A generally applicable ELISA for the detection and quantitation of the cytosolic factors of NADPH-oxidase activation in neutrophils.
An enzyme-linked immunosorbent assay (ELISA) has been developed using polyclonal antibodies raised against two cytosolic proteins of 47 kDa (p47) and 67 kDa (p67) which behave as activation factors for the superoxide-generating NADPH oxidase of neutrophils at the onset of phagocytosis. These two proteins become associated with the NADPH oxidase complex during activation. This immunological technique has been used to follow the purification steps of p47 and p67. It allows the detection of very small amounts of cytosolic factors in a crude neutrophil extract. It is straightforward and much more sensitive (about 1000 times more) than the classical assay based on the use of a cell-free system of oxidase activation and production of superoxide anion. The percentages of p47 and p67 assessed by ELISA with respect to total cytosolic protein were estimated to amount to 0.13 and 0.20%, respectively. The described method has potential applications for the titration of the cytosolic factors of oxidase activation in autosomal recessive forms of chronic granulomatous disease.
Jouan
A
A
Laboratoire de Biochimie (URA 1130 du CNRS), Département de Biologie Moléculaire et Structurale, C.E.N.G., Grenoble, France.
Pilloud-Dagher
M C
MC
Fuchs
A
A
Vignais
P V
PV
D000818:Animals; D000906:Antibodies; D000911:Antibodies, Monoclonal; D000918:Antibody Specificity; D001798:Blood Proteins / Q000032:analysis* / Q000378:metabolism*; D015153:Blotting, Western / Q000379:methods; D002417:Cattle; D002852:Chromatography, Ion Exchange; D003600:Cytosol / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D004789:Enzyme Activation; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D051379:Mice; D008807:Mice, Inbred BALB C / Q000276:immunology; D008970:Molecular Weight; D009247:NADH, NADPH Oxidoreductases / Q000097:blood*; D019255:NADPH Oxidases; D009504:Neutrophils / Q000201:enzymology* / Q000502:physiology; D010587:Phagocytosis; D011817:Rabbits / Q000276:immunology
D000906:Antibodies; D000911:Antibodies, Monoclonal; D001798:Blood Proteins; D009247:NADH, NADPH Oxidoreductases; D019255:NADPH Oxidases