CN101467041A - Assays to predict atherosclerosis and dysfunctional high-density lipoprotein - Google Patents

Abstract

This invention provides novel assays for the detection of dysfunctional HDL. The assays are good diagnostics and/or prognostics for atherosclerosis or other pathologies characterized by an inflammatory response. In certain embodiments the methods involve measurements of heme-related HDL-associated proteins (e.g., haptoglobin, hemopexin, etc.), and/or measurements of the relative distribution of HDL-associated proteins between HDL and the non-lipoprotein fractions of plasma/serum, and/or measurements of the ability of pro-inflammatory HDL to consume nitric oxide, and/or measurement of the ability of HDL to inhibit LDL aggregation.

Description

The mensuration of prediction atherosclerotic and abnormal function high-density lipoprotein (HDL)

The cross reference of related application

[0001] the application requires interests and the right of priority of the USSN 60/772,429 of the USSN 60/843,213 of on September 7th, 2006 application and application on February 10th, 2006, these two documents in all cases all integral body be attached to herein by reference.

The statement of making according to the research and development of federal funding about inventor's rights and interests

[0002] this work obtains the part support of the subsidy 1,RO1,HL7 1776 and the HL30658 of NIH.U.S. government has some rights and interests of the present invention.

Invention field

[0003] the present invention relates to atherosclerotic diagnosis.Specifically, the invention provides the improvement mensuration that is used to detect abnormal function HDL.

Background of invention

[0004] atherosclerotic is a kind of chronic inflammatory diseases of big and medium sized artery, is main causes of M ﹠ M in western countries.The use of statins reduces 1/3 with mortality ratio.Yet although the statins treatment is arranged, this sick mortality ratio still has 2/3.

[0005] oxidation of low-density lipoprotein (LDL) is the atherosis principal element of human artery (Witztum and Steinberg (2001) Trends Cardiovasc.Med., 11:93-102; Witztum and Steinberg (1991) J.Clin.Invest., 88:1785-1792).LDL in SES hold back and oxidation and the interaction between endothelial cell and the monocyte subsequently are the critical process (Navab etc. that bring out the atherosclerotic lesion development, (1996) Arterioscler.Thromb.Vasc.Biol., 16:831-842; Berliner etc., (1995) Circulation 91:2488-2496).Slight modify/LDL (MM-LDL) of oxidation contains the bioactive molecule of can inducing endothelial cell producing the inflammatory material, for example chemotactic factor (CF), adhesion molecule and growth factor.These inflammatory molecules promote monocytes to the raising and adhering to of endothelial cell (Berliner etc., (1995) Circulation 91:2488-2496).Several bioactive oxidized phospholipids (Watson etc., (1995) J.Clin.Invest., 95:774-782 in the atherosclerotic lesion of MM-LDL and animal model, have been identified; Watson etc., (1995) J.Clin.Invest., 96:2882-2891; Watson etc., (1997) J.Biol.Chem., 272:13597-13607; Watson etc., (1999) J.Biol.Chem., 274:24787-24798; Leitinger etc., (1999) Arterioscler.Thromb.Vasc.Biol., 19:1291-1298; Subbanagounder etc., (2000) Free Radic.Biol.Med., 28:1751-1761).Oxidation-L-α-1-palmityl-2-arachidonic acidic group-sn-glycerol-3-phosphocholine (ox-PAPC) and 3 kinds of component 1-palmityl-2-(5-oxidation valeryl)-sn-glycerol-3-phosphocholine (POVPC), 1-palmityl-2-glutaryl-sn-glycerol-3-phosphocholine (PGPC) and 1-palmityl-2-(5,6-epoxy isoprostane E ₂)-sn-glycerol-3-phosphocholine (PEIPC) (Watson etc., (1999) J.Biol.Chem., 274:24787-24798; Leitinger etc., (1999) Proc.Natl.Acad.Sci.U.S.A. (1999) 96:12010-12015; Subbanagounder etc., (2000) Arterioscler.Thromb.Vasc.Biol. 20:2248-2254), induces monocyte in conjunction with endothelial cell, and plays a major role in the activation of MM-LDL to endothelial cell.After finding these molecules, identified a series of similar bioactive other oxidized phospholipids that have, they by the sn-2 position oxidation unsaturated fatty acid at phosphatide form (Berliner and Watson, (2005) N.Engl.J.Med., 353:9-11).

[0006] relation of the retrocorrelation between HDL and the atherosclerotic risk is fully established.Although as if the anti-atherogenicity effect for HDL do not have single explanation, but become be clearly, the functional status that depends primarily on the HDL of its protein component is the important determinative (Navab etc. of coronary heart disease (CHD), (2001) Arterioscler.Thromb.Vasc.Biol., 21:481-488).Paraoxonase 1 (PON1), lecithin cholesterol acyltransferase (LCAT), platelet activating factor PAF-AH (PAF-AH), proteinase (elastoser sample), phospholipase D, albumin, apoJ and apoA-I are the albumen that has the anti-atherogenicity characteristic that can prevent that MM-LDL from forming among the HDL.Showed already that HDL had the effect that prevents the LDL oxidation.Shown that HDL suppresses the mild oxidation of LDL, and therefore suppressing the human artery parietal cell produces effective monocyte chemoattractant MCP-1.

