Analysis of vitamin B12 in seawater and marine sediment porewater using ELISA

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Vitamin B12 (B12) is a set of closely related organocobalt compounds required by phytoplankton. A highly sensitive and specific enzyme‐linked immunosorbent assay (ELISA) was developed for the determination of B12 in seawater and marine sediment porewater. An antibody directed against B12 was coated on the surface of a 96‐well microtiter plate, and horseradish peroxidase (HRP) was used as a labeling enzyme. In the indirect competitive immunoassay format, water samples or standards and a constant amount of HRP‐labeled B12 were added into the microtiter plate wells, HRP‐labeled and free B12 compete for binding to the plate‐bound antibody. After immunoreaction, the immunochemically adsorbed HRP‐B12 conjugate was determined by measuring the absorbance produced in a solution containing substrate tetramethylbenzidine (TMB) and hydrogen peroxide. The calibration graph for B12 was linear over the range of 0.4–100 ng/mL (0.3–74 nM; higher concentrations were not evaluated) with a detection limit of 0.2 ng/mL (3s). Coupled with C‐18 column extraction–preconcentration, the method is readily applicable to seawater levels (~1–10 pM). No interferences from humic acids, total dissolved organic matter (DOM), and salinity were observed. ELISA determined B12 in coastal seawater and surface tidal flat porewater (0–2 cm) ranged from 4.5–38 pM and 12–47 pM, respectively.