Determination of reduced disulfide groups in monoclonal antibodies

Reduction of disulfide bonds to sulfhydryl groups for direct radiolabeling of antibodies for immunoscintigraphic and therapeutic applications continues to be of considerable interest. Sensitive spectrophotometric methods have been evaluated that will enable investigators to determine submicrogram quantities of cysteine units produced, for the assurance of controlled reduction. One method, which generates a cysteine-ninhydrin complex (520 nm), has a molar extinction coefficient of 30 250 and can determine 0.04 micrograms/ml cysteine units with an absorbance of 0.01. The method has been applied to determine the quantity of cysteine groups produced by the reduction of an immunoglobin G antibody with five different reducing agents in normal to five times the previously determined optimal molar ratios. The quantities of cysteine units produced from the controlled reduction from 240 micrograms immunoglobin G ranged from 0.073 +/- 0.01 to 1.07 +/- 0.04 micrograms, which were merely 0.54 +/- 0.08% to 7.9 +/- 0.28% of the total available disulfide groups in the protein.