Horseradish peroxidase conjugation with

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.

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. 

Enzyme-Immunoassay. Fish tissue samples for testing were cut into uniform 3mm thick slices with parallel razor blades mounted on a handle. Four discs were then punched out from each slice with a stainless steel borer, 3-mm in diameter, and each disc was placed in a well of a 96-well polystyrene microtiter plate (Flow Laboratories, Inc., Hamden, CT). Samples were washed once with 0.2 ml Tris buffer. After the wash solution was aspirated, each sample was fixed in 0.2 ml of 0.3% H O -methanol fixative for 30 min. at room temperature. Samples were then transferred to clean wells and 0.2 ml of a 1 100 dilution of sheep-anti-ciguatoxin-horseradish peroxidase conjugate in Tris buffer was added to each well. The plate was then incubated at room temperature for 1 hr. The sheep-anti-cigua-toxin-horseradish peroxidase was removed by aspiration, and the tissues were immersed for 5 min. in 0.2 ml Tris buffer. Each sample was transferred to clean wells and incubated for 5 min. at room temperature with 0.2 ml of 4-chloro-l-naphthol substrate. The final steps involved removal of the tissue and addition of 0.015 ml of 3 M sodium hydroxide to stop the enzymatic reaction. Absorbance readings at 405 nm of each well were obtained in the Titertek Multi-skan (Flow Laboratories, Inc., Hamden, CT). 

After being washed with TBST for 5 min four times, the membrane was further incubated with 1 5,000 horseradish-peroxidase-conjugated anti-rabbit IgG antibody (Promega) in TBST containing 1% skim milk for 60 min. 

E. Snyder and R. Fall, Biocbem. Educ. 18, 147-148 (1990). Western Blotting with a Concanavalin A-Horseradish Peroxidase Conjugate. ... 

In glycoprotein detection systems the carbohydrate portions of proteins are oxidized with sodium metaperiodate to generate aldehydes that can react with hydrazides. A biotin hydrazide is used to attach biotin onto the oxidized carbohydrates and horseradish peroxidase-conjugated streptavidin is used for chemiluminescence-based detection (Glycoprotein Detection Module, Amersham Biosciences, Uppsala, Sweden). 

Dilute horseradish peroxidase-conjugated rabbit anti-mouse IgG antibodies in BSA5PBST according to the manufacturer s recommendation. We typically use a V400 dilution with this particular brand of antibody conjugate (see Subheading 2.2.2.). 

After blocking the nitrocellulose transfer with 3% (w/v) bovine serum albumin (BSA) in Tris-buffeied saline (TBS), first antibody should be added at a concentration no greater than a 1 1000 dilution. Incubation with first antibody should proceed for a minimum of 1 hr at room temperature, or it can be allowed to continue overnight at 4°. After washing 3 times with TBS, horseradish peroxidase-conjugated second antibody is added (1 1000 in 3% (w/v) BSA-TBS) for 1 hr at room temperature. After 3 more washes with TBS (the final wash with 10% methanol added), 4-chloro-l-naphthol is used to visualize the bound antibodies. 

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. 

The immune complex on the membrane is visualized using appropriate procedures. Color development or chemiluminescence with alkaline phosphatase or horseradish peroxidase-conjugated secondary antibodies are most commonly used. 

Fig. 1. An immuno-slot blot fin the detection of 0 -hydro]Q thy]deoxygiianosine. Calf thymus DNA was hydrm ethylated in vitro with 6.6 mAf of hydroxyethyl-nitrosourea. Aliquots containing 300, 100, 33.3, 11.1, 3.7, 1.2, and 0 finol of O -hydroxyethyldeoxyguanosine in 3 (ig ofheat-denatured DNA (corresponding to 217-0 pmol/tnol of deoxyguanosine) were blotted onto nitrocellulose and incubated with a rabbit anti-O -hydroxyethyldeoiQ guanosine serum (NPZ-146-2,1 15,000 see ref. 4). Bound antibodies were reacted with a goat-antirabbit IgG horseradish peroxidase conjugate and detected with hydrogen peroxide and 4-chloro-l-naphth6l.

Incubate with diluted horseradish peroxidase-conjugated avidin (see Notes 5 and 6). 

An indirect enzyme-linked immunosorbent assay (ELISA) was developed for maduramicin in poultry feed. The assay utilized polyclonal anti-maduramicin antibody raised in rabbits, maduramicin monoamide with 1,6-hexane diamine-conjugated ovalbumin as the coating antigen, horseradish peroxidase conjugated goat anti-rabbit IgG and 2,2 azinobis(3-ethylbenzthiazoline) sulfonic acid (ABTS) for quantitation. Standard curves ranging from 0 to 80 ng/mL maduramicin were constructed. The assay did not cross-react with monensin, lasalocid, salinomycin, lincomycin, narasin, chlortetracycline or roxarsone. Broiler feed fortified at 4 to 7 ppm maduramicin were shown to be quantifiable by ELISA at an average recovery of 98.1%. This ELISA method for maduramicin in poultry feed is comparable to the established HPLC-F method. 

Some cell lines have endogenous alkaline phosphatase activity (especially carcinoma lines). In this case, horseradish peroxidase-conjugated goat antimouse IgG can be used (substitute in step 14 of Subheading 3.1.) with appropriate substrate (e.g., 2,2 -azino-di-[3-ethyl-benzthiazoline-6-sulfonic acid] [ABTS]). 

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