Premium
Analysis of vitamin B 12 in seawater and marine sediment porewater using ELISA
Author(s) -
Zhu Qingzhi,
Aller Robert C.,
Kaushik Aleya
Publication year - 2011
Publication title -
limnology and oceanography: methods
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.898
H-Index - 72
ISSN - 1541-5856
DOI - 10.4319/lom.2011.9.515
Subject(s) - seawater , chemistry , chromatography , absorbance , detection limit , microtiter plate , horseradish peroxidase , immunoassay , substrate (aquarium) , calibration curve , adsorption , environmental chemistry , enzyme , geology , biochemistry , organic chemistry , antibody , immunology , biology , oceanography
Vitamin B 12 (B 12 ) 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 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 competitive 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 (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 B 12 in coastal seawater and surface tidal flat porewater (0–2 cm) ranged from 4.5–38 pM and 12–47 pM, respectively.