WO1998049199A1 - Estimation du risque de maladie cardio-vasculaire - Google Patents

Estimation du risque de maladie cardio-vasculaire Download PDF

Info

Publication number
WO1998049199A1
WO1998049199A1 PCT/US1998/008720 US9808720W WO9849199A1 WO 1998049199 A1 WO1998049199 A1 WO 1998049199A1 US 9808720 W US9808720 W US 9808720W WO 9849199 A1 WO9849199 A1 WO 9849199A1
Authority
WO
WIPO (PCT)
Prior art keywords
adma
binding protein
arginine
bound
binding
Prior art date
Application number
PCT/US1998/008720
Other languages
English (en)
Inventor
Robert F. Balint
John P. Cooke
Original Assignee
Panorama Research, Inc.
Cooke Pharma, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Panorama Research, Inc., Cooke Pharma, Inc. filed Critical Panorama Research, Inc.
Priority to AU72690/98A priority Critical patent/AU7269098A/en
Publication of WO1998049199A1 publication Critical patent/WO1998049199A1/fr

Links

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6806—Determination of free amino acids
    • G01N33/6812—Assays for specific amino acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/32—Cardiovascular disorders

Definitions

  • EDNO is the most potent endogenous vasodilator known, and, by its effect upon vascular resistance and cardiac contractility, is a major regulator of blood pressure (Moncada and Higgs, 1993; Cooke and Dzau, 1997). NO exerts its effects as a vasodilator, in part, by stimulating soluble guanylate cyclase to produce cGMP.
  • a deficiency of EDNO (as in the endothelial NOS knockout, or with administration of NOS antagonists), causes hypertension (Dananberg et al., 1993; Shesely et al., 1996).
  • An overproduction of NO causes hypotension and cardiovascular collapse (Rees et al.,1990; Petros et al., 1991).
  • NO is released from the endothelium in response to a wide variety of physiologic stimuli.
  • physiologists have recognized that as blood flow increases through a conduit vessel, the vessel dilates. This flow-mediated vasodilation is dependent upon the integrity of the endothelium, and is largely due to the release of EDNO in response to endothelial shear stress (Cooke et al., 1990; Cooke et al., 1991a). Endothelial cells also respond to pharmacological stimuli.
  • Most vasoconstrictors such as norepinepherine, 5-hydroxytryptamine, and angiotensin II, also stimulate NO release by the endothelium (Moncada and Higgs, 1993; Cooke and Dzau, 1997).
  • VSM vascular smooth muscle
  • EDNO also affects vascular structure by inhibiting the interaction of circulating blood elements with the vessel wall. Platelet adherence and aggregation is inhibited by EDNO (Radomski et al., 1987; Stamler et al, 1989). The adherence and infiltration of leukocytes into the vessel wall during experimental inflammation is reduced by exogenous administration of NO donors, and is enhanced by administration of NOS antagonists (Lefer et al., 1993; Gaboury et al, 1993). To summarize, in states of vascular injury or inflammation, a deficiency of NO contributes to thrombosis, leukocyte infiltration, and vascular smooth muscle proliferation.
  • Atherosclerosis is the major cause of disability in this country and is responsible for 500,000 deaths annually due to coronary artery disease and cerebral vascular attack. Atherosclerosis is accelerated by hyper-cholesterolemia, hypertension, diabetes mellitus, tobacco use, elevated levels of lipoprotein(a) ("Lp(a)”) and homocysteine. Intriguingly, all of these disorders are characterized in humans by an endothelial vasodilatory dysfunction well before there is any clinical evidence of atherosclerosis (Cooke and Dzau, 1997). In all of these conditions, the abnormality appears to be due in large part to a perturbation of the NOS pathway. In most of these conditions, the abnormality is reversed or ameliorated by the administration of the NO precursor, L-arginine (Cooke and Dzau, 1997). L-arginine is metabolized by NOS to citrulline and NO.
  • NOS antagonists reduced vascular NO synthesis, increased endothelial adhesiveness for monocytes, and accelerated lesion formation (Tsao et al., 1994; Naruse et al, 1994; Cayatte et al, 1994).
  • Cooke and others have shown that EDNO exerts its effects on atherogenesis by suppressing the expression and the signaling of endothelial adhesion molecules such as VCAM-1, and by reducing the expression of chemokines such as monocyte chemotactic protein- 1 (Marui et al., 1993; Tsao et al., in press).
  • vascular NO may represent a novel therapeutic strategy for cardiovascular disease.
  • the initial studies in humans are encouraging. Cooke and others have recently demonstrated that chronic oral administration of L-arginine in hypercholesterolemic humans or those with coronary artery disease can enhance vascular NO activity (as assessed by vascular reactivity studies and measurement of urinary nitrogen oxides), inhibit platelet aggregability, and reduce the adhesiveness of peripheral blood mononuclear cells (Bode-B ⁇ ger et al, 1994; Wolfe et al., 1995; Theilmeier et al., in press; Lerman et al., 1997).
  • ADMA a determinant of endothelial dysfunction and novel risk factor for atherosclerosis
  • ADMA asymmetric dimethylarginine
  • asymmetric dimethylarginine is an endogenous antagonist of nitric oxide synthase.
  • Vallance and Moncada demonstrated that, in uremic rats and in patients with renal failure, plasma ADMA levels were elevated 5- 10-fold from normal values of about 1 micromolar (Vallance et al., 1992a,b).
  • Plasma from uremic animals and patients (but not controls) induced the constriction of isolated vascular rings. This vasoconstriction was reversed by L-arginine.
  • infusions of ADMA into the brachial artery of normal volunteers caused a significant increase in forearm vascular resistance at concentrations of ADMA that are found in patients with renal failure (Vallance et al., 1992b).
  • ADMA dimethylarginine dimethylaminohydrolase
  • ADMA Alzheimer's disease
  • ADMA is formed primarily by methylation of protein arginine inside cells, where it plays an important role in modulating protein-RNA interactions (Liu and Dreyfuss, 1995). Free ADMA is released upon protein turnover, and is probably secreted by most tissues and either passed in the urine or metabolized in the kidney (Tojo et al., 1997). Many types of physiological stress, such as the chronic inflammatory stress associated with atheroma formation, oxidative stress from environmental toxins, and stress which might result from poor nutrition, overweight, or age, is associated with chronic cellular damage and leads to increased rates of protein turnover, which in turn may lead to increased secretion of methylated amino acids and higher circulating levels of these amino acids, including ADMA.
  • ADMA may be an important determinant of endothelial vasodilator dysfunction, and potentially, an important new risk-factor for atherosclerosis.
  • methodology must be developed to detect ADMA with greater sensitivity, specificity, and with higher throughput.
  • Cardiovascular disease assessment is improved by determining the value of the ADMA level in a subject's physiological fluid by itself, as an ADMA L-arginine ratio or in conjunction with other know indicators of cardiovascular disease.
