Activation of Antibodies with Sodium Periodate

Dissolve the antibody to be periodate-oxidized at a concentration of lOmg/ml in 0.01 M sodium phosphate, 0.15 M NaCl, pH 7.2. 

Dissolve sodium periodate in water to a final concentration of 0.1 M. Protect from light. 

Immediately add 100 pi of the sodium periodate solution to each ml of the antibody solution. Mix to dissolve. Protect from light. 

Immediately quench the reaction by the addition of sodium sulfite (Na2S03) to provide a 2X molar excess over the initial amount of periodate added. Purify the oxidized antibody by gel filtration using a desalting resin. The chromatography buffer is 0.1 M sodium phosphate, 0.15M NaCl, pH 7.2. To obtain efficient separation between the oxidized antibody and excess periodate, the sample size applied to the column should be at a ratio of no more than 5 percent sample volume to the total column volume. Collect 0.5 ml fractions and monitor for protein at 280 nm. 

Pool the fractions containing protein. Adjust the antibody concentration to lOmg/ml for the conjugation step. The oxidized antibody should be used immediately. 

---

Thus, glycoproteins such as HRP, GO, or most antibody molecules can be activated for conjugation by brief treatment with periodate. Crosslinking with an amine-containing protein takes place under alkaline pH conditions through the formation of Schiff base intermediates. These relatively labile intermediates can be stabilized by reduction to a secondary amine linkage with sodium cyanoborohydride (Figure 20.8). 

---