Comparison of Horseradish Peroxidase and Alkaline Phosphatase-Labelled Antibodies in Enzyme Immunoassays

Abstract

The periodate method was found to be most effective for preparing horseradish peroxidase-sheep anti-human and horseradish peroxidase-donkey anti-mouse immunoglobulin (IgG) conjugates. The conjugates were improved by carrying out the oxidation of the enzyme at low pH. Anti-human and anti-mouse IgG-peroxidase conjugates (0·5 mg/mL IgG and 0.7 mg/mL 19G, respectively) were used at 1:15 000 and 1:8000 dilutions, respectively, in a sandwich ELISA to detect human and mouse IgG in buffer or in a growth medium containing 20% foetal calf serum. Using the peroxidase conjugates, it was possible to detect human and mouse IgG at concentrations as low as I ng/mL. The glutaraldehyde method was found to be much more effective than the periodate method for conjugating alkaline phosphatase to the antibodies. The optimum dilutions for anti/human and anti-mouse IgG-alkaline phosphatase conjugates (0.18 mg/mL IgG and 0.3 mg/mL IgG, respectively) in ELISA were 1:500 and 1:1000, respectively. The detection limit with alkaline phosphatase conjugates was 7 ng/ml for human IgG and 4 ng/ml for mouse IgG.