By John N. Abelson, Melvin I. Simon, Robert C. Murphy, Frank A. Fitzpatrick
This quantity contains details on arachidonic acid metabolites and platelet activating issue. Assays for the quantitative dimension of prostaglandins, leukotrienes, and platelet-activating issue, together with enzyme immunoassays and mass spectrophotometric strategies, are offered. guidance and research of cytochrome P-450 metabolites of arachidonic acid are mentioned. The isolation, purification, and assay of enzymes very important within the biosynthesis and metabolism of those lipids are integrated
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Extra resources for Arachidonate Related Lipid Mediators
I 1 10 100 pg FIG. 4. Effect of the temperature on the sensitivity of the TxB2assay. The antiserum was used at 1/150,000 and the immunological reaction performed for 18 hr. 15 8 5 4 13 9 9 5 6 5 Specificity of Assay: Validation with Gas ChromatographyMass Spectrometry The optimal way of establishing the specificity of an immunoassay should be performed by an independent assay method in the relevant biological fluid. Comparison with gas chromatography-mass spectrometry represents an excellent validation criterion.
000 . . ° . 1/10,000 Antiserum . . . . . . . . . 1/100,000 dilution FIG. I. Titration of TxB2 antiserum. Titration was performed at room temperature as described in the text. The time for the immunological reaction are indicated in the box. Results are expressed in Bo (percentage of bound tracer in the absence of competitor) as a function of the final dilution of antiserum. 5-2 ng/ml depending on the eicosanoid and on the antiserum). See considerations on stability in Ref.
Samples and standards are followed for 20 min in order to allow equilibration to take place. 5% formic acid. The sample is loaded on a PrepSep cartridge that has been preconditioned by washing with 10 ml of methanol followed by 10 ml of water. The PrepSep cartridge is washed with water (5 ml), methanol/water (5 ml; 2 : 3, v/v), and dried under vacuum from a water pump for 5 min. 7 ml) and the ethyl acetate evaporated. A solution of methoxyamine hydrochloride in pyridine (50/~1; 1:99, w/v) is added to the residue and allowed to stand overnight at room temperature.