Development of enzyme immunoassays for leukotrienes using acetylcholinesterase.

ABSTRACT

We have developed sensitive solid phase enzyme immunoassays (EIA) to analyze quantitatively leukotrienes (LTs) using acetylcholinesterase from Electrophorus electricus as a label for LTB4, LTC4 and LTE4. However, because of problems specific to LTs, we used different coupling procedures to prepare LTs conjugates necessary for the production of antibodies and for the preparation of enzymatic tracers. For the immunogens, all LTs were coupled to bovine serum albumin using glutaraldehyde (ethylene diamine was used to add an amino group to LTB4). Immunizations in rabbits were done following classical procedures. For the enzymatic tracers, succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate was selected to conjugate the LTs via their amino groups to acetylcholinesterase. Titers of the different antisera ranged from 130,000 (LTE4), 140,000 (LTC4) to 150,000 (LTB4) and sensitivities (IC50) were 5.5 pg, 4.3 pg and 2.4 pg, respectively. Cross reactivities were also examined against other LTs. Sensitivities and specificities of the different systems were dependent on the conditions of incubation (temperature). Validation of the technique was done (i) after spiking known amounts of LTC4 in plasma and measuring the substance added after prior extraction and purification, (ii) by analyzing the supernatant of human neutrophils suspended in buffer or in plasma, (iii) by measuring LTE4 in urine. Due to the background provided by these complex matrixes, quantitation was performed after addition of [3H]LTs for recovery, protein precipitation, extraction by Sep-PakR and purification by HPLC. Measurement of LTs can be done in biological fluids with the same ease and advantages as other enzyme immunoassays that we have previously developed for eicosanoids analysis.