Radiolabelling of monoclonal antibodies: optimization of conjugation of DTPA to F(ab')2-fragments and a novel measurement of the degree of conjugation using Eu(III)-labelling.
F(ab')2 fragments (at concentrations of 5-30 mg/ml) derived from monoclonal antibodies raised against human prostate specific acid phosphatase were derivatized with a bicyclic anhydride of diethylenetriaminepentaacetic acid (cDTPAA) in the molar ratios of cDTPAA/F(ab')2 of 1:1, 5:1, 10:1 or 50:1. The most optimal product, aimed at radioimaging of prostatic cancer was obtained when the antibody fragment concentration was at least 10 mg/ml and the molar ratio of cDTPAA to F(ab')2 was 5:1. cDTPAA was added dissolved in dimethylsulfoxide (DMSO). Under these conditions, 1.8-2.2 DTPA molecules/F(ab')2 molecule were bound, giving a coupling efficiency of 37%-44%, and the labelling efficiency with 111In (3 mCi/1 mg protein) was 95% +/- 3% (n = 7). The antibody fragment completely retained its immunoreactivity measured by radioimmunoassay and showed no aggregation when studied using sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE). For evaluation of the degree of conjugation of DTPA to the antibody fragment, a novel technique was developed relying on the use of EuCl3, and the measurement of europium fluorescence employing time resolved fluorometry. Results by EuCl3 labelling were identical to those obtained by the conventional 111InCl3 labelling method.
Hartikka
M
M
Biocenter, University of Oulu, Finland.
Vihko
P
P
Södervall
M
M
Hakalahti
L
L
Torniainen
P
P
Vihko
R
R
D000135:Acid Phosphatase / Q000276:immunology; D000911:Antibodies, Monoclonal; D000951:Antigens, Neoplasm / Q000276:immunology; D005063:Europium; D006801:Humans; D007140:Immunoglobulin Fab Fragments; D007553:Isotope Labeling / Q000379:methods*; D004369:Pentetic Acid / Q000031:analogs & derivatives; D017430:Prostate-Specific Antigen
D000911:Antibodies, Monoclonal; D000951:Antigens, Neoplasm; D007140:Immunoglobulin Fab Fragments; C039777:cyclic DTPA anhydride; D005063:Europium; D004369:Pentetic Acid; D000135:Acid Phosphatase; D017430:Prostate-Specific Antigen