Antibodies partially reduced

The use of a 500-fold molar excess of 2-mercaptoethylamine over the concentration of antibody presence was found to result in a partially reduced antibody in which two disulfides were reduced to yield four thiols (Sun et al., 2005). This strategy can be used to retain a biospecific antibody construct for subsequent discrete conjugation at the hinge region between the heavy chains. 

The following protocol is based on the methods recommended by Thermo Fisher for use of the cyanine dye DyLight 649. Antibody reduction is based on the methods of Sun et al. (2005), which results in partially reduced bispecific immunoglobulin containing available thiols in the hinge region for labeling. 

The following protocol is adapted from Myers et al. (1989). It involves activation of ricin with MBS and conjugation with a partially reduced antibody (Figure 21.14). 

Conjugation of MBS-Activated Ricin with Partially Reduced Antibody... 

Mix the MBS-activated ricin with the partially reduced antibody in a molar ratio of 15 1 (or 6.24mg activated ricin per mg of reduced antibody). This represents a volume ratio (at lOmg/ml for both proteins) of 1ml ricin solution mixed with 160pi antibody solution. 

Goupling of antibodies to CNBr-Sepharose 323 Linking of antibodies via carbohydrate side-chains 326 Coupling of partially reduced antibodies via -SH bonds 327 Covalent coupling of antibodies to Protein A/G 328... 

For complete reduction of all disulfides in the presence of a denaturant, react for 16 hours at 0°C and 2 hours at room temperature. For partial reduction of disulfides, the reaction time may be reduced to 2 hours at 37°C, particularly for antibody thiol reduction, if only partial reduction of thiols in the hinge region is done. 

Free daunomycin and not PEG-conjugated hposomes containing daunomycin, disappear rapidly from the circulation. Plasma clearance of the hposome was reduced 66-fold by PEG-conjugation. Coupling 29 0X26 monoclonal antibodies per PEG-liposome partially reversed the effect of PEG-conjugation on plasma clearance. 

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