Affinity chromatography conjugates

One limitation to this method should be noted. If the antibody-enzyme conjugate is prepared using antibody fragments such as Fab or F(ab )2, then nickel-chelate affinity chromatography will not work, since the requisite Fc portion of the antibody necessary for complexing with the metal is not present. 

Figure 20.15 An affinity chromatography support containing iminodiacetic acid groups chelated with nickel may be used to remove excess enzyme after reactions to produce antibody-enzyme conjugates. The nickel chelate binds to the antibody in the Fc region, retaining the conjugate while allowing free enzyme to pass through the gel unretarded.

Instead of dialysis or gel filtration, an affinity chromatography on Concanavalin A Sepharose (cf. Protocol 3.6.2.4) is recommended, because on the one hand, no conjugated antibody is removed, and on the other hand, the sugar used for elution stabilizes the enzyme-antibody conjugate in solution. 

Purify the conjugate by affinity chromatography on Con-canavalin A Sepharose (Protocol 3.6.2.4), add glycerol to 50% final concentration and store aliquots at -20 °C. The aliquots are stable for months. 

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