Chemistry: analytical and immunological testing – Involving an insoluble carrier for immobilizing immunochemicals
Patent
1989-01-06
1992-07-07
Kepplinger, Esther L.
Chemistry: analytical and immunological testing
Involving an insoluble carrier for immobilizing immunochemicals
436504, 436548, 436 18, 436174, 436804, 436826, 435 724, 435962, 435968, 424 852, 424 857, G01N 33543
Patent
active
051282704
DESCRIPTION:
BRIEF SUMMARY
The present invention relates, in a general manner, to assays of monokines. More specifically, the present invention relates to the assay of endogenous monokines in a biological fluid, in particular a blood sample.
Monokines belong to the group of cell mediators known as cytokines. Among cytokines, there are distinguished, in effect;
lymphokines, soluble mediators secreted by lymphocytes and activating either macrophages or other lymphocytes, and
monokines, mediators secreted by monocytes or macrophages and acting either on cells of the lymphocytic line or on cells of mesenchymal origin.
The monokines to which the present invention relates consist, in particular, of tumor necrosis factor (TNF.alpha.) and interleukin-1 (IL-1.beta.). These two cell mediators are, in effect, known to have marked similarities in their behavior, such as their kinetics of release, or in their biological action ((1) see bibliographic references).
Apart from their aspect of anticancer or antiviral factors, it is known that TNF.alpha. and IL-1.beta. are secreted in different pathological conditions, especially, as regards TNF.alpha., in inflammatory states and during septic and pancreatic shock. More generally speaking, the secretion of TNF.alpha. and IL-1.beta. discloses a macrophage activation. Accordingly, the possibility of following this secretion of TNF.alpha. or of IL-1.beta. by a simple, reliable and rapid assay can be a source of very enlightening information concerning the functional aspect of macrophages in many pathological situations.
When the immunoassay of cytokines in a blood sample is contemplated, certain problems are encountered. Often, the sensitivity of the assays is regarded as insufficient for detecting these mediators directly in plasma or in blood serum, the quantities present to be assayed being too small. The problem can also arise from the nature of the values obtained, utilizable only with difficulty, where appropriate, the serum or plasma values varying little or in a non-significant manner from subject to subject, or between healthy subjects and sick subjects. This applies especially to lymphokines (IFN.gamma. (2) and IL-2 (3 a-b)).
As regards TNF.alpha., as will be seen later, (Example V: Clinical assessment), RIA immunoassay, which is, however, very sensitive, does not permit the direct detection in plasma of concentrations below 20 pg/ml, even with assay incubation times of 24 hours. Furthermore, if this time is reduced to 4 hours, which is more in accordance with practical requirements, this concentration detection limit rises to approximately 100 pg/ml.
As regards IRMA assays, which require incubations of only 2 hours, they do not, however, permit the direct detection of the presence of monokines in plasma.
The concentrations of TNF.alpha., obtained by direct RIA assay in plasma, are on average of the order of 50 pg/ml for healthy subjects, and do not exceed 200 pg/ml in most pathological conditions. These direct RIA assays in plasma are, however, utilizable only with difficulty.
In effect, values are obtained which vary little from one subject to another, even if the difference may be significant between a healthy subject and a subject suffering from a pathological condition in some cases. In addition, it is generally considered that the quantities to be assayed must be markedly greater (4- to 5-fold at least) than the detection limit value of the assay in order to offer a satisfactory discrimination of assaying, that is to say the possibility of large variations from subject to subject. These two conditions are hence not complied with.
Traditionally, the solution has consisted in assaying the cytokines in a blood sample by resorting to a stage of cell culturing in order to stimulate cytokine secretion ((2) IFN.gamma., (3 a-b) IL-2, (4) TNF.alpha., (5) IL-1).
The assays then consist in assaying the release of cytokines into culture media stimulated by the addition of a mitogenic agent, for example in assaying monokines on the supernatant of monocytes of peripheral blood cultured in the presence
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De Groote Donat
Dehart Isabelle
Delacroix Dominique
Franchimont Paul
Gysen Philippe
Bidwell Carol E.
Ire-Medgenix S.A.
Kepplinger Esther L.
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