The influence of periodate oxidation on monoclonal antibody avidity and immunoreactivity.
Eight monoclonal antibodies of different classes and isotypes and rabbit IgG were oxidized under a variety of conditions with 5-50 mM periodate. The number of aldehyde groups generated per immunoglobulin were measured by reduction with tritiated sodium borohydride or coupling of fluoresceinthiosemicarbazide. There were up to 25.5 aldehyde groups detected on the periodate-oxidized antibody 96.5, measured by borohydride reduction whereas the same conditions led to only 9.6 aldehydes per IgG on the antibody L6 of the same IgG2A isotype. Fluoresceinthiosemicarbazide bound to oxidized antibodies but not to the same extent as tritium. On mildly oxidized IgMs it was possible to generate more than 200 aldehyde groups per antibody molecule. Depending on the conditions and the antibody used periodate oxidation could lead to antibody crosslinking. The avidities of the modified antibodies were determined by Scatchard analyses and inhibition assays. A new mathematical method to evaluate the immunoreactivities of modified antibodies relative to the unlabeled native antibody from inhibition binding data was established. Periodate concentrations higher than 50 mM decreased the avidities and immunoreactivities of all IgGs tested. This effect is more pronounced if the oxidation is performed at pH 5.6 and 25 degrees C instead of pH 4.6 and 0 degree C. The BR96 antibody is inactivated even under mild oxidation conditions.
Abraham
R
R
Bristol-Myers Squibb Pharmaceutical Research Institute, Seattle, WA 98121.
Moller
D
D
Gabel
D
D
Senter
P
P
Hellström
I
I
Hellström
K E
KE
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000915:Antibody Affinity; D006863:Hydrogen-Ion Concentration; D007074:Immunoglobulin G / Q000276:immunology; D010084:Oxidation-Reduction; D010504:Periodic Acid / Q000494:pharmacology*; D011817:Rabbits
D000911:Antibodies, Monoclonal; D007074:Immunoglobulin G; D010504:Periodic Acid; C009288:metaperiodate