Conjugation using glutaraldehyde

The one- and two-step procedures for enzyme activation and conjugation using glutaraldehyde can be found in Chapter 20, Section 1.2. 

The enzymes commonly used as labels in ELISA and other immunochemical reactions include horse radish peroxidase (HRP) and alkaline phosphatase (AP). The enzyme can be covalently coupled to the antibody using glutaraldehyde conjugation to reactive amino groups on the enzyme (lysines) in a phosphate buffered aqueous solution at neutral pH, as shown in Fig. 19 (103). Alternatively, carbohydrates present in the immunoglobulin structure can be cleaved by periodate treatment (see Fig. 20) and bound to free amino groups on the enzyme through a Schiff base reaction (103). 

IgG antibody may be labeled with HRP using glutaraldehyde in a two-step procedure (2) this produces small conjugates, but their activity is low. Heterobifunctional reagents such as succinimidyl 4-(7V-maleidomethyl)-cyclohexane-1 -carboxylate may also be used to link the thiol group of Fab fragments to an amino group in the enzyme (3). 

Fig. 7.21. Enzyme coupling via p-benzoquinone (pBQ) activation (pBQ also reacts with carbohydrates) and coupling to a basic polymer (I). At pH 6, only one of two potential sites of pBQ reacts, whereas at pH 8 the second site is activated. The complex is conjugated to denatured DNA using glutaraldehyde. Alternatively, haptenated basic polymers can be linked by 1,2,7,8-diepoxyoctane (carcinogenic ) to DNA (II).

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