Analysis of vitamin B12 in seawater and marine sediment porewater using ELISA
Limnol. Oceanogr. Methods 9:515-523 (2011) | DOI: 10.4319/lom.2011.9.515
ABSTRACT: 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.