Tracking of antibody reduction fragments by capillary gel electrophoresis during the coupling to microparticles surface.
Due to their high specificity and efficiency, antibodies are ideal ligands for target-specific ultrasound contrast agents. The present study focuses on the chemical stability of antibodies during functionalisation with sulfosuccinimidyl-pyridyldithiopropionamidohexanoate (SPDP), a heterobifunctional linker, which exposes free thiol groups upon treatment with a reducing agent. Thiolated antibodies can then react with thiol-reactive group, such as maleimide present on the microbubble surface to form stable covalent complexes. The immunoglobulin structure relies on several intra- and inter-chain disulfide bridges which might be affected by reducing agents. A capillary electrophoresis-sodium dodecyl sulfate (CE-SDS) method with UV detection was applied to address the effect of the functionalisation process on the structural integrity of the antibodies and revealed that antibody disulfide bonds are prone to reduction as function of the reducing agents. Depending on the coupling conditions, various IgG fragments were identified reflecting different combinations between the light and heavy chains. Furthermore, two commonly used reducing agents, namely triscarboxyethylphosphine (TCEP) and 1,4-dithiothreitol (DTT) were compared under various preparation conditions. Results showed that reduction conditions based on DTT as a reducing agent under acidic pH were more appropriate to preserve intra- and inter-disulfide bridges of SPDP-modified antibodies.
Cherkaoui
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S
Bracco Research SA, 31 route de la Galaise, 1228 Plan-les-Ouates/Geneva, Switzerland. samir.cherkaoui@brg.bracco.com <samir.cherkaoui@brg.bracco.com>
Bettinger
T
T
Hauwel
M
M
Navetat
S
S
Allémann
E
E
Schneider
M
M
D000906:Antibodies / Q000737:chemistry*; D003287:Contrast Media / Q000737:chemistry; D004229:Dithiothreitol / Q000737:chemistry; D016503:Drug Delivery Systems / Q000379:methods*; D019075:Electrophoresis, Capillary / Q000379:methods*; D007128:Immunoglobulin Fragments / Q000737:chemistry*; D007074:Immunoglobulin G / Q000032:analysis; D008301:Maleimides / Q000737:chemistry; D045423:Microbubbles; D010084:Oxidation-Reduction; D010720:Phosphines / Q000737:chemistry; D013388:Succinimides / Q000737:chemistry
D000906:Antibodies; D003287:Contrast Media; D004229:Dithiothreitol; D007128:Immunoglobulin Fragments; D007074:Immunoglobulin G; D008301:Maleimides; D010720:Phosphines; D013388:Succinimides; C080938:tris(2-carboxyethyl)phosphine; C043592:maleimide; C018151:N-succinimidyl 3-(2-pyridyldithio)propionate