Competitive enzyme-linked immunosorbent assay for the determination of catecholamine, dopamine in serum.
A competitive enzyme-linked immunosorbent assay for dopamine (DA) has been optimized and characterized. DA is sensitive to oxygen and light according to a function of the pH on the DA oxidation process. The phenolic groups in DA are readily oxidisable to a quinoid form and thus, free DA deteriorates in alkaline media. Thus, effect of factors such as pH, enzyme-label with substrate, ionic strength and reaction time was considered on performance of ELISA. Assay was performed with 5 microg mL(-1) of BSA-DA and 1/7500 dilution of anti-DA antibody. A dose-response curve was constructed, and a limit of detection and a dynamic range for DA were accomplished to 1.0x10(-9) M (0.19 microg L(-1)) and five orders (3.2x10(-8) M to 3.2x10(-3) M) of magnitude, respectively. The correlation diagram of the absorbance obtained both in buffer and in serum has shown good agreement with correlation coefficient (R(2) = 0.9947): Abs. (in serum) = 0.6128 x Abs. (in buffer) + 0.2926. The cross-reactivity was examined with the structurally similar compounds. And the results demonstrated that epinephrine and 3-methoxytyramine showed cross-reactivity (18.9% each), whereas 3,4-dihydroxyphenylacetic acid and homovanillic acid showed low cross-reactivity (<1%). And percent recoveries of DA in serum were quite satisfactory. This provides usefulness of the present assay to monitor DA in serum.
Kim
Jisun
J
Department of Chemistry, Seoul Women's University, Seoul 139-774, Republic of Korea.
Jeon
Moonsun
M
Paeng
Ki-Jung
KJ
Paeng
Insook Rhee
IR
D002138:Calibration; D002395:Catecholamines / Q000097:blood* / Q000737:chemistry / Q000276:immunology; D003365:Costs and Cost Analysis; D003429:Cross Reactions; D004298:Dopamine / Q000097:blood* / Q000737:chemistry / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay / Q000191:economics / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013997:Time Factors
D002395:Catecholamines; D004298:Dopamine