Antibody-enzyme conjugates chromatography

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. 

The enzyme-labeled antibody can be evaluated by enzyme-linked immunosorbent assay. Immobilize the appropriate antigen on the wells of a microtiter plate or strip (at a concentration of 2-10 ]Xg/ mL), incubate various dilutions of the conjugate for a few hours, wash the wells, add substrate, and measure the amount of product formed (see Chapter 15). This approach may also be used for monitoring conjugate purification in chromatography fractions. 

SJ Harrington, R Varro, TM Li. High-performance capillary electrophoresis as a fast in-process control method for enzyme-labelled monoclonal antibody conjugates. J Chromatography 559 385-390, 1991. 

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