Antibodies horseradish peroxidase

Imagawa, M., Yoshitake, S., Hamguchi, Y., Ishikawa, E., Niitsu, Y., Urushizaki, I., Kanazawa, R., Tachibana, S., Nakazawa, N., and Ogawa, H. (1982) Characteristics and evaluation of antibody-horseradish peroxidase conjugates prepared by using a maleimide compound, glutaraldehyde, and periodate./. Appl. Biochem. 4, 41-57. 

Key Words Antibody horseradish peroxidase conjugation labeling periodate oxidation. 

Antifluorescein antibody/horseradish peroxidase conjugate. Available from Amersham International (RPN 3022). 

Aptamer based biosensors, for example for recombinant human erythropoietin (as model analyte), can be made more sensitive by amplification with a boronic acid tethered gold nanoparticle that is then associated with an alkaline phosphatase to produce a redox active probe molecule. A similar re-usable bio-immuno-sensor has been suggested for carcinoembryonic antigen. A phenylboronic acid is assembled on gold to (reversibly) bind the antibody horseradish peroxidase conjugate. Interaction with the antigen slows down the hydrogen peroxide reduction. An HIV-1 immunoassay based on electroluminescence has been proposed by Zhou etaV In this process the... 

Secondary antibody, horseradish peroxidase-coupled secondary antibody (Boehnnger Mannheim) The appropriate dilution should be determined Usually a range of 1 in 1000 to 1 in 5000 works best. The antibody should be diluted in 0 05% Tween-20/PBS. 

The sensitivity of enzyme assays can also be exploited to detect proteins that lack catalytic activity. Enzyme-linked immunoassays (ELlSAs) use antibodies covalently finked to a reporter enzyme such as alkafine phosphatase or horseradish peroxidase, enzymes whose products are readily detected. When serum or other samples to be tested are placed in a plastic microtiter plate, the proteins adhere to the plastic surface and are immobilized. Any remaining absorbing areas of the well are then blocked by adding a nonantigenic protein such as bovine serum albumin. A solution of antibody covalently linked to a reporter enzyme is then added. The antibodies adhere to the immobilized antigen and these are themselves immobilized. Excess free antibody molecules are then removed by washing. The presence and quantity of bound antibody are then determined by adding the substrate for the reporter enzyme. 

Conventional ion-selective electrodes have been used as detectors for immunoassays. Antibody binding measurements can be made with hapten-selective electrodes such as the trimethylphenylammonium ion electrode Enzyme immunoassays in which the enzyme label catalyzes the production of a product that is detected by an ion-selective or gas-sensing electrode take advantage of the amplification effect of enzyme catalysis in order to reach lower detection limits. Systems for hepatitis B surface antigen and estradiol use horseradish peroxidase as the enzyme label and... 

Secondary antibody and determination. A secondary antibody labeled with an enzyme is added which binds to the primary antibody that is bound to the coating antigen. If the primary antibody were produced in a rabbit, an appropriate secondary antibody would be goat anti-rabbit immunoglobulin G (IgG) conjugated with horseradish peroxidase (HRP) (or another enzyme label). Excess secondary antibody is washed away. An appropriate substrate solution is added that will produce a colored or fluorescent product after enzymatic conversion. The amount of enzyme product formed is directly proportional to the amount of first antibody bound to the coating antigen on the plate and is inversely proportional to the amount of analyte in the standards. 

Immunodetection is performed by chemiluminescence (ECL , Amersham Pharmacia Biotech) using horseradish peroxidase-conjugated secondary antibodies (Amersham Pharmacia Biotech). 

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.

Table 2. Responses of a grating coupler sensor coated with multilayers of monoclonal antibodies to the respective analytes. The response values are related to that of an adsorbed antibody monolayer (B2M p2-microglobulin, HCG human choriogonadotropin, HRP horseradish peroxidase.

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. 

Due to the relatively high-molecular-weight of the enzyme, conjugates formed with antibodies and P-gal tend to be much bulkier than those associated with AP or horseradish peroxidase. For this reason, antibody conjugates made with P-gal may have more difficulty penetrating tissue structures during immunohistochemical staining techniques than those made with the other enzymes. 

Wilson, M.B., and Nakane, P.K. (1978) Recent developments in the periodate method of conjugating horseradish peroxidase (HRPO) to antibodies. In Immunofluorescence and Related Staining Techniques (W. Knapp, K. Holuber, and G. Wick, eds.), pp. 215-224. Elsevier/North-Holland Biomedical Press, Amsterdam. 

L. Alfonta, A.K. Singh, and I. Willner, Liposomes labeled with biotin and horseradish peroxidase a probe for the enhanced amplification of antigen-antibody or oligonucleotide-DNA sensing processes by the precipitation of an insoluble product on electrodes. Anal. Chem. 73, 91-102 (2001). 

Fig. 8. Schematic presentation of a enzyme linked immunosorbent assay (ELISA). An antigen ( ) is immobilized on the surface of a microtiter plate and incubated with its antibody (abl). A second antibody (ab2) with a covalently linked enzyme ( , e.g., horseradish peroxidase) binds to the primary one and catalyzes a color reaction with its enzyme. All incubations are separated by washing steps...

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. 

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