Simplified sulfidoleukotriene ELISA using LTD4-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 detection limit of this assay was 6.3 +/- 1.2 pg sLT/100 microliters (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 commercial radioimmunoassay (r = 0.97; n = 43) confirmed the validity of this 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 contrast to another sLT ELISA using the same antibody but based on competition with solid-phase LTE4-BSA, our assay is about 7-fold more sensitive. It also requires about 40 times less leukotriene for conjugate production than the previous method for the same number of determinations. The assay allows measurement of sLTs generated by small numbers of basophils 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.
Furukawa
K
K
Institute of Clinical Immunology, University of Bern, Switzerland.
Tengler
R
R
de Weck
A L
AL
Maly
F E
FE
D000469:Alkaline Phosphatase; D000485:Allergens / Q000494:pharmacology*; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D039741:Antigens, Dermatophagoides; D001491:Basophils / Q000187:drug effects / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005786:Gene Expression Regulation / Q000187:drug effects*; D006023:Glycoproteins / Q000494:pharmacology; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000187:drug effects / Q000378:metabolism*; D017998:Leukotriene D4 / Q000276:immunology*; D051379:Mice; D015203:Reproducibility of Results; D012130:Respiratory Hypersensitivity / Q000097:blood / Q000209:etiology; D013189:SRS-A / Q000096:biosynthesis / Q000097:blood*; D012680:Sensitivity and Specificity
D000485:Allergens; D000911:Antibodies, Monoclonal; D039741:Antigens, Dermatophagoides; D006023:Glycoproteins; D013189:SRS-A; D017998:Leukotriene D4; D000469:Alkaline Phosphatase