  • immunoassays are provided for the determination of the level of ADMA in the physiological fluid.
  • the level of DDAH enzyme activity may be determined, by itself or in conjunction with ADMA, as an indication of the ability of the patient to hydro lyze ADMA and maintain an acceptable level of ADMA in plasma.
  • Antibodies are provided which may be used in the immunoassays and effectively distinguish ADMA from close congeners.
  • Kits may be provided which provide the various reagents for the ADMA assay and or the DDAH assay, conveniently in conjunction with reagents for at least one of L-arginine, HDL, LDL, VLDL, Lp(a), triglycerides, homocysteine, chylomicron size distribution, or the like.
  • Figure 1 is a graph of the plasma concentrations of ADMA as found in hypercholesterolemic and normo-cholesterolemic humans, as determined as described below;
  • Figure 2. is a graph of flow-mediated vasodilation of hypercholesterolemic subjects before and after intravenous infusion of the reagents;
  • Figure 3. is a bar graph of urinary nitrate excretion in hypercholesterolemic subjects before and after treatment as compared to normocholesterolemic control subjects;
  • Figure 4. provides molecular structures of Arginine, ADMA and SDMA.
  • ADMA is determined directly or vicariously, by determining the activity of DDAH.
  • ADMA may be determined in any appropriate physiological fluid by an immunoassay, employing antibodies which provide for detection of ADMA with minimal interference from congeners, such as L-arginine and SDMA.
  • Methods for detecting ADMA in physiological fluids include extraction, chemical derivatization, isolation by reverse-phase HPLC, GC-mass spectrometry, and fluorescence, although other methods may also be available. (Chen et al, 1997).
  • the subject invention includes novel immunoassays employing antibodies which provide for sensitive detection of ADMA, without significant interference from congeners. These immunoassays may be modeled on immunoassays available today, where ADMA is substituted for the available analytes.
  • the labels involved may be enzymes, fluorescers, chemiluminescers, particles, which particles may be opaque or clear, or may be conjugated with the previously indicated labels.
  • the assay may involve spectrophotometric detection, fluorimetric detection, or visual detection, and may use any of a wide variety of reagents that are commercially available or may be developed along with methodologies described in the literature.
  • ADMA arginine and symmetric dimethylarginine
  • ADMA differs from arginine only in having two methyl groups on one of the guanidino nitrogens (see Figure 4).
  • concentration of arginine is approximately 100 micromolar, or approx. 100 times that of ADMA.
  • ADMA should comprise >90% of antibody-bound analyte.
  • a useful antibody must have >1000-fold higher affinity (equilibrium association constant, Ka) for ADMA than for arginine, despite the small chemical difference.
  • SDMA is even more similar to ADMA than is arginine, having the two methyl groups on different guanidino nitrogens instead of on the same nitrogen (see Figure 4).
  • concentration of SDMA is comparable to that of ADMA, or approx. 1 micromolar.
  • a useful antibody must have > 10-fold higher affinity for ADMA than for SDMA to insure that >90% of bound analyte is ADMA.
  • the antibodies which are employed may be antisera from any convenient source, e.g. bovine, caprine, ovine, canine, equine, rodent, or the like, where the antisera may be purified by selecting out antibodies strongly binding to L-arginine and SDMA.
  • the immunogen for production of the antibodies may be prepared by conjugating the ADMA at the alpha-amino group or the carboxyl group, particularly in the latter case using a linker for bonding to the antigen.
  • monoclonal antibodies may be produced in accordance with known ways.
  • mice may be immunized with the ADMA conjugate, splenocytes isolated and immortalized, and then screened for affinity for ADMA, against binding to L-arginine and SDMA.
  • Clones of interest may be expanded and grown or their DNA isolated and the genes expressing the anti-ADMA heavy and light chains isolated and manipulated for expression in an appropriate host cell or host. Indeed, as described below, the genes may be mutated to further enhance binding affinity.
  • binding proteins which have the general structure of the variable region of an antibody. These binding proteins will have a Ka of at least about 5 x 10 6 , usually at least 10 7 , and preferably 10 8 , or higher. These binding proteins will comprise a region having substantially the structure and organization of the variable region ("Fv")of an antibody, where the heavy and light chain may be covalently or non-covalently joined. Other portions of an antibody may also be present, such as the first constant region.
  • variable-region V-region repertoire library cloned from pooled non-immune human blood as the source (Hoogenboom et al., 1991; Marks et al, 1991a,b).
  • a library of ⁇ 4xl0 10 antibody Fab fragments displayed on filamentous bacteriophage was panned against immobilized ADMA to select ADMA-binding antibodies. To favor the selection of antibodies with high afinity for free ADMA but low affinity for arginine or SDMA, it was necessary to modify the standard panning strategy.
  • soluble arginine and SDMA had to be present in the phage suspensions to inhibit antibodies with high affinities for these from binding to the immobilized ADMA.
  • two completely different structural forms of immobilized ADMA had to be used in tandem to favor selection of antibodies with high affinity for free ADMA.
  • the presence of soluble arginine and SDMA favored selection of antibodies with high affinity for the composite epitopes of immobilized ADMA conjugates, but only weak affinity for free ADMA.
  • selection of antibodies with high affinity for free ADMA could only be favored by tandem selection on two completely different structural forms of immobilized ADMA.
  • affinities for free ADMA 10 7 - 10 8 M '1 and affinities for arginine and SDMA of ⁇ 10 4 - 10 5 M “1 and ⁇ 10 6 - 10 7 M “1 , respectively.
  • the affinities for ADMA as compared to arginine are at leastlO 2 greater, preferably at least 10 3 , and as compared to SDMA, are at least 10 fold greater.
  • ADMA a compound which competes with ADMA for binding to the binding protein, that is, has at least one common epitope
  • any ADMA in the physiological specimen measuring the amount of the binding protein that binds to the ADMA mimic.
  • This can be achieved by having the ADMA mimic bound to a solid surface, such as a wall of a vessel, e.g. a microtiter plate or a particle, which can be separated from the medium.
  • One can use channeling where by bringing two of the binding proteins together, one obtains a different signal, e.g. fluorescence energy transfer.
  • one has a polyepitopic reagent which has at least two epitopes competitive with ADMA and two different binding proteins, one with a fluorescer which acts as a donor and one with a fluorescer which acts as a receiver.
  • a fluorescer which acts as a donor
  • a fluorescer which acts as a receiver
