Use of progesterone-3(O-carboxymethyl oxime)-horseradish peroxidase in a sensitive microtitre-plate EIA and its application to a visual membrane EIA of progesterone.
A simple method of visual membrane enzyme-immunoassay (EIA) for the detection of progesterone is described. When two types of progesterone-horseradish peroxidase (HRP) tracers were challenged for binding, in the presence of progesterone, to the monoclonal anti-progesterone antibody, 15A, coated on the microtitre plate, the HRP conjugated at the C-3 position (A-ring) of progesterone competed more effectively with progesterone to the binding site of the monoclonal antibody (mAb) than HRP conjugated at the C-11 position of the C-ring. By using this combination of mAb, 15A, and progesterone-3(O-carboxymethyloxime)-HRP (P-3CMO-HRP), we developed a visual membrane EIA system in which free progesterone in the sample could be quantified by the degree of color development. In this system, free progesterone competed with P-3CMO-HRP for binding sites of mAb immobilized on the nitrocellulose membrane. The stable grey color was formed on the surface of membrane for progesterone-negative and no color for progesterone-positive sample using 3,3'-diaminobenzidine (DAB) with Co2+ as an insoluble substrate solution. To examine whether tetramethylbenzidine (TMB) can substitute for DAB in membrane EIA, an experiment was conducted where TMB was used as an insoluble substrate.
Yoon
D Y
DY
Genetic Engineering Research Institute, Korea Institute of Science and Technology (KIST), Taejon.
Song
E Y
EY
Kwon
D H
DH
Choi
M J
MJ
Byun
S M
SM
Choe
I S
IS
Chung
T W
TW
D015100:3,3'-Diaminobenzidine / Q000737:chemistry; D000911:Antibodies, Monoclonal / Q000276:immunology; D000937:Antigen-Antibody Reactions; D001560:Benzidines / Q000737:chemistry; D001667:Binding, Competitive; D002413:Cations, Divalent / Q000737:chemistry; D002863:Chromogenic Compounds / Q000737:chemistry; D003035:Cobalt / Q000737:chemistry; D003101:Collodion / Q000737:chemistry; D006735:Horseradish Peroxidase / Q000276:immunology; D007124:Immunoenzyme Techniques; D008567:Membranes, Artificial; D011374:Progesterone / Q000031:analogs & derivatives* / Q000032:analysis* / Q000737:chemistry / Q000276:immunology
D000911:Antibodies, Monoclonal; D001560:Benzidines; D002413:Cations, Divalent; D002863:Chromogenic Compounds; D008567:Membranes, Artificial; C094926:progesterone-11HS-horseradish peroxidase; C086104:progesterone-3-(O-carboxymethyl oxime)-horseradish peroxidase; D015100:3,3'-Diaminobenzidine; C021758:3,3',5,5'-tetramethylbenzidine; D003035:Cobalt; D011374:Progesterone; D003101:Collodion; D006735:Horseradish Peroxidase