A 1-hour enzyme-linked immunosorbent assay for quantitation of acrolein- and hydroxynonenal-modified proteins by epitope-bound casein matrix method.
A simple and rapid enzyme-linked immunosorbent assay (ELISA) method for quantitation of acrolein and 4-hydroxy-2-nonenal (HNE)-modified proteins was developed. Microtiter plate wells were precoated and blocked simultaneously with epitope-bound bovine caseins as matrix proteins, and aldehyde-modified proteins were quantitated by a competition assay with a monoclonal antibody specific for acrolein-modified lysine or HNE-modified histidine epitopes. Minimal reaction times required for the coating/blocking; first monoclonal antibody and the peroxidase-conjugated second antibody binding steps were 3, 3, and 7 min, respectively, the former two steps being found to be or akin to diffusion-rate-limiting reactions. The convenient ELISA should find an application for analyses of the intricate processes involved in oxidative stress and carcinogenic insult. The epitope-attachment methodology may also be advantageous for the quantitation of various other biologically important haptenic molecules.
Satoh
K
K
Second Department of Biochemistry, Hirosaki University, School of Medicine, Hirosaki, 036, Japan. kisatoh@mail.cc.hirosaki-u.ac.jp
Yamada
S
S
Koike
Y
Y
Igarashi
Y
Y
Toyokuni
S
S
Kumano
T
T
Takahata
T
T
Hayakari
M
M
Tsuchida
S
S
Uchida
K
K
D000171:Acrolein; D000447:Aldehydes; D000818:Animals; D000911:Antibodies, Monoclonal; D002364:Caseins; D002417:Cattle; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D000939:Epitopes; D006801:Humans; D007120:Immunochemistry; D010047:Ovalbumin / Q000032:analysis / Q000737:chemistry / Q000276:immunology; D011506:Proteins / Q000032:analysis* / Q000737:chemistry* / Q000276:immunology; D051381:Rats
D000447:Aldehydes; D000911:Antibodies, Monoclonal; D002364:Caseins; D000939:Epitopes; D011506:Proteins; D000171:Acrolein; D010047:Ovalbumin; C027576:4-hydroxy-2-nonenal