Self-labeling of human polymorphonuclear leucocyte myeloperoxidase with 125iodine.
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 with 125Iodine:chloramine T, lactoperoxidase, and an original technique of 'self labeling' based on the ability of the enzyme to oxidize and bind 125I 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 microCi/micrograms MPO (85 MBq), a satisfactory level of immunoreactivity, and a low-specific binding (less than or equal to 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.
Deby-Dupont
G
G
Laboratoire de Radioimmunologie, Université de Liège, Belgique.
Pincemail
J
J
Thirion
A
A
Deby
C
C
Lamy
M
M
Franchimont
P
P
D002700:Chloramines; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism; D007202:Indicators and Reagents; D007457:Iodine Radioisotopes; D007784:Lactoperoxidase; D009504:Neutrophils / Q000201:enzymology*; D009195:Peroxidase / Q000097:blood* / Q000302:isolation & purification; D011863:Radioimmunoassay / Q000379:methods*; D014105:Tosyl Compounds
D002700:Chloramines; D007202:Indicators and Reagents; D007457:Iodine Radioisotopes; D014105:Tosyl Compounds; C016300:chloramine-T; D006861:Hydrogen Peroxide; D007784:Lactoperoxidase; D009195:Peroxidase