  • the two different binding proteins are bound to the polyepitopic reagent there will be energy transfer, so that by using excitation light to excite the donor, one reads the emission wavelength of the receiver.
  • a similar assay can be used with enzymes, where the product of one enzyme is the substrate of the other enzyme.
  • One may have ADMA conjugated to a label, e.g. an enzyme, where binding of the protein to the conjugated ADMA changes the enzyme activity.
  • binding protein Rather than labeling the binding protein, one can label a protein which binds to the binding protein.
  • the anti-(binding protein) may be an antibody specific for an epitope of ADMA or the binding protein may be conjugated to a small molecule for which there is a strong binding protein, e.g. biotin and avidin, digoxin and antidigoxin, etc.
  • Enzyme-linked immunosorbant assays of one sort or another have been widely used for more than 25 years for the detection and measurement of analytes (primarily proteins) in bodily fluids (Harlow and Lane, 1988). Their principal advantages stem from the unparalleled affinities and specificities of monoclonal antibodies (mAbs) for analyte tagging, and the unparalleled catalytic power of enzymes for signal amplification. More recently the immunoassay repertoire has been expanded by the development of homogeneous solution phase assay formats which can be processed in fewer steps than ELISA with improved kinetics and sensitivity (Kopetzki et al., 1994; Coty et al., 1994; Henderson et al., 1986).
  • the preferred format for the latter is based on readily assayable enzymes which have been modified in some way to make their activities sensitive to the presence of the target analyte.
  • the analyte is directly conjugated to the enzyme at a site which does not inhibit its activity except when the analyte adduct is bound by antibody (Coty et al., 1994; Henderson et al., 1986).
  • the inhibitory antibody is displaced from the enzyme, producing a proportional increase in activity which can be measured directly with chromogenic substrates.
  • ADMA In conventional ELISA haptenized ADMA is immobilized on the surface of microtiter plate wells (Harlow and Lane, 1988). Antibody is adsorbed out of solution by binding to the immobilized hapten, and after washing, the bound antibody is detected by binding a secondary antibody-enzyme conjugate which reacts with a chromogenic substrate, producing a signal which is propotional to the amount of bound anti-ADMA antibody (Harlow and Lane, 1988). Free ADMA in a clinical specimen is determined by diluting the specimen into the antibody solution prior to exposure of the antibody solution to the immobilized ADMA hapten.
  • the resultng signal will be reduced by an amount proportional to the concentration of ADMA in the specimen, due to the fraction of antibody prevented from binding to the immobilized hapten by binding to free ADMA in the specimen.
  • a series of two-fold dilutions of the specimen bracketing the expected concentration range is tested in triplicate and compared to standards of known ADMA concentration. The sensitivity of the assay is greatest when the antibody is used at a concentration no greater than ⁇ 0.1 x Kd. Under this condition, the specimen dilution which gives a 50% reduction of signal has an ADMA concentration equivalent to the Kd.
  • Fluorescence Polarization Immunassay is another homogeneous solution phase immunoassay for small molecule analytes, which has the unique advantage that a positive signal is generated by competition.
  • FPIA detects the difference between antibody-bound and free fluorescently-labelled ligand as the polarization of emitted light when excited by plane-polarized light. Small molecule ligands tumble so fast during the excited state that emitted light is nearly isotropic, whereas, the ⁇ 500-fold larger antibody-bound ligand hardly rotates at all during the excited state and therefore emits highly polarized or anisotropic light.
  • Analyte is measured by its ability to displace fluorescently-labelled analyte from analyte-specific antibody, or compete with label for binding to antibody, and thereby lower the polarization of emitted light, and increase its intensity at certain angles relative to the incident light.
  • Sensitivity is a function of antibody affinity and the size difference between bound and free ligand. With nanomolar affinities and a ⁇ 500-fold size difference between free and antibody-bound analyte, sub-picomolar concentrations may be detectable.
  • FPIA has the advantage that it is simple and homogeneous. FPIA is based on the principle that when a fluorescently-labeled molecule is excited with plane-polarized light, it emits light which is polarized to a degree which is proportional to the size of the molecule (Burke et al., 1996). This is because size is inversely proportional to rotation rate in solution and the farther an excited molecule rotates before emission, the less polarized the emitted light will be. Fluorescently-labeled small molecules rotate rapidly, thus, emitted light is less polarized, whereas, the same molecules rotate much more slowly when bound to an antibody, and therefore emitted light is much more polarized.
  • ADMA may be conjugated to many fluorophores such as fluorescein by reaction of the free primary amino group of ADMA with an activated derivative of the fluorophore such as flurescein isothiocyanate (FITC).
  • FITC flurescein isothiocyanate
  • Free ADMA is measured as a function of its concentration-dependent ability to compete with fluorescein- ADMA for binding to antibody, thereby reducing polarized emission.
  • Additional immunoassay formats which could be used with our anti-ADMA antibodies include, but are not limited to, radioimmunoassay (RIA; Lauritzen et al, 1994), cloned enzyme donor immunoassay (CEDIA; Coty et al., 1994), biomolecular interaction analysis (BIA; Fagerstam et al., 1992), and fluorescence resonance energy transfer immunoassay (FRET; Youn et al., 1995).
  • RIA radioimmunoassay
  • CEDIA cloned enzyme donor immunoassay
  • BIOA biomolecular interaction analysis
  • FRET fluorescence resonance energy transfer immunoassay
  • ELISA fluorescence resonance energy transfer immunoassay
  • most immunoassays for small, monovalent molecules are competitive inhibition assays in which the specimen analyte competes with labeled ligand for binding to antibody. In RIA the ligand is radioactive.
  • the ligand is conjugated to the ⁇ -fragment of ⁇ -galactosidase, such that antibody-binding inhibits enzyme activity.
  • competitive inhibition of the antibody-ligand interaction by specimen analyte results in an increase in chromogenic enzyme activity.
  • the ligand is labeled with a fluorophore which can transfer its energy detectably to a fluorophore attached to the antibody only when both are in close proximity.
  • BIA does not assay by competitive inhibition of binding, but rather directly monitors the interaction of specimen analyte with immobilized antibody using a phenomenon called surface plasmon resonance, whereby the refractive index at the antibody-bound surface is detectably altered when analyte binds to the antibody.
  • the assay may employ a fluorescence activated cell sorter and fluorescent particles employed.
