SIMPLIFIED SULFIDOLEUKOTRIENE ELISA USING LTD(4)-CONJUGATED PHOSPHATASE FOR THE STUDY OF ALLERGEN-INDUCED LEUKOTRIENE GENERATION BY ISOLATED MONONUCLEAR-CELLS AND DILUTED WHOLE-BLOOD

By using leukotriene D4 (LTD4)-labeled alkaline phosphatase (LTD4-AP) and a mouse monoclonal anti-sulfidoleukotriene (sLT) antibody (1A-LDR1 ), we developed a solid-phase competition ELISA for sLTs. The detectio n limit of this assay was 6.3 +/- 1.2 pg sLT/100 mul (mean +/- SEM; n = 10). Intraassay variations at 15, 50 and 150 pg sLT/well (9.8, 13.5 and 12.3%, respectively), the corresponding interassay variations (32. 1, 11.9 and 18.8%, respectively) and the good correlation with a comme rcial radioimmunoassay (r = 0.97. n = 43) confirmed the validity of th is ELISA. Furthermore, the assay had a low background and gave a 70-90 % recovery of LTD4 added to medium containing up to 10% serum. In cont rast to another sLT ELISA using the same antibody but based on competi tion with solid-phase LTE4-BSA, our assay is about 7-fold more sensiti ve. It also requires about 40 times less leukotriene for conjugate pro duction than the previous method for the same number of determinations . The assay allows measurement of sLTs generated by small numbers of b asophils present in isolated mononuclear cells or diluted whole blood in response to allergen, and allows simple and rapid determination of basophil reactivity to suspected allergens.