Immunofiltration purification for urinary leukotriene E4 quantitation

Analytical Biochemistry
J Y WestcottS E Wenzel

Abstract

Leukotriene E4 (LTE4) is a major leukotriene metabolite in urine. Urinary LTE4 concentration is often utilized as an index of total leukotriene synthesis. A novel method employing immunofiltration for the purification of urinary LTE4 was developed. This immunofiltration method is based upon the addition of excess anti-LTE4 antibody to urine which binds LTE4. Separation of bound LTE4 (high M(r)) from high levels of unbound contaminants (low M(r)) is then accomplished by filtration through a 10,000 M(r) cut-off filter. The LTE4-antibody complex is separated by precipitation of the antibody with methanol which is subsequently removed by centrifugation. Following evaporation of the methanol, enzyme immunoassay is utilized for quantitation. This methodology was validated by determining the recovery of tritiated and unlabeled LTE4 added to urine and buffer and by comparison of results obtained with urine samples measured after HPLC purification (correlation r2 = 0.72). Reproducibility of the assay was assessed by analyzing the same sample on two different days (standard deviation of 18%). The mean urinary LTE4 levels in healthy subjects and asthmatics measured utilizing this method were found to be identical to levels determined by H...Continue Reading

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Citations

Nov 21, 1998·Clinical and Experimental Allergy : Journal of the British Society for Allergy and Clinical Immunology·R Dworski, J R Sheller
Apr 15, 2004·Clinical and Experimental Allergy : Journal of the British Society for Allergy and Clinical Immunology·N L A MissoP J Thompson
Aug 7, 1999·Clinical Reviews in Allergy & Immunology·J Y Westcott
May 28, 2009·Journal of Separation Science·Elisabeth Viktoria ReiterEbrahim Razzazi-Fazeli
Jul 8, 1998·Prostaglandins & Other Lipid Mediators·J Y WestcottS E Wenzel

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