Plasma sampling and freezing procedures influence vitellogenin measurements by enzyme-linked immunoassay in the fathead minnow (Pimephales promelas)

• Published in: Environmental toxicology and chemistry 2006-02, Vol.25 (2), p.337-348
• Main Authors: Brodeur, Julie C., Woodburn, Kent B., Zhang, Fagen, Bartels, Michael J., Klecka, Gary M.
• Format: Article
• Language: English
• Subjects: Animal, plant and microbial ecology; Animals; Antibodies; Applied ecology; Aprotinin; Biological and medical sciences; Blood; Chromatography; Chromatography, Liquid; Citrates; Cryopreservation; Cyprinidae - physiology; Degradation; Ecotoxicology, biological effects of pollution; ELISA; Environmental impact; Environmental science; Enzyme-linked immunoassay; Enzyme-Linked Immunosorbent Assay; Enzymes; Fathead minnow; Female; Fish; Freeze thaw cycles; Freezing; Frozen; Fundamental and applied biological sciences. Psychology; General aspects; Immunoassay; Ionization; Liquid chromatography; Male; Mass Spectrometry; Mathematical analysis; Melting; Molecular Structure; Pimephales promelas; Plasma; Polyethylene glycol; Presses; Reproducibility of Results; Sampling; Sensitivity and Specificity; Thawing; Toxicology; Vitellogenin; Vitellogenins - blood; Vitellogenins - chemistry
• ISSN: 0730-7268; 1552-8618
• DOI: 10.1897/05-368R.1

The present study compared three different methods for measuring plasma vitellogenin (VTG) in fathead minnow (FHM; Pimephales promelas): A procedure using liquid chromatography with electrospray ionization combined with tandem mass spec‐trometry (LC/ESI‐MS/MS), and two commercial enzyme‐linked immunoassay (ELISA) kits using either anti‐carp or anti‐FHM antibodies. The influence on plasma VTG measurements of using the protease‐inhibitor aprotinin during blood sampling and of submitting the plasma samples to a freeze–thaw cycle before analysis also was evaluated. The addition of aprotinin to the blood during sampling significantly reduced the plasma VTG concentrations measured by ELISA, whereas the VTG values measured after plasma samples were submitted to a freeze–thaw cycle were significantly higher than those measured before freezing. This inflating effect of freezing on VTG measurements made by ELISA could be prevented if plasma samples were frozen diluted in citrate buffer containing 16 mg/ml of polyethylene glycol (PEG). In contrast, measurements of VTG made by LC/ESI‐MS/MS were unaffected by freezing and, conceptually, are independent from enzymatic degradation. Although the use of aprotinin and PEG effectively reduced the influence of enzymatic and physical degradation caused by freezing and thawing on VTG measurements made by ELISA, it did not improve agreement between the three analytical techniques evaluated. More information is needed regarding the molecular structure and the existence of possible multiple forms of VTG before this protein can be measured adequately in FHM.