1. Dissolve 2 mg of peroxidase in 500 |L of water.
2. Add 100 |L of freshly prepared 0.1 M sodium periodate and stir the solution for 20 min at room temperature protecting it from light. (The color changes from orange to green).
3. Dialyze the modified enzyme against 1 mM sodium acetate buffer, pH 4.4 (2 L) overnight at 4°C (see Note 2).
4. Dissolve 4 mg of IgG in 500 |L of 10 mM sodium carbonate buffer, pH 9.5.
5. Adjust the pH of the dialyzed enzyme solution to 9.0-9.5 by adding 10 |L of 0.2 M sodium carbonate buffer, pH 9.5, and immediately add the IgG solution. Stir the mixture for 2 h at room temperature.
6. Add 50 |L of freshly prepared sodium borohydride solution (4 mg/ mL) and stir the mixture for 2 h at 4°C.
7. Fractionate the mixture by gel filtration on a column (approx 1.5 x 85 cm) of Sepharose CL-6B in PBS. Determine the A280 and A403 (see Note 3).
8. Pool the fractions in the first peak (both A280 and A403 peaks coincide), add BSA to give a final concentration of 5 mg/mL, and store the conjugate in aliquots at -20°C (see Notes 4 and 5).
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