  • the assay would provide that the number of fluorescent particles counted would be related to the amount of ADMA present. For example, by having a competitive assay between particles to which ADMA is conjugated and ADMA in the specimen for fluorescently labeled binding proteins, the degree to which the particles are labeled with the fluorescent binding proteins will be proportional to the amount of ADMA in the specimen. One would then compare the number of fluorescent particles counted with the specimen as compared to a control value.
  • the following table organizes various assays which may find use in the subject invention.
  • Competitor is the immobilized antigen and bound label is mversely proportional to analyte.
  • Polarization of fluorescent label is inversely proportional to analyte.
  • Donor fluorescence is proportional to analyte, optional acceptor fluorescence is mversely proportional analyte
  • the physiological sample may be subject to prior treatment, depending on the nature of the sample and the nature of the assay.
  • anticlotting factors may be included, alternatively, the red blood cells may be removed, the blood may be citrated or heparinized, etc.
  • the sample may be concentrated or diluted, components precipitated out, the pH modified, particular buffers added, or the like.
  • the untreated or treated sample may then be combined with the other reagents appropriate for the assay, incubated as appropriate and then assayed.
  • ADMA concentration in plasma is greater than about 2 ⁇ M, particularly greater than about 1.5 ⁇ M
  • the subject may be considered to have a serious cardiovascular disease risk.
  • the normal urinary excretion will generally be 13.5 ⁇ 3.1 mg per 24 hours.
  • Of particular interest will be the history of the subject, e.g. smoker, diabetic, etc., the total cholesterol, the HDL/LDL ratio, triglycerides, homocysteine and the like.
  • prophylactic treatment such as providing excess L- arginine or L-lysine in the subject's diet, generally in addition to the normal diet, a supplement of from about 5 to 12 g/day.
  • Other prophylactic regimens may also be employed, such as lower fat diets, increased fiber in the diet and the like.
  • the ADMA assay may be used as an initial assay, whereby a positive result, particularly a borderline result, in the range of 1 -2 ⁇ M, more usually in the range of 1.5 - 2 ⁇ M, would warrant further tests to corroborate the existence of the risk for cardiovascular disease.
  • ADMA Alzheimer's disease
  • the other factors which are considered today aid in further enhancing the accuracy of the cardiovascular risk assessment.
  • Other common factors include L-arginine, HDL, LDL, VLDL, Lp(a), triglycerides, homocysteine, and chylomicron size distribution.
  • the levels of these various factors present in a physiological fluid, where the factors may be determined individually or compared as ratios, may be used in conjunction with the ADMA determination in cardiovascular risk assessment.
  • Kits may be provided where the reagents for the ADMA assay are made available in conjunction with the reagents for the determination of the other factors.
  • the ratio of low-density lipoprotein (LDL) to high-density lipoprotein (HDL) is a commonly-used ratio to assess risk of cardiovascular disease.
  • LDL low-density lipoprotein
  • HDL high-density lipoprotein
  • the ratio of arginine to ADMA appears to be a much better predictor of endothelial vasodilatory dysfunction than is the LDL-to-HDL ratio.
  • the arginine/ ADMA ratio may be a more accurate predictor for cardiovascular disease than traditional risk factors because it is more reflective of the vascular response to hypercholesterolemia.
  • the vascular response to hypercholesterolemia varies widely due to genetic background, but chronically elevated ADMA correlates much better with vascular dysfunction because it is a potentially causative precondition.
  • Disclosure of techniques used for determining various of the factors include: HDL, U.S. Patent Nos. 5,034,332; 5,451,370 and 5,460,974; LDL, U.S. Patent Nos. 4,126,416; 5,401,466 and 5,417,863; triglycerides, U.S. Patent Nos. 4,245,041 and 5,221,615; and homocysteine, U.S. Patent Nos. 4,940,658 and 5,631,127.
  • kits which are provided will usually have the necessary reagents, depending upon the purchaser to have the equipment.
  • a kit will have at least labeled or unlabeled anti-ADMA, where in the latter case a labeled anti-(anti-ADMA) may be employed.
  • Surfaces of vessels, microtiter plates, centrifuge tubes or the like may be coated with the appropriate reagent (s) for detecting one or more of the factors.
  • Particles may be provided, which are coated with the appropriate reagents, e.g. antibodies, for isolating the analyte, analyte-receptor complexes, or the like.
  • Reagents for developing a detectable signal e.g. enzyme substrates may be included.
  • V-region encoding fragements were amplified from peripheral blood lymphocyte RNA by reverse transcription and polymerase chain reaction (RT-PCR) using degenerate primers containing all the known 5' and 3' sequences of human V-regions. These libraries of V-region encoding fragements were then ligated into a phagemid vector for expression in the E.
  • Fab fragments fused to the amino terminus of the phage minor coat protein (glllp) via the Fd C-terminus.
  • Fab fragments are comprised of complete light chain and heavy chain Fd, which is comprised of the V-region plus first constant region.
  • Fab and glllp in the expression product three additional elements were encoded by the vector: (1) a 12-residue epitope tag (c-myc) for ELISA detection by anti-epitope antibody, (2) six-histidine tag for purification by affinity chromatography, and (3) a supressible stop codon (amber) for free Fab production in non-supressing hosts without the need for subcloning.
  • the phage library was prepared by quantitative infection of E. coli strain TGI cells (an amber-supressing host) expressing the Fab-glllp fusions with helper phage, which is essentially wildtype phage containing an antibiotic resistance gene.
  • Phage particles which are produced by the infected cells contain Fab on their surface and the Fab-encoding phagemid inside.
  • the library used for the present work was comprised of phage representing ⁇ 4xl0 10 independent Fab clones. Phage were panned against immobilized ADMA according to established procedures (McCafferty and Johnson, 1996; McCafferty, 1996). ADMA was conjugated through the alpha-amino group to epsilon-amino groups of exposed lysines on bovine serum albumin (BSA) via a suberate linker, and this conjugate was immobilized on a polystyrene surface.
  • BSA bovine serum albumin
  • a suspension containing ⁇ 10 13 phage particles was exposed to the immobilized ADMA conjugate for 1-2 hours to allow binding equilibration.
  • the suspension contained BSA and suberate at 3% and 1 millimolar, respectively, to inhibit capture of Fab having high affinity for these components of the conjugate.
  • Bound phage were washed and eluted with triethylamine. From the first round, 6.5x10 5 phage were recovered. These were amplified in E. coli strain TGI back up to ⁇ 10 13 and subjected to two more rounds of panning, i.e., binding, washing, elution, and amplification.
