Antibody-enzyme conjugation cross-linkers

The two-step nature of SPDP cross-linking provides control over the conjugation process. Complexes of defined composition can be constructed by adjusting the ratio of enzyme to secondary molecule in the reaction as well as the amount of SPDP used in the initial activation. The use of SPDP in conjugation applications is extensive, perhaps making it the most popular cross-linker available. It is commonly used to form immu-notoxins, antibody—enzyme conjugates, and enzyme-labeled DNA probes. A standard activation and coupling procedure can be found in Chapter 5, Section 1.1. 

Succinimidyl-4-(N-maleimidomethyl)cyclohexane-l-carboxylate (SMCC) is a heterobifunctional cross-linker with significant utility in cross-linking proteins, particularly in the preparation of antibody-enzyme and hapten-carrier conjugates (Hashida and Ishikawa, 1985 Dewey et al., 1987). The NHS ester end of the reagent can react with primary amine groups on proteins to form stable amide bonds. The maleimide end of SMCC is specific for coupling to sulfhydryls when the reaction pH is in the range of 6.5-7.5 (Smyth et al., 1964) (Fig. 158). 

The following is a generalized protocol for the activation of a protein with sulfo-SMCC with subsequent conjugation to a sulfhydryl-containing second molecule or protein. Specific examples of the use of this cross-linker to make antibody—enzyme or hapten—carrier conjugates may be found in Chapter 10, Section 1.1, and Chapter 9, Section 5, respectively. 

SMCC is a heterobifunctional cross-linker with significant utility in cross-linking proteins, particularly in the preparation of antibody—enzyme (Chapter 10) and hapten-carrier (Chapter 9) conjugates (Hashida and lshikawa, 1985 Dewey et al.,... 

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