Development of Enzyme-Immunoassay Techniques for Measurement of Eicosanoids
Enzyme immunoassays of eicosanoids have been developed in recent years. Although the assays are quite different in the enzyme label and the type of development, they represent a major conceptual change for the immunoanalysis of these compounds. We have coupled covalently acetylcholine esterase from electrophorus electricus to various eicosanoids, including PGE2, PGD2, PGF2α, 6-keto-PGF1α, TXB2, dinor-TXB2, ll-dehydro-TXB2, and LTC4. Using 96-well microtiter plates coated with the second antibody, we could obtain results equal to or better than those obtained with corresponding [125I]-radioiodinated tracers when available. The high sensitivity of this method combined to semiautomation may prove to be a useful tool to investigate the level of these substances from various biological fluids. In addition, this enzyme has been coupled successfully to other substances, suggesting that it could be used as a general label to produce nonisotopic tracers.
Widespread interest concerning the various oxygenated metabolites of arachidonic acid has led to the development of various techniques to monitor the in vitro or in vivo production of these compounds. Two analytical techniques have been complementary in obtaining quantitative information on these compounds. The first, gas chromatography coupled to mass spectrometry (GC/MS), has allowed structural information on all these molecules (1). GC/MS has been used quantitatively with internal standards; further, extreme sensitivities by means of negative-ion chemical ionization have permitted the use of GC/MS to monitor these substances in biological fluids. Due to the specificity of the simultaneous measurement of several ions, GC/MS is used as a reference for all quantitative assays. However, the cost of equipment and the high degree of expertise required to use this technique do not promote its widespread use (2). In addition, GC/ MS does not lend itself to the analysis of a serial number of samples.
The second technique, based on immunoanalysis of these compounds, is the more widespread one. However, a major limitation is its validation for specificity to ensure proper use as a reliable quantitative technique (see later). A number of radioimmunoassays (RIA) have therefore been developed for prostaglandins, thromboxanes, and the recently described leukotrienes. Further, the need to get high sensitivity by increasing the specific radioactivity of the tracer has been circumvented by the use of iodinated tracers for these molecules which, in addition to their high specific activity, avoid quenching problems and decrease counting time (3). However, the use of RIA entails certain disadvantages; among them are the limited half-life of reagents susceptible to radiolysis, increasing expenses in developed countries inherent in the rigid rules imposed for radioactive isotopes, cost of tubes, scintillation cocktails associated with radioactive disposal, and high personnel requirement per sample due to its limited automation.
All these reasons explain why, during these recent years, users of immunoassays have stressed the need to obtain nonradioactive labels. Immunoassays for eicosanoids have not escaped these evolutions, and since 1981, a consequent number of enzyme immunoassays (EIA) have been developed. In this paper, we briefly review these different approaches, including our own experience.
KeywordsEnzyme Immunoassay High Specific Activity Urinary Metabolite Enzyme Label Bovine Serum Albumin Conjugate
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