Development of an immunoblot assay with infrared fluorescence to quantify paraoxonase 1 in serum and plasma

Philip W. Connelly, Graham F. Maguire, Clive M. Picardo, John F. Teiber, Dragomir Draganov

Research output: Contribution to journalArticle

26 Citations (Scopus)

Abstract

Paraoxonase 1 (PON1) requires calcium for activity and is inactivated in the presence of EDTA. Because of this, studies to date have used serum or heparinized plasma for both activity and mass assays of PON1. Whole serum and EDTA plasma were analyzed by SDS-electrophoresis and Western blot using anti-PON1 monoclonal antibody 4C10. Because PON1 has one disulfide and one free cysteine residue, the samples were reduced with dithiothreitol before electrophoresis. Western blot identified a major PON1 band with a molecular mass of ∼45 kDa and two minor bands of ∼40 and 35 kDa in both serum and EDTA plasma. This established that PON1 is inactive, but structurally intact, in EDTA plasma and suggested that a mass assay could be developed based on SDS-electrophoresis and Western blot. Linearity was established for plasma and for a PON1 standard. Quantification was based on the major PON1 band at 45 kDa. The correlation between serum and plasma PON1 mass was 0.9553. The between-run variation was determined with a serum pool to be 7.8%. The mass of PON1 in serum was significantly correlated with arylesterase activity (r = 0.85). Thus, we have demonstrated the feasibility of measuring PON1 mass in either serum or EDTA plasma.

Original languageEnglish (US)
Pages (from-to)245-250
Number of pages6
JournalJournal of Lipid Research
Volume49
Issue number1
DOIs
StatePublished - Jan 2008

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Aryldialkylphosphatase
Assays
Fluorescence
Infrared radiation
Plasmas
Serum
Edetic Acid
Electrophoresis
Western Blotting
Dithiothreitol
Molecular mass
Disulfides
Cysteine
Monoclonal Antibodies

Keywords

  • High density lipoproteins
  • Lipoproteins
  • Quantitation
  • Western blot

ASJC Scopus subject areas

  • Endocrinology

Cite this

Development of an immunoblot assay with infrared fluorescence to quantify paraoxonase 1 in serum and plasma. / Connelly, Philip W.; Maguire, Graham F.; Picardo, Clive M.; Teiber, John F.; Draganov, Dragomir.

In: Journal of Lipid Research, Vol. 49, No. 1, 01.2008, p. 245-250.

Research output: Contribution to journalArticle

Connelly, Philip W. ; Maguire, Graham F. ; Picardo, Clive M. ; Teiber, John F. ; Draganov, Dragomir. / Development of an immunoblot assay with infrared fluorescence to quantify paraoxonase 1 in serum and plasma. In: Journal of Lipid Research. 2008 ; Vol. 49, No. 1. pp. 245-250.
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