Conjugation using cross-linkers

SMPT, succinimidyloxycarbonyl-a-methyl-a-(2-pyridyldithio)toluene, contains an NHS ester end and a pyridyldisulfide end similar to those of SPDP, but its hindered disulfide makes conjugates formed with this reagent more stable (Thorpe etal., 1987) (Chapter 5, Section 1.2). The reagent is especially useful in forming immunotoxin conjugates for in vivo administration (Chapter 11, Section 2.1). A water-soluble analog of this cross-linker containing an extended spacer arm is also commercially available as Sulfo-LC-SMPT (Pierce). 

Dissolve a cross-linked protein or peptide that has been conjugated with the use of a disulfide containing cross-linker at a concentration of 1—10 mg/ml in 0.01 M sodium phosphate, 0.15 M NaCl, pH 7.4. Alternative buffer conditions and pH levels may be used however, a pH between 7.0 and 8.1 usually works best. 

Purify the reduced IgG from excess 2-mercaptoethylamine and reaction byproducts by dialysis or gel filtration using Sephadex G-25. All buffers should contain 1—10 mM EDTA to preserve the free sulfhydryls from metal-catalyzed oxidation. The sulfhydryl containing half-antibody may now be used in conjugation protocols that use—SH-reactive heterobifunctional cross-linkers (Chapter 10, Section 1.1). 

The amidine bond formed is quite stable at acid pH however, it is susceptible to hydrolysis and cleavage at high pH. Typical reaction conditions for using imidate cross-linkers is a buffer system consisting of 0.2 M triethanolamine in 0.1 M sodium borate, pH 8.2. After conjugating two proteins with a bifunctional imidoester crosslinker, excess imidoester functional groups may be blocked with ethanolamine. 

Figure 121 Homobifunctional cross-linkers may be used in a two-step process to conjugate two proteins or other molecules. In the first step, one of the two proteins is reacted with the cross-linker in excess to create an active intermediate. After removal of remaining cross-linker, a second protein is added to effect the final conjugate. Two-step reaction schemes somewhat limit the degree of polymerization obtained when using homobifunctional reagents.

Difluorobenzene derivatives are small homobifunctional cross-linkers that react with amine groups. Conjugation using these compounds results in bridges of only about 3 A in length, potentially providing information concerning very close interactions between macromolecules. 

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. 

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