Horseradish peroxidase substrates, chromogens

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. 

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. 

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.

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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