Competitive enzyme-linked immunosorbent assay for the determination of catecholamine, dopamine in serum Original Research Article

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 μg 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 1.0 × 10−9 M (0.19 μg L−1) and five orders (3.2 × 10−8 M to 3.2 × 10−3 M) of magnitude, respectively. The correlation diagram of the absorbance obtained both in buffer and in serum has shown good agreement with correlation coefficient (R2 = 0.9947): Abs. (in serum) = 0.6128 × 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.