Preparation of Activated Enzymes for Conjugation

Of the total number of commercial diagnostic assays utilizing antibody—enzyme conjugates, GO is employed in less than 1% of clinical tests. The enzyme remains, however, an important tool in many assays developed for research use. One particular advantage to the enzyme is that there is no endogenous GO activity in mammalian tissues, making it an excellent choice for immunohistochemical staining procedures. 

Antibody conjugates with GO can be made using the cross-linking agents glu-taraldehyde (Chapter 10, Section 1.2) or SMCC (Chapter 10, Section 1.1). The heterobifunctional reagent SMCC provides the best control over the conjugation process and usually results in high-activity preparations. 

Whatever the method of conjugate creation, the most important considerations are retention of activity in the complex and prevention of extensive oligomer generation, which may cause precipitation. The following sections discuss some of the more common methods for producing enzyme conjugates. The list, however, is by no means inclusive of every possible procedure used in the literature. 

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Currently, a common form of activated mPEG used for preparation of therapeutic enzymes is mPEG-succinate-N-hydroxysuccinimide ester (SS-PEG) (11). It reacts with proteins in short periods of time under mild conditions, producing extensively modified conjugates with well preserved biological activity. However, the ester linkage between the polymer and the succinic acid residue has limited stability in aqueous media (5,12). 

Pool the fractions containing protein. Adjust the enzyme concentration to lOmg/ml for the conjugation step (see next section). The periodate-activated enzyme may be stored frozen or freeze-dried for extended periods without loss of activity. Do not store the preparation in solution at room temperature or 4°C, since precipitation will occur over time due to self-polymerization. 

Tussen, R, and Kurstak, E. (1984) Highly efficient and simple methods for the preparation of peroxidase and active peroxidase—Antibody conjugates for enzyme immunoassays. Anal. Biochem. 136, 451-457. 

Many types of mono-, bi-, and multifunctional coupling reagents are available for labeling antibodies or antigens with an enzyme. Glutaraldehyde, carbodiimide, N-succinimidyl-3-(2-pyridyldithio)propionate, and periodate oxidation of carbohydrate moieties to form active dialdehydes are several commonly used approaches in the preparation of enzyme conjugates (104-106). 

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