IMMUNOFILTRATION PURIFICATION FOR URINARY LEUKOTRIENE E4 QUANTITATION

Leukotriene E4 (LTE4) is a major leukotriene metabolite in urine. Urin ary LTE4 concentration is often utilized as an index of total leukotri ene synthesis. A novel method employing immunofiltration for the purif ication of urinary LTE4 was developed. This immunofiltration method is based upon the addition of excess anti-LTE4 antibody to urine which b inds LTE4. Separation of bound LTE4 (high M-r) from high levels of unb ound contaminants (low M-r) is then accomplished by filtration through a 10,000 M-r cut-off filter. The LTE4-antibody complex is separated b y precipitation of the antibody with methanol which is subsequently re moved by centrifugation. Following evaporation of the methanol, enzyme immunoassay is utilized for quantitation. This methodology was valida ted by determining the recovery of tritiated and unlabeled LTE4 added to urine and buffer and by comparison of results obtained with urine s amples measured after HPLC purification (correlation r(2) = 0.72). Rep roducibility 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 met hod were found to be identical to levels determined by HPLC/immunoassa y. The ease and accuracy of this assay make it amenable for the analys is of large numbers of samples. (C) 1997 Academic Press.