Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1998-10-06
2004-11-30
Chin, Christopher L. (Department: 1641)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S007500, C435S007230, C435S007240, C435S007920, C435S375000, C435S287200, C436S506000, C436S507000, C436S512000, C436S513000, C436S548000, C436S177000
Reexamination Certificate
active
06824986
ABSTRACT:
FIELD OF THE INVENTION
The invention relates to methods for monitoring immunological function in a human or animal. More particularly, the present invention relates to accurately measuring the production of cytokines in vivo.
BACKGROUND OF THE INVENTION
There have been many attempts to measure endogenous cytokines in blood and other body fluids. However, in reviewing these reports, it is apparent that there is wide variation in the reported results with regard to cytokine concentration in the blood and to fluctuations of cytokine concentration in the blood. Measurement of cytokine production in vivo is an important part of many experimental and therapeutic studies. Interpretation of results has been difficult, however, for several reasons:
1. Studies that examine cytokine secretion ex vivo are relatively easy to perform, but may not reflect cytokine production in vivo.
2. Cytokine levels in serum or other biological fluids can be measured in some cases; however, levels are often negligible because many cytokines have a very short in vivo half-life. In addition, increased absorption of cytokines by receptors on activated cells may accelerate catabolism of cytokines, so that serum levels will not correlate directly with secretion.
3. Cytokine mRNA levels can be measured directly after removal of cells or organs, and presumably, accurately reflect in vivo cytokine mRNA levels; however, protein production and secretion doesn't always vary directly with mRNA levels.
4. Cytokine-containing cells have been identified by staining procedures; however, the small number of such cells in the absence of in vitro restimulation and subjectivity in identifying such cells by staining techniques place limits on the usefulness of these techniques. In addition, it is not known if all cytokine-secreting cells have identical levels of stored, intracellular cytokine, so that intensity of staining and rate of secretion do not necessarily correlate directly.
5. ELISPOT assays for cytokine-secreting cells can be performed on cells right after they are removed from an animal; however, enumeration of cytokine-secreting cells with this technique is somewhat subjective, and this technique may detect only those cells that secrete the largest amount of cytokine, may favor selective detection of those cells that contain stores of a preformed cytokine, and, even if the assay involves only a short incubation period, may not reflect in vivo cytokine production because of the disruption of normal architecture, the possibility that some activated cells may be difficult to remove intact from organs, the possibility that the trauma of preparing single cell suspensions may modify cytokine secretion, etc.
Many reports indicate that cytokines (i.e., IL-2) are not detectable in normal subjects using immunoassays. Cytokines are difficult to measure in serum for several reasons: 1) rapid renal excretion; 2) catabolism; 3) cellular utilization; and 4) binding to molecules, such as soluble receptors that mask the cytokine active site.
It would be of great benefit if one could easily, accurately and reproducibly measure in vivo cytokine production by sampling body fluids, such as blood. This would create a useful window not only into the immune system but into a myriad of physiologically interacting processes. Such a tool would be useful in a variety of settings, allowing the collection of data of importance to basic medical sciences, clinical medicine, epidemiology and the forensic sciences.
U.S. Pat. No. 4,486,530, David et aL, issued Dec. 4, 1984, discloses a “Two-site” or “sandwich” immunometric assay technique for determination of the presence and/or concentration of antigenic substances in fluids using monoclonal antibodies. These are described and compared to conventional assays using polyclonal antibodies. Also described are inhibition assays using complexes of antigens with a monoclonal antibody. This reference discloses methods for detecting and/or determining the concentration of antigenic substances in fluids such as serum.
U.S. Pat. No. 5,587,294, Tamarkin et al., issued Dec. 24, 1996, discloses methods for measuring endogenous cytokines in blood. The method accurately measures the cytokines in the blood in the presence of substances that bind the cytokines thereby causing the measurement of the cytokines by conventional methods to give inaccurate results. The Tamarkin et aL patent also describes the non-invasive measurement of cytokines in biological fluids such as saliva and nasal secretions. Finally, the procedure described in Tamarkin et al., allows one to monitor the level of cytokines in the blood during treatment of a human or animal with cytokines.
Others have shown the prolongation of in vivo effects of exogenous cytokines by injection of cytokine--anti-cytokine antibody complexes. (
J. ImmunoL
151:1235, 1993). This reference describes a technique whereby animals are injected with preformed complexes of a cytokine and an anticytokine MAb. The antibody acts as a carrier protein for the cytokine, slowly dissociating and thereby increasing the period of time during which active cytokine is present in an animal. This paper does not mention the use of this technique for determining cytokine production in vivo. It does reference other publications that mention that endogenously produced anti-cytokine antibodies may enhance the half-life and activity of endogenously produced cytokines in vivo; however, these papers also did not mention this as a possible technique for measuring endogenous cytokine production. The reference does not disclose the idea of labeling an injected anticytokine antibody to facilitate detection of cytokine/anti-cytokine antibody complex.
U.S. Pat. No. 5,612,034, Pouletty et al., issued Mar. 18, 1997, provides first and second compounds, where the first compound is administered to a mammalian host into blood for covalent bonding to blood components, where the components have an extended lifetime in the blood stream. The first compound comprises an active functionality and an agent of interest or a first binding entity. A second compound may be subsequently administered to the patient, which comprises a second binding entity, complementary to the first binding entity and an agent of interest. By virtue of binding to long-lived blood components, the half-life of the agent of interest is greatly extended in vivo.
It would be of great benefit if one could easily, accurately and reproducibly measure the concentrations of various endogenous cytokines in blood in vivo. This would provide information not only of the immune system but of a variety of physiologically interacting processes. Such tools would be useful in a variety of settings, allowing the collection of data of importance to basic medical sciences, clinical medicine, epidemiology and the forensic sciences.
What is needed is a reliable method of measuring endogenous cytokine production by sampling blood, which is only minimally affected by cytokine catabolism, utilization, excretion, or binding to endogenous cytokine binding proteins.
SUMMARY OF THE INVENTION
The present invention provides methods for measuring the endogenous level of a target analyte which may be a hormone, drug or other analyte in a human or animal. The target analyte is preferably a macromolecule, more preferably a protein, and most preferably a cytokine.
The present invention is an immunoassay for use in detecting and monitoring endogenous cytokine production. Prior methods have been unable to accurately measure cytokine production because of rapid excretion, catabolism, and utilization of cytokines as well as the binding of cytokines to endogenous cytokine binding proteins, which can interfere with detection. The present invention obviates all of these difficulties.
The present invention provides the capability of measuring “basal,” as well as “stimulated,” cytokine production. The present invention provides a new tool for monitoring these chemical communication signals and their dysregulation in the face of challenges by pathogens, chemicals, therapeutic
Finkelman Fred D.
Morris Suzanne C.
Chin Christopher L.
Frost Brown Todd LLC
Gabel Gailene R.
University of Cincinnati
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