Protein Protocols Protein Protocols

Alkaline Phosphates Labeling of IgG Antibody 343
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Alkaline Phosphatase Labeling of IgG Antibody
G. Brian Wisdom
1. Introduction
Alkaline phosphatase (EC 3.1.3.1) from bovine intestinal mucosa is a valuable label.
It is stable, has a moderate size (140 kDa), a high turnover number, and can be assayed
using a variety of different substrates which are measured by changes in absorbance,
fluorescence, or luminescence. The most common method of labeling immunoglobulin
G (IgG) antibody with this enzyme uses the homobifunctional reagent
glutaraldehyde.
The chemistry of glutaraldehyde is complex. It reacts with the amino 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. Self-coupling
can be a problem unless the proteins are 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 (>10 6 Da) as several molecules of each
component are linked. This is the most simple labeling procedure to carry out and,
although the yields of enzyme activity and immunoreactivity are small, the conjugates
obtained are stable and practical reagents.
Alkaline phosphatase may also be coupled using heterobifunctional reagents containing
the N-hydroxysuccinimide and maleimide groups, for example, succinimidyl
4-(N-maleidomethyl)-cyclohexane-1-carboxylate. However, because the enzyme has
no free thiol groups this approach is usually used for the labeling of F(ab') 2 fragments
of IgG via their thiols (2).
2. Materials
1. Alkaline phosphatase from bovine intestinal mucosa, 2000 U/mg or greater (with
4-nitrophenyl phosphate as substrate); this is usually supplied at a concentration
of 10 mg/mL. If ammonium sulfate, Tris, or any other amine is present, it must be
removed (see Note 1). There are numerous commercial sources of labeling-grade enzyme.
2. IgG antibody. This should be the pure IgG fraction or, better, affinity-purified antibody
from an antiserum or pure monoclonal antibody (see Chapters 137–144).
3. Phosphate buffered saline (PBS): 20 mM sodium phosphate buffer, pH 7.2, containing
0.15 M NaCl.
From: The Protein Protocols Handbook, 2nd Edition
Edited by: J. M. Walker © Humana Press Inc., Totowa, NJ
343