Horseradish peroxidase conjugate preparation

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. 

Immunoassays for oreanophosphates. High-titer antibodies to fenitrothion (coupled through the phosphate group) were obtained of the other conjugates, those through the C6 spacer arm produced the best antibody responses. Horseradish peroxidase conjugates prepared in this manner were both enzymlcally active and able to bind to the antibodies. 

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. 

Dilute the antifluorescein/horseradish peroxidase conjugate 1000-fold in freshly prepared antibody binding buffer. The final volume should be at least equivalent to that used for hybridization. Incubate the blots in diluted conjugate with gentle agitation at room temperature for 60 min see Note 9). 

Figure 2. Screening of a pea cDNA library for CA immunopositive clones. Nitrocellulose filters prepared from 86 mm plates containing approximately 10,000 recombinant plaques were incubated with the CA antisera. Specifically bound antibody was detected with horseradish peroxidase conjugated goat anti-rabbit IgG. A positive signal on a primary plate is shown (A) as well as the tertiary stage of plaque purification (B). A control filter with no positive signals is also presented (C).

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... 

Methods based on chemiluminescent and bioluminescent labels are another area of nonisotopic immunoassays that continue to undergo active research. Most common approaches in this category are the competitive binding chemiluminescence immunoassays and the immunochemiluminometric assays. Chemiluminescence and heterogenous chemiluminescence immunoassays have been the subject of excellent reviews (91, 92). Detection in chemiluminescence immunoassays is based on either the direct monitoring of conjugated labels, such as luminol or acridinium ester, or the enzyme-mediated formation of luminescent products. Preparation of various derivatives of acridinium esters has been reported (93, 94), whereas a variety of enzyme labels including firefly or bacterial luciferase (70), horseradish peroxidase (86, 98), and alkaline phosphatase are commercially available. 

In each of these methods, undiluted serum, urine, acid-deproteinized milk, or a buffered saline extract of muscle was mixed with sulfamethazine-horseradish peroxidase and added to antibody-coated wells of a microtiter plate. A sulfamethazine-bovine serum albumin conjugate prepared by the glutaraldehyde procedure (56) was used for antibody production. Results showed that screening of serum was of value since sulfamethazine concentrations in serum directly correlated with those in swine tissues. Thus, for example, a level of 100 ppb of sulfamethazine in... 

Primary amine groups on proteins consisting of N-terminal a-amines and lysine side-chain e-amines are typically present in abundant quantities for modification or conjugation reactions. Occasionally, however, a protein or peptide will not contain sufficient amounts of available amines to allow for an efficient degree of coupling to another molecule or protein. For instance, horseradish peroxidase (HRP), a popular enzyme to employ in the preparation of antibody conjugates, only possesses two free amines that... 

One hundred microliters of commercial goat antirabbit globulin conjugated to horseradish peroxidase (diluted 1/500 diluting buffer) is added to every well. Incubation is done for 30 min at 37°C, followed by washing, and 100 pi of peroxidase substrate in freshly prepared substrate buffer is added to each well. 

An even more indirect method which does not require the use of a fluorescence microscope is to use enzyme linked antibodies, e.g. the peroxidase anti-peroxidase (PAP) method of Stemberger et al. (1970). Here, after the antigen has been reacted with a rabbit antibody preparation this is conjugated to sheep anti-rabbit IgG which in turn is conjugated to a complex of rabbit anti-horse-radish peroxidase and horseradish peroxidase. This then reacts with diaminobenzidine and hydrogen peroxide when a brown colour indicates the presence of the antigen. 

Preparations are incubated with appropriate reagents to allow visualization based upon the detection system associated with the secondary antibody. The secondary antibody may be conjugated to a enzyme (e.g., alkaline phosphatase, horseradish peroxidase). Incubation with the appropriate substrate to the enzyme will result in the production of an insoluble colored product that can be detected upon microscopic analyses of the cells. Secondary antibodies can also be conjugated to fluorochromes (e.g., fluorescein, rhodamine) that can be detected using a microscope equipped to detect fluorescence. Immunohisto-chemistry has proven to be a powerful tool in biochemical toxicology allowing for in situ assessments of protein responses to toxicant exposure. 

---