Analysis of vitamin B ₁₂ in seawater and marine sediment porewater using ELISA

Vitamin B12 (B ₁₂) is a set of closely related organocobalt compounds required by phytoplankton. A highly sen- sitive and specific enzyme-linked immunosorbent assay (ELISA) was developed for the determination of B ₁₂ in seawater and marine sediment porewater. An antibody directed against B ₁₂ was coated on the surface of a 96- well microtiter plate, and horseradish peroxidase (HRP) was used as a labeling enzyme. In the indirect compet- itive immunoassay format, water samples or standards and a constant amount of HRP-labeled B ₁₂ were added into the microtiter plate wells, HRP-labeled and free B ₁₂ compete for binding to the plate-bound antibody. After immunoreaction, the immunochemically adsorbed HRP-B ₁₂ conjugate was determined by measuring the absorbance produced in a solution containing substrate tetramethylbenzidine (TMB) and hydrogen peroxide. The calibration graph for B ₁₂ 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 (3Symbol.-1.c02). Coupled with C-18 column extraction-precon- centration, 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 B ₁₂ in coastal seawater and surface tidal flat porewater (0-2 cm) ranged from 4.5-38 pM and 12-47 pM, respectively.