Peroxidase conjugation

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.

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. 

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. 

Avrameas, S. and Guilbert, B. (1972) Enzyme-immunoassay for the measurement of antigens using peroxidase conjugates. Biochemie 54, 837. 

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). 

Peroxidase-conjugated anti-GST antibody (GE Healthcare Bio-Sciences, Chalfont St. Giles, US) Store at 4°C. 

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. 

Incubate with the peroxidase-conjugated secondary antibody (30 min)... 

Goat anti-rabbit horseradish-peroxidase-conjugated antibody (Dianova, Hamburg, Germany). 

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

Dilute rabbit anti-IgG-peroxidase conjugate 1 5000, using 4 pL of antibody in 20 mL of dilution buffer, approx 5 min before use, and leave it in ice (see Note 9). 

Add 50 pL of diluted antirabbit IgG-peroxidase conjugate (from step 15) into each well, and then seal the plates, and incubate at room temperature for another 90 min with continuous shaking... 

Peroxidase-conjugated anti-AFP (Dako cat. no. P128) diluted 1/1000 in assay diluent. Make up fresh for each assay batch. 

Dry around the sections again and apply rabbit antirat IgG peroxidase conjugate diluted 1 100 in PBS and incubate as in step 6. 

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