Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Patent
1993-06-01
1996-01-30
Scheiner, Toni R.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
435 79, 435 71, 435 793, 435 75, 436530, 436531, 436544, 5303882, 53038823, 53038824, G01N 3353, G01N 33543
Patent
active
054879779
DESCRIPTION:
BRIEF SUMMARY
The present invention concerns a method for the determination of sulfidoleukotrienes in tissues and biological fluids and its use in the diagnosis of allergies and other inflammatory diseases.
The release of inflammatory mediators by various types of blood or tissue cells upon interaction with various stimulants is a common feature of inflammatory processes occurring in various acute or chronic diseases, such as rheumatic or kidney diseases. In allergic reactions, the release of histamine by blood basophils and/or tissue mast cells has long been considered a major feature. The determination of histamine in supernatants from suspensions of isolated blood leukocytes from allergic patients in vitro, following interaction with allergens to which they are sensitive, is a procedure which has been extensively used in allergy research. However, the fact that such determinations require numerous manipulations, cannot be effected in whole blood, but only on isolated cells, and that histamine determination requires cumbersome and expensive fluorometric or radioimmunoasssays have prevented up to now the routine diagnosis of allergies to rest upon blood cellular assays.
The only current diagnostic method in vitro widely used is the serologic determination of allergen-specific IgE antibodies, by various types of radio-immunological or immunoenzymatic assays (e.g. RAST assay). Such assays, however, only detect the occurrence of antibodies, but do not reflect the most relevant pathophysiological feature of the allergic reaction, namely the production of inflammatory mediators by the reactive cells upon interaction with the responsible allergen(s). For that reason, practical and fiable cellular assays would be most desirable for the routine diagnosis of allergic and other inflammatory diseases. The object of the present invention is to provide a series of novel cellular assays enabling to achieve that purpose.
The sulfidoleukotrienes (sLT) LTC4, LTD4 and LTE4 are inflammatory mediators, which were previously collectively denominated Slow Reactive Substance of Anaphylaxis (SRS-A). They are synthesized in many cell types, such as tissue mast cells, blood basophils, macrophages, eosinophils and kidney mesangial cells. They play an important role in pathological events of inflammation and allergic reactions, particularly in IgE-mediated allergic reactions (Schleimer et al. J. Allergy clin. Immunol., 74, 473-481, 1984). It is of particular interest that blood basophils generate sLT in response to allergens in an IgE-dependent manner (Mita et al. Prostaglandins, 869-886, 1986), and that such basophils generate sLT in response to non-specific stimulants when pre-treated with the cytokines IL-3, IL-5 and GM-CSF (Bischoff et al. J. Exp. Med. 172, 1577, 1990). Theoretically, therefore, on the basis of current knowledge, determination of basophil sLT production in response to suspected non-specific stimulants could be of interest in the diagnosis of allergies. In practice, however, this goal has not yet been achieved, essentially because of multiple technical difficulties. According to the present invention a method is provided which comprises a combination of various, in part novel and original features, by which a routine in vitro cellular diagnostic assay for allergies and other inflammatory diseases can be established.
The subject of the present invention is thus a method for detecting the sulfidoleukotrienes sLT of the group LTC4, LTD4 and LTE4 by a single immunoenzymatic ELISA assay, based on the interaction between one or several monoclonal anti-sLT antibodies and a sLT conjugated to a revealing enzyme, which method is characterized in that a biological sample, wherein a content of sLT is assumed, is contacted with a monoclonal anti sLT-antibody bound to a carrier, the sLT present in the sample are bound to the said antibody, subsequently a conjugate of a sLT with a revealing enzyme is added to the charged carrier and eventually the amount of the conjugate bound to the carrier is evaluated, which amount is in an inverse correl
REFERENCES:
patent: 4559310 (1985-12-01), Cantor et al.
Reinke et al., 1991, A Monoclonal Antibody Against the Sulfidopeptide Leukotrienes LTC.sub.4, LTD.sub.4, and LTE.sub.4.
Harlow et al., 1988, Antibodies A Laboratory Manual Cold Spring Harbor Laboratory, pp. 584-587, 591-593, 343.
Johns et al., 1974 Microcalorimetry as a potential tool in the study of antibody-antigen reaction systems incorporating a cellular element I Immunol Meth 4:83-106.
Bischoff et al, 1990, Interleukin 5 modifies histamine release and leukotriene generation by human basophils in response to diverse agonists J Exp Med. 172:1577-82.
Corey, 1982, Chemical studies on slow reacting substances/leurotrienes Experentia 38:1259-75.
Margot Reinke et al., "A Monoclonal Antibody Against the Sulfidopeptide Leukotrienes LTC.sub.4, LTD.sub.4 and LTE.sub.4 ", Biochimica et Biophysica ACTA 1081:274-278, 1991.
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