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| | LTA4 hydrolase assay |
| Description: | LTB4 formed was quantified with the HTRF assay in which free LTB4 competes with LTB4-XL665 conjugate (acceptor) for anti-LTB4 monoclonal antibody labeled with Europium cryptate (donor), thereby inhibiting the fluorescence energy transfer.The LTB4 HTRF 384-well assay was carried out by incubating LTB4 samples or standards with LTB4-XL665 conjugate (7.5 ng/well) and anti-LTB4 monoclonal antibody-Europium cryptate conjugate (0.5 ng/well) in 50 mM phosphate, 0.4 M KF and 0.1% casein, buffer (pH 7.0) for two hours at ambient temperature. Plates were read in a RubyStar plate reader (BmG Labtechnologies Inc., NC) simultaneously at 620 nm and 665 nm to obtain signal ratios of 665 nm/620 nm. Results of energy transfer were expressed as delta F (%) which equaled [(signal ratio of sample−signal ratio of negative control)/(signal ratio of negative control)]×100%. Negative controls were control samples without LTB4 or LTB4-XL665. |
| Affinity data for this assay | |
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