反应详情

温度

-70 °C

Aliquots (10 mL) of thawed urine are centfifuged (10,000×g), and the supernatant adjusted to pH 5.4 with 100 μL glacial acetic acid. As a recovery standard, 3 nCi of [14,15,19,20-3H]-LTC4 (12 pg) is added. Samples are applied to a 3 gm particle C18 precolumn, and washed with 2 volumes of 0.1% NH4OAc buffer pH 5.4. Peptide leukotrienes are then eluted onto a C18 analytical HPLC column, and separated with a 66% MeOH/34% 0.1% NH4Ac pH 5.4 (v/v) mobile phase containing 1 mM EDTA. Fractions eluting with the retention time of synthetic LTC4 (obtained from daily calibration with standards) are collected for estimation of [3H]-LTC4 recovery by scintillation counting. Prior experiments established that recoveries of [3H]-LTC4 and [3H]-LTE4 from dog urine after RP-HPLC are comparable (86.8±1.9% and 83.1±6.1% respectively). In some experiments synthetic LTE4 (0.5 ng/mL) and/or 0.1 nCi [3H]-LTE4 (0.4 pg) are added to certain samples to identify the exact retention time of LTE4. Fractions (0.75 min, 0.75 mL) eluting before, during and after the predicted retention time of synthetic LTE4 (from daily calibration) are collected into sequential wells in a polypropylene microtitre plate, aliquots (200 μL) are removed to identify the retention time of added [3H]-LTE4), and the remainder frozen to -70° C. and lyophilised in a vacuum centrifuge. Fractions are redissolved in 50 μL of 20 mM Na2PO4 pH 7.2 containing 0.9% NaCl, 0.02% sodium azide, 0.1 mM phenyl methyl sulphonyl fluoride and 1% gelatin and mixed with 2-3 nCi of [14,15,19,20-3H]-LTE4 (5.2-7.8 pg) and an anti-LTC4 mouse monoclonal antibody (21% cross-reactivity with LTE4 ; final dilution 1/150,000) and incubated for 2 h at 21° C. Free ligand is precipitated by addition of dextran coated charcoal and centrifugation. An aliquot of the supernatant is removed and the concentration of LTE4 immunoactive material estimated by comparison of the unknown bound [3H]-LTE4 against a standard curve derived by serial dilution of a synthetic LTE4 stock solution (4000-7.8 pg/tube). LTE4 concentration is calculated as the immunoreactive material (pg) in n co-eluting fractions--n×average background immunoreactive material (pg) in pre- and post-LTE4 fractions, corrected for [3H]-LTC4 recovery, and the fraction volume removed for estimating the retention time of added [3H]-LTE4. Urinary LTE4 excretion (ng/hour) is then calculated from the concentration and excretion volume, and related to values obtained during the first collection on a case by case basis. % inhibition of baseline LTE4 is calculated for the 5-6 and 6-7 h time points, and the mean value obtained for the treatment group. An ED50 is then calculated using these values and the infusion dose by non-linear regression analysis (4 parameter fit).

1. washwashed with 2 volumes of 0.1% NH4OAc buffer pH 5.4
2. washPeptide leukotrienes are then eluted onto a C18 analytical HPLC column
3. customseparated with a 66% MeOH/34% 0.1% NH4Ac pH 5.4 (v/v) mobile phase
4. additioncontaining 1 mM EDTA
5. washFractions eluting with the retention time of synthetic LTC4 (
6. customobtained from daily calibration with standards)
7. customare collected for estimation of [3H]-LTC4 recovery by scintillation
8. additionIn some experiments synthetic LTE4 (0.5 ng/mL) and/or 0.1 nCi [3H]-LTE4 (0.4 pg) are added to certain samples
9. washFractions (0.75 min, 0.75 mL) eluting before, during and after the predicted retention time of synthetic LTE4 (from daily calibration)
10. customare collected into sequential wells in a polypropylene microtitre plate, aliquots (200 μL)
11. customare removed
12. dissolutionFractions are redissolved in 50 μL of 20 mM Na2PO4 pH 7.2
13. additioncontaining 0.9% NaCl, 0.02% sodium azide, 0.1 mM phenyl methyl sulphonyl fluoride and 1% gelatin
14. additionmixed with 2-3 nCi of [14,15,19,20-3H]-LTE4 (5.2-7.8 pg)
15. customFree ligand is precipitated by addition of dextran coated charcoal and centrifugation
16. customAn aliquot of the supernatant is removed
17. concentrationLTE4 concentration
18. customthe fraction volume removed
19. concentrationUrinary LTE4 excretion (ng/hour) is then calculated from the concentration and excretion volume
20. customobtained during the first collection on a case by case basis
21. customis calculated for the 5-6 and 6-7 h time points
22. customthe mean value obtained for the treatment group