By John N. Abelson, Melvin I. Simon, Robert C. Murphy, Frank A. Fitzpatrick

This quantity comprises 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 thoughts, are offered. training and research of cytochrome P-450 metabolites of arachidonic acid are mentioned. The isolation, purification, and assay of enzymes vital within the biosynthesis and metabolism of those lipids are incorporated

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Meese, O. Furst, P. G. Kuhl, and H. W. Seyherth, Anal. Biochem. 164, 156 (1987). 29 K. A. Waddell, S. E. Barrow, C. Robinson, M. A. Orchard, C. T. Dollery, and I. A. Blair, Biomed. Mass Spectrom. U , 68 (1984). 3o C. R. Pace-Aseiak and S. Micallef, J. Chromatogr. 310, 233 (1984). 31 j. D. Sweatt, I. A. Blair, E. J. Cragoe, and L. E. Limbird, J. Biol. Chem. 261, 8660-8666 (1986). [2] NEGATIVE-ION MASS SPECTROMETRY OF LIPIDS 19 leased arachidonate was converted to PGD2, PGE2, and PGF2~. Quantification of the PGs by capillary column G C - N C I - M S allowed the size of the initial pool of released arachidonate to be quantified in the presence of a relatively high background of TxB2.

With growing concerns of the use of radioactivity in the laboratory and its inherent costs, an enzyme label is an interesting option. Furthermore, the techniques described are technically straightforward and have a potential for automation, representing a major advantage over radioimmunoassay. Although the acetylcholinesterase tracers provide very high sensitivity, all guidelines of validation for immunoassays in a particular biological fluid should be strictly applied before one can rely on the quantitative information provided by this technique.

Add 20 ml potassium phosphate buffer 5 x 10-z M to 200 p~g of monoclonal IgG, and distribute 200 /xl of this solution into each well using the automatic dispenser. (Note: Considerable time can be saved by using an automatic dispensing system and a microplate washer. ) Cover with plastic film. Allow this to incubate at least 18 hr at room temperature prior to the saturation step. Saturation of Plates. 3 g/liter sodium azide. Cover with microtiter [3] ENZYME IMMUNOASSAY OF EICOSANOIDS 27 plate, plastic film, and leave for at least 18 hr at 4 ° before use.

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