Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving virus or bacteriophage
Reexamination Certificate
1997-07-25
2002-11-05
Wortman, Donna C. (Department: 1648)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving virus or bacteriophage
C435S006120, C435S007800, C435S007940, C436S501000, C436S518000
Reexamination Certificate
active
06475717
ABSTRACT:
The invention relates to a method for detecting and determining mediators and/or their derivatives.
Mediators are, e.g., interleukins, such as IL-1, TNF, IL-8 or IL-4, cytokinos, such as GM-CSF, G-CSF or Meg-CSF, or alternatively erythropoietin. Mediators are important signal proteins which are secreted by particular cells of the body, such as lymphocytes, and can act in a regulatory manner on these same cells or on other cells of the body. Such mediators can exert this regulatory function at very low concentration. In pathological states of disease, the natural cooperation of different mediators can be disturbed. Under these circumstances, mediators can, compared to average concentrations of these mediators in average healthy persons, be present at higher or lower concentration. From the diagnostic point of view, it is important to determine the concentration of particular mediators in body fluids or other sources, such as organ homogenates. Moreover, patients can be treated with particular mediators for therapeutic or prophylactic purposes. Here, too, it is of great interest from the diagnostic point of view to determine the concentration of the mediator which has been administered, or of other mediators whose concentrations are altered through the influence of the administered mediator. Thus, for example, the appearance of IL-4 in the persons with allergic diseases or infections can be altered in a pathological manner as compared with healthy persons. The reason for this can be the increased appearance, often occurring in such diseases, of T-lymphocytes of the TH2 subpopulation, which, as compared with the TH1 subpopulation, preferentially produces inter-leukin-4 (S. Romagnani, Immunology Today, Vol. 12, No. 8, 256-257, 1991; Else and Grencis, Parasitology Today, Vol. 7, No. 11, 313-316, 1991). In addition, it is important from the diagnostic point of view to determine mediators in supernatants of cell cultures of lymphocytes or other cells.
Mediators, such as those listed above, exert their positive or negative effect via receptors located in the membrane. These receptors bind, via a defined binding site, to a defined epitope of a mediator. Subsequently, a signal transduction takes place via the receptor and/or associated molecules into the cell in which a biological effect takes place as a result. The biological effect of such a mediator is consequently strictly linked to optimal binding to the binding site on the receptor located in the membrane. For example, substances or mediators which do not bind directly to the binding site, but instead to other epitopes on the receptor, may not trigger any signal transduction.
It has now been found, surprisingly, that recombinant, soluble receptors for these mediators can be used for detecting the presence and the concentration of mediators in liquids.
The invention therefore relates to a method for detecting and determining mediators and/or their derivatives in liquids where a recombinant, soluble receptor for the mediator to be detected is brought into contact with a sample, which can contain the mediator, and the mediator bound to the receptor is detected directly or indirectly by an antibody which is specific for the mediator.
In an advantageous method, dimers or multimers of the mediator, or derivatives thereof, are determined.
In a further advantageous method, the receptor, or derivatives thereof, is bound to a solid phase.
In another advantageous method, a recombinant fusion protein comprising a receptor and the Fc moiety of antibodies, or derivatives thereof, is bound to the solid phase via Fc-specific antibodies.
In a particularly advantageous method, the receptor, or derivatives thereof, is bound to the solid phase via specific antibodies, and, subsequently, sample material containing the corresponding mediator to be determined is applied, and the mediator is detected using a labeled antibody or antiserum having specificity for the mediator.
In a very particularly advantageous method, a recombinant fusion protein comprising a receptor and the Fc moiety of antibodies, or derivatives thereof, is bound to the solid phase via Fc-specific antibodies and, subsequently, sample material containing the corresponding mediator to be determined, or derivatives thereof, is applied, and the bound mediator is detected using a labeled antibody or antiserum having specificity for the mediator.
In another advantageous method, conjugates comprising a receptor, or derivatives thereof, coupled to substances which are suitable for the detection in suitable measuring systems are used to detect a mediator, and/or derivatives thereof, which is bound to a solid phase, for example via specific antibodies.
The invention furthermore relates to the use of such a method for screening for agonists or antagonists of the mediator or of the receptor.
The invention also relates to the use of such a method for determining the affinity between a mediator and its receptor.
The invention likewise relates to the use of such a method for identifying and analyzing modified mediators (“muteins”) or parts of the mediators (for example oligopeptides).
In this context, it is advantageous to use the method for identifying and analyzing substances which influence the interaction of pathogenic organisms (for example viruses or bacteria) with their cellular receptors. It is particularly advantageous to use such a method for identifying substances which influence the interaction of cellular adhesion molecules.
In a particularly advantageous method, the receptor is a cytokine receptor, a growth hormone receptor, a hormone receptor, a neurotransmitter receptor, or a cellular receptor for a pathogenic organism, e.g. a virus.
In a very particularly advantageous method, the receptor is the interleukin-4 receptor or a derivative thereof, an erythropoietin receptor, or a derivative thereof, the interleukin 1 receptor type I or type II or a derivative thereof, the interleukin 7 receptor or a derivative thereof, the GM-CSF receptor or a derivative thereof or the IL-8 receptor or a TNF receptor or a derivative thereof.
The invention also relates to a method of this type for the diagnostic detection of interleukin-4 in diseases exhibiting an increased appearance of TH2 T-cells, for example allergic diseases and infections.
The use of the natural, biologically important, receptor binding site for detecting the corresponding mediator is particularly advantageous since only those mediator molecules are determined which would also bind to the natural receptor, located in the membrane, and consequently are biologically active.
For the purposes of this invention, receptors are also understood to mean all variants and derivatives which are capable of binding the corresponding mediator specifically.
The detection can be carried out by binding the recombinant soluble receptor, under suitable conditions, to the solid phase of the detection system, for example the synthetic material of a microtitration plate. Such solid phases are known per se to the person skilled in the art. Advantageous solid phases are: magnetic particles, particles composed of natural or synthetic polymers, e.g. so-called latex particles or ion-exchange resins or synthetic polymers in the form of covalent or convex articles, such as, e.g., microtitration plates or spheres. Magnetizable particles, latex particles or microtitration plates are particularly advantageous. Microtitration plates are very particularly advantageous. Although the receptor is not present in the form in which it is located within the membrane, it surprisingly binds the corresponding epitope on the corresponding mediator with the affinity of the natural, membrane-bound receptor. The bound mediator can then be detected by methods which are known per se to the person skilled in the art. For this, the bound mediator can be coupled with one or more monoclonal antibodies, which are directed against a further epitope on the mediator, or an antiserum, which is directed against several further epitopes on the mediator.
In order to detect a bound antiserum or
Enssle Karlheinz
Kurrle Roland
Langner Klaus-Dieter
Lauffer Leander
Pauly Josef-Urban
Dade Behring Marburg GmbH
Finnegan, Henderson Farabow, Garrett and Dunner L.L.P.
Wortman Donna C.
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