Glutaraldehyde 2- step conjugation procedure

Amounts of proteins and glutaraldehyde used in the one-step conjugation procedure (Avrameas et al., 1978) ... 

Fig. 11.7. One- and two-step conjugation procedures by glutaraldehyde. The one-step procedure, in which reagents and proteins are mixed, is poorly controlled and generally leads to excessive polymerization of antibody or of enzyme and antibody. In the two-step procedure, used primarily for POase which is relatively unreactive with GA, the enzyme is first treated with an excess of eross-linker, which is removed prior to admixture with the antibody so that no polymers of the latter are formed. However, if the enzyme has several activated groups, it can act as a bridge between antibodies and cause large polymers.

The following procedure utilizes the one-step glutaraldehyde method. A two-step method may be used to somewhat limit polymerization of the conjugate (Chapter 20, Section 1.2). Varying the pH and the amount of glutaraldehyde added to the reaction can control the yield and molecular weight of the conjugates formed. 

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

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). 

HRP is also conjugated to (strept)avidin using the two step glutaraldehyde procedure and is used in this form in the Labeled... 

Alkaline phosphatase can also be coupled to antigens and antibodies using a two-step glutaraldehyde procedure. In the case of coupling to IgG, a two-step procedure does not seem to be of any advantage. However, in the case of labeling of staphylococcal protein A with alkaline phosphatase, a two-step procedure was found to be the only way to obtain workable conjugates, and with fibronectin it was the most efficient way. ... 

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