Self-labeling of human polymorphonuclear leucocyte myeloperoxidase with125iodine |
| |
Authors: | G. Deby-Dupont J. Pincemail A. Thirion C. Deby M. Lamy P. Franchimont |
| |
Affiliation: | (1) Laboratoire de Radioimmunologie, B23, Université de Liège, Sart-Tilman, B-4000 Liège 1, Belgique;(2) Service d'Anesthésiologie CHU, B35, Université de Liège, Sart-Tilman, B-4000 Liège 1, Belgique;(3) Laboratoire de Biochimie et de Radiobiologie, Institut de Chimie B6, Université de Liège, Sart-Tilman, B-4000 Liège 1, Belgique;(4) Centre de Transfusion de Liège, rue Dos Fanchon, B-4000 Liège, Belgique |
| |
Abstract: | In order to obtain a radioimmunoassay (RIA) technique for the measurement of human plasma myeloperoxidase (MPO), we purified the enzyme from polymorphonuclear granulocytes (neutrophils), and compared three methods of labeling it with125Iodine: chloramine T, lactoperoxidase, and an original technique of self labeling based on the ability of the enzyme to oxidize and bind125I in the presence of H2O2. The chloramine T technique produced a degraded protein, as well shown by a high non-specific binding of tracer to antibody. The lactoperoxidase technique did not succeed in labeling MPO with an adequate specific activity. In contrast, the self-labeling method gave a stable tracer with a specific activity of 23 Ci/gmg MPO (85 MBq), a satisfactory level of immunoreactivity, and a low-specific binding (3%). After labeling, purification of tracer was achieved by gel filtration chromatography in phosphate buffer (0.05 M; pH7) to which 0.1% poly-L-lysine was added. The labeled molecule remained stable for 40 days and could be used for RIA with a polyclonal antibody raised in rabbits. |
| |
Keywords: | Human leucocytes myeloperoxidase-iodination-radioimmunoassay |
本文献已被 SpringerLink 等数据库收录! |
|