Optimisation of small-scale coupling of A5B7 monoclonal antibody to carboxypeptidase G2
| Autor: | J.M.B. Boyle, G.T. Rogers, Roger G. Melton, Roger F. Sherwood, Kd Bagshawe, P. J. Burke |
|---|---|
| Rok vydání: | 1993 |
| Předmět: |
Stereochemistry
Immunology Succinimides Antineoplastic Agents Sulfides Immunoglobulin Fab Fragments Acetic acid chemistry.chemical_compound Carboxypeptidase-G2 Methods Immunology and Allergy Prodrugs Sulfhydryl Compounds Maleimide biology Antibodies Monoclonal gamma-Glutamyl Hydrolase Enzyme assay Carboxypeptidase G chemistry CpG site Drug Design Thioglycolates Reagent biology.protein Binding Sites Antibody Conjugate |
| Zdroj: | Journal of Immunological Methods. 158:49-56 |
| ISSN: | 0022-1759 |
| DOI: | 10.1016/0022-1759(93)90257-8 |
| Popis: | Conjugates of F(ab′) 2 fragment of the monoclonal antibody A5B7 coupled to carboxypeptidase G 2 (CPG 2 ) have been produced using the heterobifunctional reagents 2-mercapto-[ S -acetyl]acetic acid, N -hydroxysuccinimide ester (SATA) and m -maleimidobenzoyl- N -hydroxysuccinimide ester (SMPB). The effect of various levels of modifying reagent on enzyme activity and antigen binding activity were determined, and it was shown that whilst CPG 2 is relatively sensitive to modification, insertion of three maleimide groups per CPG 2 resulted in the loss of 30% of enzyme activity; 5B7 F(ab′) 2 was insensitive to modification, little or no activity being lost. The coupling efficiency of the reation was shown to be fairly constant over a wide range of substitution levels. There was thus no advantage to be gained in using high substitution levels, which may result in loss of enzyme activity. The formation of undesired high molecular weight aggregates could be controlled by adjustment of the protein concentration during the final coupling step. |
| Databáze: | OpenAIRE |
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