Horseradish peroxidase Conjugates

Jansen H., Iwamoto G. and Jackson G. (1998). Central connections of the ovine olfactory bulb formation identified using wheat germ agglutinin-conjugated horseradish peroxidase. Brain Res Bull 45, 27-39. 

Behzadi, G., Kalen, P., Parvopassu, R Wiklund, L. (1990). Afferents to the median raphe nucleus of the rat retrograde cholera toxin and wheat germ conjugated horseradish peroxidase tracing, and selective D-[3H]aspartate labelling of possible excitatory amino acid inputs. Neuroscience 37, 77-100. 

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. 

Secondary antibody conjugate horseradish peroxidase (HRP)- or alkaline... 

Chemiluminescent detection is also used, mainly to detect antigen capture on antibody arrays by sequential incubations with biotinylated secondary antibodies and Streptavidin-conjugated horseradish peroxidase (HRP) [61]. Phosphorylation of arrayed kinase substrates in the presence of radiolabeled adenosine triphosphate (ATP) has been detected by autoradiography [62]. 

Electrophoresis and immunolo gv Proteins were resolved by SDS-PAGE on 7.5-15% polyacrylamide gradient gels. For Western blots proteins were transferred to nitrocellulose paper and stained immunologically with antibodies to Fac polypeptides and Protein A-conjugated horseradish peroxidase. 

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). 

Burns J, Hambridge M, Taylor CR. Intracellular immunoglobulins. A comparative study of three standard tissue processing methods using horseradish peroxidase and fluorochrome conjugates. J. Clin. Pathol. 1974 27 548-557. 

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. 

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. 

Reduction of the cystamine-labeled oligo using a disulfide reducing agent releases 2-mer-captoethylamine and creates a thiol group for conjugation (Figure 27.6). DNA probes labeled in this manner have been successfully coupled with SPDP-activated alkaline phosphatase (Chapter 26, Sections 1.2 and 2.5), maleimide-activated horseradish peroxidase (HRP) (Chapter 26, Section 1.1), NHS-LC-biotin (Chapter 11, Section 1 and Chapter 27, Section 2.3), and the fluorescent tag AMCA-HPDP (Chapter 9, Section 3 and Chapter 27, Section 2.5). 

Boorsma, D.M., and Kalsbeek, G.L. (1976) A comparative study of horseradish peroxidase conjugates prepared with a one-step and a two-step method. Histochem. Cytochem. 23, 200-207. 

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. 

Mannik, M., and Downey, W. (1973) Studies on the conjugation of horseradish peroxidase to Fab fragments./. Immunol. Meth. 3, 233. 

Yoshitake, S., Imagawa, M., and Ishikawa, E. (1982b) Efficient preparation of rabbit Fab -horseradish peroxidase conjugates using maleimide compounds and its use for enzyme Immunoassay. Anal. Lett. 15(B2), 147-160. 

The form of the enzyme with the greatest oxidation potential is known as horseradish peroxidase, compound 1 (HRP-I), which consists of a radical cation stabilized throughout the highly conjugated protoporphyrin IX ring system. In the presence of vindoline, HRP-I is reduced to HRP-H, an Fe(IV) form of the enzyme. The vindoline cation radical 55 thus formed eliminates a second elec-... 

A very interesting approach was presented recently by Niemeyer et al. [113]. They prepared covalent DNA-streptavidin conjugates to which biotinylated alkaline phosphatase, beta-galactosidase, and horseradish peroxidase, as well as biotinylated anti-mouse and anti-rabbit immunoglobulins, were coupled. Immobilization of DNA-streptavidin conjugates was performed by hybridization with the complementary oligonucleotides, bound to the surface. It was demonstrated... 

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