  • soluble arginine was included at 1 micromolar to prevent capture of Fab which cross-react with arginine. From the final round of panning l. ⁇ xlO 10 phage were recovered. An aliquot of this phage population was used to infect E. coli strain
  • ADMA-suberate-BSA conjugate 57% of these clones were found to give a positive ELISA signal with no detectable inhibition by soluble arginine, BSA, or suberate. However, none of the ELISA-positive clones showed inhibition by soluble ADMA at concentrations up to 1 micromolar. Thus, the presence of soluble components of the conjugate, including a structural analog of ADMA (arginine), biased the selection in favor of antibodies which had high affinities for the composite epitope of ADMA-suberate-BSA, but which had low affinities for each of the conjugate components alone, including ADMA.
  • ADMA was linked, again through the alpha-amino group, to tosyl-activated magnetic beads (Dynabeads, Dynal Corp.).
  • the phage population selected after three rounds of panning on ADMA-suberate-BSA were then subjected to four rounds of panning against ADMA-Dynabeads, without intervening amplification.
  • the suspension contained 10 micromolar arginine and 100 nanomolar SDMA to inhibit capture of Fab with high affinities for these ADMA analogs. These concentrations were selected on the basis of the required level of discrimination for an antibody having a desired Kd ( 10 "8 M). Approximately 2000 clones were recovered from this process. Soluble Fab from 600 of these clones were screened by competitive ELISA against immobilized ADMA-suberate-BSA in the presence and absence of 100 nanomolar soluble, free ADMA. Four clones were found to be inhibited by >50% by 100 nanomolar ADMA, and two of these clones, F and G, were inhibited by >80% by 100 nanomolar ADMA and by 20%-50% by 10 nanomolar ADMA.
  • anti-ADMA Fab clones F and G have Kd in the 10 "7 -10 "8 M range. Both clones showed no detectable inhibition by up to 10 micromolar arginine and by up to 1 micromolar SDMA. Thus, anti-ADMA Fab clones F and G are judged to have the necessary affinities and specificities to allow accurate determination of ADMA in clinical specimens without detectable interference by arginine or SDMA.
  • Fab were purified from the supernatants of large-scale bacterial cultures of clones F and G. Ultrafiltration was used to increase the concentration approximately 40-fold and replace the bacterial growth medium with phosphate-buffered saline (PBS). Fab were then affinity-purified by the six-histidine tag at the carboxy terminus of each encoded by the expression vector, using immobilized metal ion affinity chromatography (IMAC; Janknecht et al, 1991). Fab yields were typically 0.5-1 mg per liter and were judged to be >90% pure by silver-stained SDS-PAGE.
  • IMAC immobilized metal ion affinity chromatography
  • the purified Fab were retested for ELISA performance and conditions were optimized. When microtiter wells were coated with 1 micro gram
  • ADMA-suberate-BSA, lxlO "9 M Fab gave an adequate signal (1-2 OD405 in 30' with 0.1-0.2 OD background) with anti-myc-tag mouse antibody, horseradish peroxidase-conjugated (FIRP) rabbit anti-mouse antibody, and ABTS, a chromogenic
  • Kd may be obtained from the slope (-1/Kd).
  • the complex concentration is equal to the total Fab concentration times (A- A b )/(A f -A b ) , where A f is the A405 of the Fab in the absence of ADMA and A b is the A405 of the Fab in an excess of ADMA, i.e. a concentration of >100 x Kd, e.g., 10 "5 M.
  • Free ADMA is equal to the total ADMA minus the complex.
  • ADMA is determined by the standard method, which involves removal of serum proteins, fluorescent labeling with o-phthalaldehyde, reversed phase high performance liquid chromatography (HPLC), and post-column, in-line fluorometric detection (Chen et al., 1997).
  • the same specimens are assayed by competition ELISA essentially as described in Example 1 with the Fab concentration at lxlO "9 M.
  • a series of dilutions of each specimen ranging from 1 :10 to 1:100 is assayed in triplicate, and the results are compared to those of the standard method and a standard curve of pure ADMA from 10 '9 M to 10 "7 M.
  • ADMA was conjugated through its alpha-amino group to Oregon Green (OG) and purified by reversed phase HPLC.
  • the fluorescence polarization of the free conjugate was determined with a polarizing fluorometer at 498nm excitation maximum and 524nm emission maximum over a range of concentrations from 10 "10 M to 10 "8 M.
  • the polarization of ADMA-OG was found to be fairly constant over the entire range with a value of 20 ⁇ 5 at 10 "9 M being typical.
  • polarization was determined after equilibration with various concentrations of Fab ranging from 10 "10 M to 10 "6 M. As expected the minimum value was equivalent to that of the free conjugate and was reached between 10 "9 M and 10 "10 M.
  • the complex concentration is equal to the total ADMA-OG concentration times (P - P f )/(P b - P f ), where P f is the polarization of free ADMA-OG and P b is the polarization of ADMA-OG in the presence of an excess of Fab, i.e., >100 x Kd, e.g., 10 "5 M.
  • Free Fab is equal to total Fab minus the complex. When the Fab is at 5 x 10 '8 M ( ⁇ Kd) and ADMA-OG is at 10 "9 M, polarization is 50% of maximum. This is the region of maximum sensititvity of the assay.
  • a range of dilutions from 1 :2 to 1:100 are assayed and compared to a standard curve ranging from 10 "6 M to 10 "8 M. Again, the results are observed to agree with the standard assay as well as with the competition ELISA.
  • the subject invention provides for an early warning risk factor for cardiovascular disease.
  • an early indication of risk of cardiovascular disease is given. This determination may be further buttressed with other symptoms and other assays, as well as lifestyle and the like.
  • the subject invention provides for highly sensitive assays which have low interference from congeners, so as to have low false positives and negatives. Binding proteins are provided which allow for successful assays for ADMA without significant interference from L-arginine and SDMA.
  • Bode-B ⁇ ger SM B ⁇ ger RH, Creutzig A, Tsikas D, Gutzki FM, Alexander K, Frolich JC.
  • L-arginine infusion decreases peripheral arterial resistance and inhibits platelet aggregation in healthy subjects.
  • Clin Sci 1994;87(3):303-10 Bode-B ⁇ ger SM, B ⁇ ger RH, Thiele W, Junker W, Frolich JC. Elevated L-arginine/dimethylarginine ratio contributes to enhanced systemic NO production by dietary L-arginine in hypercholesterolemic rabbits. Biochem Biophys Res Comm 1996;219(2):598-603
  • VCAM-1 Vascular cell adhesion molecule- 1
  • Naruse K Shimizu K, Muramatsu M, Toki Y, Miyazaki Y, Okumura K, Hashimoto H,
  • Prostaglandin H2 does not contribute to impaired endothelium-dependent relaxation and long-term inhibition of nitric oxide synthesis promotes atherosclerosis in hypercholesterolemic rabbit thoracic aorta.
  • Arterioscler Thromb 1994;14:746-752 Petros A Bennett D, Vallance P: Effect of nitric oxide synthase inhibitors on hypotension in patients with septic shock. Lancet 1991;338:1557 Radomski MW, Palmer RMJ, Moncada S. Comparative pharmacology of endothelium-derived relaxing factor, nitric oxide, and prostacyclin in platelets.

