Enzymes creating maleimide-activated

Figure 20.4 Reduction of the disulfide bonds within the hinge region of an IgG molecule produces half-anti-body molecules containing thiol groups. Reaction of these reduced antibodies with a maleimide-activated enzyme creates a conjugate through thioether bond formation.

The number of sulfhydryls created on the immunoglobulin using thiolation procedures such as this one is more critical to the yield of conjugated enzyme molecules than the molar excess of maleimide-activated enzyme used in the conjugation reaction. Therefore, it is important to use a sufficient excess of Traut s reagent to obtain a sufficient number of available sulfhydryls. 

Figure 20.5 Antibodies may be modified with 2-iminothiolane at their amine groups to create sulfhydryls for conjugation with SMCC-activated enzymes. The maleimide groups on the derivatized enzyme react with the thiols on the antibody to form thioether bonds.

The amine groups on these fragments also may be modified with thiolating agents, such as SATA or 2-iminothiolane, to create sulfhydryl residues suitable for coupling to maleimide-activated enzymes (Section 1.1, this chapter) (Figure 20.13). Amine groups further may be utilized... 

Figure 20.13 The thiolation reagent SATA can be used to create sulfhydryl groups on Fab fragments. After deprotection of the acetylated thiol of SATA with hydroxylamine, conjugation with a maleimide-activated enzyme can take place, producing thioether linkages.

Other Matrices. A large number of other matrices have been employed, including starch (33), cross-linked dextrins (34,35), a vinyl maleimide polymer (36), chitin (37), mannan (38), and insolubilized proteins (39). The number of potential matrices is almost limitless, and most of the matrices used for immobilization of enzymes have potential application in bioselective adsorption. Among these are nylon (40), metal oxides (41), maleic anhydride-ethylene co-polymers (42), and polystyrene derivatives (43). Few of these have been used because of their potential for nonspecific adsorption either by charge, as with the metal oxides, or by hydrophobic interactions (as would be the case with polystyrene). The derivatized matrix must be free of nonspecific adsorption. Proper derivatization can eliminate nonspecific adsorption or, as with cyanogen bromide activation, create nonspecific adsorption in matrices having no prior nonspecific adsorption properties. 

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