Homobifunctional reagents conjugation

In two-step protocols, one of the proteins to be conjugated is reacted with the homobifunctional reagent and excess crosslinker and by-products are removed. In the second stage, the activated protein is mixed with the other protein or molecule to be conjugated, and the final conjugation process occurs (Figure 4.2). 

Figure 4.2 Homobifunctional crosslinkers 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 crosslinker in excess to create an active intermediate. After removal of remaining crosslinker, 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, but can t entirely prevent it.

The problems of indeterminate conjugation products are amplified in single-step reaction procedures using homobifunctional reagents (Chapter 4). Single-step procedures involve the addition of all reagents at the same time to the reaction mixture. This technique provides the least control over the cross-linking process and invariably leads... 

Conjugation with OPDM (Table 11.15) is still not very widely used, even if it gives better results than other homobifunctional reagents. With the use of Fab -BGase conjugates in EIA, antigens can be detected in the femto- to attomole (10 -l0- ) range (Adachi et al., 1978). 

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