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Molecular Biology (AREA)
  • Immunology (AREA)
  • Biomedical Technology (AREA)
  • Urology & Nephrology (AREA)
  • Hematology (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • General Health & Medical Sciences (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biochemistry (AREA)
  • Microbiology (AREA)
  • Food Science & Technology (AREA)
  • Biotechnology (AREA)
  • Analytical Chemistry (AREA)
  • Biophysics (AREA)
  • Cell Biology (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Organic Chemistry (AREA)
  • Bioinformatics & Computational Biology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Genetics & Genomics (AREA)
  • Peptides Or Proteins (AREA)

Abstract

La présente invention concerne l'estimation améliorée d'un risque cardio-vasculaire par détermination du taux N,N-diméthyl arginine ('ADMA') asymétrique dans le fluide physiologique d'un sujet. Des taux accrus d'ADMA, supérieurs à la normale, indiquent un risque de maladie cardio-vasculaire. On obtient des protéines de liaison présentant une affinité élevée pour l'ADMA et une affinité sensiblement réduite pour des congénères rencontrés dans des fluides physiologiques, de façon à réduire la fréquence de résultats erronés. Les résultats observés avec l'ADMA peuvent être étayés par la détermination d'autres indicateurs, ce qui permet d'obtenir facilement un kit comportant les réactifs pour le dosage de l'ADMA ainsi que d'autres réactifs.
PCT/US1998/008720 1997-05-01 1998-04-30 Estimation du risque de maladie cardio-vasculaire WO1998049199A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU72690/98A AU7269098A (en) 1997-05-01 1998-04-30 Cardiovascular disease risk assessment

