationship between the final signal output and concentration of the analyte (dose-response) may be one of direct or inverse proportionality, and is dependent on the specific assay format. In addition, a number of different reporter enzymes may be used (e.g., horseradish peroxidase, alkaline phosphatase, p-galactosidase), along with a number of different signaling systems (e.g., substrates that yield chromogenic or fluorescent or chemiluminescent products, activation of signaling enzymes, amplification by biotin-avidin system or polymerase chain reaction). 

Chromogenic detection of horseradish peroxidase POase is widely used in enzyme immunoassays (EIA) and many suitable chromogens (which are oxidized by the enzyme in the presence of the peroxide or urea peroxide substrates) have been developed. Peroxide is the usual substrate, particularly on solid phases since its reduction results in the formation of inert water near the solid phase (Fig. 7.9). It should be realized that POase has a very pronounced optimum concentration of H2O2 substrate (Tijssen et al., 1982). Activity is low at low substrate concentrations, but inhibition is considerable at high substrate concentrations. The universally used POase, C isozyme, has an optimum in solution of 0.003% peroxide but higher concentrations are usually required on a solid phase. 

Fig. 3. Microtiter plate colorimetric assay format. Specific probe is bound to the welts of the plate and hybridizes to the amplified, biotinylated DNA target. Unbound primers are removed by washing and the DNA is detected with avidin-horseradish peroxidase and a chromogenic substrate. Reprinted with the permission of Herman et al. (H5) and The American Association of Clinical Chemistry.

Bos E, van der DoelanA, van RooyN, Schuurs A (1981) 3,3 ,5,5 -tetramethylbenzidine as an Ames test negative chromogen for horseradish peroxidase in enzyme-immunoassay. J Immunoassay 2 187-204... 

Fig. 6.4 Enzyme labeling. Horseradish peroxidase (HRP) is attached to an antibody and serves as an enzyme label. HRP changes a colorless chromogen (DAB) into a brown substance seen in the microscope. The catalyst for the HRP reaction is hydrogen peroxide (H2O2) that is converted to water (H2O)...

Kuhlmann WD, Peschke P (1986) Glucose oxidase as label in histological immunoassays with enzyme-amplification in a two-step technique coimmobilized horseradish peroxidase as secondary system enzyme for chromogen oxidation. Histochemistry 85 13-17. 

Horseradish peroxidase (HRP) - the enzyme most commonly used to label antibodies its reachon product, called a chromogen, is seen in bright-field microscopes. 

A protein-free filtrate of a 0.5-mL sample of whole blood, serum, or plasma is prepared by precipitating proteins with zinc hydroxide. The glucose in an aliquot of this is reacted with a mixture of the enzymes glucose oxidase and horseradish peroxidase, and the hydrogen peroxide produced is coupled with the chromogenic hydrogen donor o-dianisidine to form a color with absorption maximum at 540 nm. 

Immunoassay. All samples were analyzed for trlazlne herbicides using RES-I-MUNE Immunoassay kits (ImmunoSystems Inc., Scarborough, Maine). The kits use polyclonal antibodies coated on the walls of polystyrene test tubes and an atrazine-enzyme conjugate prepared by covalently binding atrazine to horseradish peroxidase by a modified carbodilmide technique (12-13. Other reagents in the immunoassay kits include three standards (negative control, 0.1 ug/L atrazine solution and 1,0 ug/L atrazine solution), substrate, chromogen, and a "stop" solution of 2.5 N (normal) sulfuric acid. 

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