Periodate-Oxidized HRP to Antibodies by Reductive Amination

Conjugation of Periodate-Oxidized HRP to Antibodies by Reductive Amination 

The following protocol assumes that HRP has already been periodate-oxidized by the method of Section 1.3, above. 

Dissolve the IgG to be conjugated at a concentration of 10 mg/ml in 0.2 M sodium bicarbonate, pH 9.6, at room temperature. The high-pH buffer will 

for 2 h at room temperature prior to use. A volume of HRP solution equal to the volume of antibody solution will be required. 

Mix the antibody solution with the enzyme solution at a ratio of 1 1 (v/v). Since an equal mass of antibody and enzyme is present in the final solution, this will result in a 3.75 molar excess of HRP over the amount of IgG. For conjugates consisting of greater enzyme-to-antibody ratios, proportionally increase the amount of enzyme solution as required. Typically, molar ratios of 4 1 to 15 1 (enzyme antibody) give acceptable conjugates useful in a variety of ELISA techniques. 

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