Free sulfhydryl groups on whole antibody can be generated using specific reagents discussed under antibodies/antibody modification for conjugation of this webpage.
Conjugation to PDP-PEG-PE forms a disulfide bonds with thiolated antibodies. Pyridyl dithiol is perhaps the most popular type of thiol-disulfide exchange functional group. It will readily undergo an interchange reaction with a free sulfhydryl to yield a single mixed disulfide product. This is due to the fact that the pyridyl disulfide contains aleaving group that is easily transformed into a non reactive compound not capable of participating in further mixed disulfide formation. The PDP ragent is highly efficient in forming disulfide bonds with sulfhydryl containing molecules. In addition, the PDP leaving group has unique spectral properties that allow the measurement of sulfhydryl coupling by monitoring the increase in absorbance at 343 nm.
Conjugation of DSPE-PEG PDP to thiolated antibody
Maleimide PE derivatives generate thioether linkages with thiolated antibodies. The double bond of maleimide undergos an additional reaction with thiol forming stable thioether bonds. One of the carbon adjacent to the maleimide double bond undergoes an nucleophilic attack by the thiolate anion to generate the additional product. Maleimide reactions are specific for thiols in the pH range of 6.5-7.5. At pH 7.0 the reaction of the maleimide with sulfhydryls proceeds at a rate 1000 times greater than its reaction with amines. At higher pH values, some cross reactivity with amino groups takes place. When sufficient quantities of –SH is being alkylated, the reaction may be followed specrophotometrically by the decrease in absorbance at 300 nm as the double bond reacts and disappears.
After the sulfhydryl has been added to the maleimide the ring opening reaction happens faster as the pH becomes higher. Also, hydrolysis depends on the type of functional chemical group next to maleimide. For instance SMCC provides increased stability to maleimide hydrolysis probabaly due to its stearic effects and its lack of aromatic character. However, the adjacent phenyl ring of MBS allows for much greater rates of hydrolysis to occur.
Conjugation of DSPE-PEG maleimide to thiolated antibody
Conjugation of DOPE MBP to thiolated antibody
Conjugation of DOPE MCC to thiolated antibody
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