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US4527897P 1997-05-01 1997-05-01
US60/045,278 1997-05-01

Publications (1)

Publication Number Publication Date
WO1998049199A1 true WO1998049199A1 (fr) 1998-11-05

Family

ID=21936974

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1998/008720 WO1998049199A1 (fr) 1997-05-01 1998-04-30 Estimation du risque de maladie cardio-vasculaire

Country Status (2)

Country Link
AU (1) AU7269098A (fr)
WO (1) WO1998049199A1 (fr)

Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000046395A1 (fr) * 1999-02-05 2000-08-10 Panorama Research, Inc. Criblage de dimethyl-l-arginine asymetrique
WO2002014873A2 (fr) * 2000-08-18 2002-02-21 Boeger Rainer H Procede et moyen permettant de detecter la probabilite de l'apparition ou de l'aggravation futures de maladies liees a un trouble du metabolisme de no
WO2002036738A2 (fr) * 2000-10-30 2002-05-10 Kalobios, Inc. Maturation d'affinite par selection concurrentielle
WO2003102031A1 (fr) * 2002-05-31 2003-12-11 Unither Pharma, Inc. Proteines et procedes utiles pour l'evaluation du risque de maladie cardiovasculaire
JP2004279412A (ja) * 2003-02-24 2004-10-07 Sanwa Kagaku Kenkyusho Co Ltd 低分子化合物の免疫学的測定・検出方法
WO2006103416A1 (fr) * 2005-03-30 2006-10-05 Ucl Business Plc Dosage de la méthylarginine asymétrique dans un échantillon biologique
US7611844B2 (en) * 2002-11-15 2009-11-03 Board Of Trustees Of The Leland Stanford Jr. University Methods for detecting asymmetric dimethylarginine in a biological sample
US8481690B2 (en) 2008-08-07 2013-07-09 Idexx Laboratories, Inc. Methods for detecting symmetrical dimethylarginine
US9084671B2 (en) 2002-06-21 2015-07-21 Advanced Cardiovascular Systems, Inc. Methods of forming a micronized peptide coated stent
US10775365B2 (en) 2015-02-20 2020-09-15 Idexx Laboratories, Inc. Homogenous immunoassay with compensation for background signal
US11035861B2 (en) 2013-09-05 2021-06-15 Idexx Laboratories, Inc. Methods for detecting renal disease
US11422136B2 (en) 2017-10-19 2022-08-23 Idexx Laboratories, Inc. Detection of symmetrical dimethylarginine

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4126416A (en) * 1977-11-21 1978-11-21 The Trustees Of Boston University Method for determining the level of LDL cholesterol in blood plasma
US4245041A (en) * 1977-12-07 1981-01-13 American Monitor Corporation Triglycerides assay and reagents therefor
US4940658A (en) * 1986-11-20 1990-07-10 University Patents, Inc. Assay for sulfhydryl amino acids and methods for detecting and distinguishing cobalamin and folic acid deficency
US5034332A (en) * 1990-08-06 1991-07-23 Wisconsin Alumni Research Foundation Assay for high density lipoprotein cholesterol

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4126416A (en) * 1977-11-21 1978-11-21 The Trustees Of Boston University Method for determining the level of LDL cholesterol in blood plasma
US4245041A (en) * 1977-12-07 1981-01-13 American Monitor Corporation Triglycerides assay and reagents therefor
US4940658A (en) * 1986-11-20 1990-07-10 University Patents, Inc. Assay for sulfhydryl amino acids and methods for detecting and distinguishing cobalamin and folic acid deficency
US5034332A (en) * 1990-08-06 1991-07-23 Wisconsin Alumni Research Foundation Assay for high density lipoprotein cholesterol

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
DATABASE SCISEARCH 1 January 1900 (1900-01-01), RITZ E, NOWICKI M: "ADMA and Mortality in Dialysis-Role of Asymmetric Dimethyl-L-Arginine", XP002910434, Database accession no. 03768651 *
JIN J.-S., D'ALECY L. G.: "CENTRAL AND PERIPHERAL EFFECTS OF ASYMMETRIC DIMETHYLARGININE, AN ENDOGENOUS NITRIC OXIDE SYNTHETASE INHIBITOR.", JOURNAL OF CARDIOVASCULAR PHARMACOLOGY, RAVEN PRESS, NEW YORK, NY, vol. 28., 1 January 1996 (1996-01-01), NEW YORK, NY, pages 439 - 446., XP002910432, ISSN: 0160-2446, DOI: 10.1097/00005344-199609000-00014 *
VALLANCE P., ET AL.: "ACCUMULATION OF AN ENDOGENOUS INHIBITOR OF NITRIC OXIDE SYNTHESIS IN CHRONIC RENAL FAILURE.", THE LANCET, THE LANCET PUBLISHING GROUP, GB, vol. 339., 7 March 1992 (1992-03-07), GB, pages 572 - 575., XP002910433, ISSN: 0140-6736, DOI: 10.1016/0140-6736(92)90865-Z *

