G. Brian Wisdom
Alkaline phosphatase is coupled to immunoglobin G antibody in a one-step procedure using the homobifunctional reagent glutaral-dehyde, which reacts with amino groups in the two proteins. The procedure is simple to perform and requires minimal equipment.
Key Words: Antibody; alkaline phosphatase; conjugation; labeling; glutaraldehyde.
Alkaline phosphatase (EC 126.96.36.199) from bovine intestinal mucosa has proven its worth as an enzyme label for many years. It is stable, has a moderate size (140 kDa), a high turnover number, and can be assayed using a variety of different substrates. Its activity is easily detected by eye in, for example, immunoblots, and it can be quantified by changes in absorbance, fluorescence, or luminescence for use in enzyme-linked immunosorbent assays.
The most common method of labeling immunoglobin G (IgG) antibody with this enzyme uses the homobifunctional reagent glu-
taraldehyde. The chemistry of glutaraldehyde is complex; it reacts with the amino groups and, to a lesser extent, the thiol groups of proteins and when two proteins are mixed in its presence, stable conjugates are produced without the formation of Schiff bases. Excessive self-coupling can be minimized by mixing the proteins at appropriate concentrations. In the method (1) described, there is usually little self-coupling of the enzyme or the IgG antibody, however, the size of the conjugate is large (>106 Da) because several molecules of each component are linked. This is the simplest enzyme labeling procedure to perform and, although the yields of enzyme activity and immunoreactivity are relatively small, the conjugates obtained are stable and practical reagents.
Alkaline phosphatase may also be coupled to antibody using heterobifunctional reagents containing the N-hydroxysuccinimide and maleimide groups, for example, succinimidyl 4-(N-maleido-methyl)-cyclohexane-1-carboxylate. However, because the enzyme has no free thiol groups, this approach is usually used for the labeling of Fab' fragments of IgG via their thiols (2). Alternatively a three-step procedure may be used in which thiol groups are introduced into either the enzyme or the antibody prior to the reactions of the maleimide and N-hydroxysuccinimide groups of the heterobifunctional linker.
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