Cited By (20)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000046395A1 (fr) * 1999-02-05 2000-08-10 Panorama Research, Inc. Criblage de dimethyl-l-arginine asymetrique
US6358699B1 (en) 1999-02-05 2002-03-19 Cooke Pharma Assay for asymmetrical NG, NG dimethyl-l-arginine
WO2002014873A2 (fr) * 2000-08-18 2002-02-21 Boeger Rainer H Procede et moyen permettant de detecter la probabilite de l'apparition ou de l'aggravation futures de maladies liees a un trouble du metabolisme de no
WO2002014873A3 (fr) * 2000-08-18 2003-08-07 Rainer H Boeger Procede et moyen permettant de detecter la probabilite de l'apparition ou de l'aggravation futures de maladies liees a un trouble du metabolisme de no
WO2002036738A2 (fr) * 2000-10-30 2002-05-10 Kalobios, Inc. Maturation d'affinite par selection concurrentielle
WO2002036738A3 (fr) * 2000-10-30 2002-07-04 Horizon Biotechnologies Inc Maturation d'affinite par selection concurrentielle
WO2003102031A1 (fr) * 2002-05-31 2003-12-11 Unither Pharma, Inc. Proteines et procedes utiles pour l'evaluation du risque de maladie cardiovasculaire
US9084671B2 (en) 2002-06-21 2015-07-21 Advanced Cardiovascular Systems, Inc. Methods of forming a micronized peptide coated stent
US7611844B2 (en) * 2002-11-15 2009-11-03 Board Of Trustees Of The Leland Stanford Jr. University Methods for detecting asymmetric dimethylarginine in a biological sample
JP2004279412A (ja) * 2003-02-24 2004-10-07 Sanwa Kagaku Kenkyusho Co Ltd 低分子化合物の免疫学的測定・検出方法
GB2439877A (en) * 2005-03-30 2008-01-09 Ucl Business Plc Assay for measuring asymmetric methylarginine in a biological sample
WO2006103416A1 (fr) * 2005-03-30 2006-10-05 Ucl Business Plc Dosage de la méthylarginine asymétrique dans un échantillon biologique
GB2439877B (en) * 2005-03-30 2010-04-28 Ucl Business Plc Assay for measuring asymmetric methylarginine in a biological sample
US8481690B2 (en) 2008-08-07 2013-07-09 Idexx Laboratories, Inc. Methods for detecting symmetrical dimethylarginine
US9091684B2 (en) 2008-08-07 2015-07-28 Idexx Laboratories, Inc. Methods for detecting symmetrical dimethylarginine
US9970927B2 (en) 2008-08-07 2018-05-15 Idexx Laboratories, Inc. Methods for detecting symmetrical dimethylarginine
US11035861B2 (en) 2013-09-05 2021-06-15 Idexx Laboratories, Inc. Methods for detecting renal disease
US10775365B2 (en) 2015-02-20 2020-09-15 Idexx Laboratories, Inc. Homogenous immunoassay with compensation for background signal
US11913942B2 (en) 2015-02-20 2024-02-27 Idexx Laboratories, Inc. Homogenous immunoassay with compensation for background signal
US11422136B2 (en) 2017-10-19 2022-08-23 Idexx Laboratories, Inc. Detection of symmetrical dimethylarginine

Also Published As

Publication number Publication date
AU7269098A (en) 1998-11-24

Similar Documents

Publication Publication Date Title
AU2020277178B2 (en) Evaluation and treatment of bradykinin-mediated disorders
EP1070139B1 (fr) Criblage de dimethyl-l-arginine asymetrique
KR101643783B1 (ko) 막성 신장병증에 대한 진단법
WO1998049199A1 (fr) Estimation du risque de maladie cardio-vasculaire
EP2403876A1 (fr) Dosage pour la détection de la vitamine d et anticorps correspondants
KR102698737B1 (ko) 절단된 고분자량 키니노겐의 검출을 위한 면역검정법
Timofeeva et al. Systemic lupus erythematosus: Molecular cloning and analysis of 22 individual recombinant monoclonal kappa light chains specifically hydrolyzing human myelin basic protein
Hiwasa et al. Serum SH3BP5-specific antibody level is a biomarker of atherosclerosis
JP2013007757A (ja) ヒトシスタチンｃを評価するための比濁イムノアッセイ
JP4290424B2 (ja) タンパク質ｅｓｍ−１を検出するためのキット及び前記キットを使用する検出方法
US20060034819A1 (en) Proteins and methods useful for assessing risk of cardiovascular disease
Timofeeva et al. Systemic lupus erythematosus: Molecular cloning and analysis of recombinant monoclonal kappa light chain NGTA1-Me-pro with two metalloprotease active centers
ES2648788T3 (es) Método para evaluar el riesgo de un sujeto de tener una enfermedad cardiovascular
US20140221507A1 (en) Biomarker of Renal Impairment
Eisenberg et al. Concordance of creatine kinase-MB activity and mass.
JP4856381B2 (ja) ヒトオロト酸ホスホリボシルトランスフェラーゼタンパク質の測定法
Cheng et al. Detection of calcitonin gene-related peptide based on increased antigen-driven interaction with antibody variable regions
US7527938B2 (en) Methods for diagnosis and treatment of chronic immune diseases
Khait The evolution and characteristics of protein-protein interaction interfaces
US20080193952A1 (en) Inducible Nitric Oxide Synthase Binds, S-Nitrosylates, and Activates Cyclooxygenase-2
JP2004061345A (ja) 尿中のプラスミノーゲンアクチベーターインヒビター−１測定による糖尿病性腎症の進展度の判定方法
KR20240133761A (ko) 절단된 고분자량 키니노겐의 검출을 위한 면역검정법
JPH11346782A (ja) ヒトｐｌｔｐタンパク質に対するモノクローナル抗体、このモノクローナル抗体を用いたヒトｐｌｔｐの測定方法、及びこの測定方法を行うための測定キット

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AU CA JP

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
121 Ep: the epo has been informed by wipo that ep was designated in this application
NENP Non-entry into the national phase

Ref country code: JP

Ref document number: 1998547379

Format of ref document f/p: F

122 Ep: pct application non-entry in european phase
NENP Non-entry into the national phase

Ref country code: CA