[0007] according to background and context, HDL can be used as anti-inflammatory molecular or short scorching molecule exists.The acute phase response of rabbit and philtrum (APR) can be short scorching form by the anti-inflammatory formal transformation with HDL, and promptly HDL loses the protective capability of the inflammation that its anti-LDL induces, and in fact HDL promotes the inflammation that LDL induces with its short scorching state.Be not limited to concrete theory, it is generally acknowledged that HDL plays antiinflammatory action under basic condition, but in the APR process, the activities of antioxidant enzymes forfeiture, apoA-I is impaired, and the albumen that combines with HDL is replaced and/or is exchanged, and produces short scorching HDL.For example, showed already, in heredity to atherogenicity meals in the mouse of atherosclerotic susceptible with HDL by anti-inflammatory change into short scorching (but can be not so in the antiatherogenic mouse in heredity) (Navab etc., (1997) J.Clin.Invest., 99:2005-2019).Since then, several researchs shown short scorching HDL in animal model exist situation and character (100:464-474 summarizes in Navab etc. for Castellani etc., (1997) J.Clin.Invest., (2005) Ann.Med., 37:173-178).

[0008] HDL of C57BL/6J mouse (the atherosclerotic susceptible strain of in heredity meals being induced) is an anti-inflammatory to the mouse of the normal diet of ingesting (chow diet), but the mouse to the atherogenicity meals of ingesting is short scorching (Shih etc., (1996) J.Clin.Invest., 97:1630-1639).By contrast, antiatherogenic C3H/HeJ (C3H) the mouse normal diet of no matter ingesting still is the atherogenicity meals, and its HDL is (ibid) of anti-inflammatory.Atherosclerotic all mouse models that the appearance of being studied up to now has macrophage enrichment pathology all have short scorching HDL.These mouse comprise the C57BL/6J mouse (ibid) of the atherogenicity meals of ingesting, the mistake of normal diet of ingesting is expressed the transgenic mice (Castellani etc. of apoA-II, (1997) J.Clin.Invest., 100:464-474), the invalid mouse of PON1 (ibid) of atherogenicity meals ingests, invalid mouse (the Navab etc. of apoE, (1997) J.Clin.Invest., 99:2005-2019), invalid mouse (the Castellani etc. of the invalid PON1 of adding of comprehensive apoE of normal diet ingest, (1997) J.Clin.Invest., 100:464-474), invalid mouse (the Navab etc. of ldl receptor (LDLR) of high fat diet ingest, (1997) J.Clin.Invest., 99:2005-2019) with the transgenic mice (Leitinger etc. that express sPLA2 excessively, (1999) Arterioscler.Thromb.Vasc.Biol., 19:1291-1298).All mouse all be high fat of blood but occur in the different mouse model of atherosclerotic heredity without any mouse, find HDL be anti-inflammatory (Navab etc., (2005) Ann.Med., 37:173-178).On the other hand, other 7 all to occur with macrophage enrichment pathology be that the atherosclerotic mouse model of feature has short scorching HDL (ibid), and the anti-inflammatory of prompting HDL function or short inflammatory matter are than the more responsive existence of HDL cholesterol levels or do not have atherosclerotic indicator.The character of HDL and function have become attractive target (Castellani etc., (1997) J.Clin.Invest., the 100:464-474 that is used to form new treatment; Navab etc., (2005) Ann.Med., 37:173-178; Ridker (2002) Circulation, 105:2-4; Ansell etc., (2003) Circulation, 108:2751-2756).

[0009] although numerous albumen and enzymatic activity and HDL are associated, knows hardly and the short relevant concrete proteinogram of scorching HDL.

Summary of the invention

[0010] determines that the quality of HDL and function the most important thing is effectively to detect the atherosclerotic risk population and seek novel therapies.Numerous parameters and the component of existing abnormal function HDL inspection HDL are loaded down with trivial details relatively and expensive.

[0011] the invention provides the simple novel mensuration that is used for predicted anomaly function HDL.

[0012] in certain embodiments, the present invention relates to distinguish the evaluation of the proteinogram of urging scorching HDL and normal/anti-inflammatory HDL.This provides and has been used for the early detection atherosclerotic and is the biomarker that has situation and/or easy ill physique (neurological susceptibility) of other illness of feature with the inflammatory reaction, and therefore is provided for treating the New Policy of intervention.

[0013] in multiple embodiments, mensuration comprises that detection protoheme correlativity HDL is in conjunction with albumen (for example hoptoglobin, Hemopexin etc.), and/or detect HDL in conjunction with the relative distribution of albumen between the non-lipoprotein fraction (the non-lipoproteinfractions) of HDL and plasma, and/or detect short scorching HDL and consume nitric oxide production ability, and/or detect HDL and suppress the ability that LDL assembles (aggregation).

[0014] in certain embodiments, the invention provides the method that in mammal, detects or quantitatively urge scorching HDL.This method generally includes to be provided from mammiferous biological sample, and wherein said sample contains HDL; And detection and more than 2 kinds of HDL combination, more than 3 kinds or more than 4 kinds or more than 5 kinds, more than 6 kinds, more than 7 kinds or 8 kinds of different albumen, wherein said albumen is selected from the albumen of m/z ratio about 9.3, the albumen of m/z ratio about 14.9, the albumen of m/z ratio about 15.6, the albumen of m/z ratio about 15.8, the albumen of m/z ratio about 16.2, the albumen of m/z ratio about 16.5, the albumen of m/z ratio about 18.6 and the albumen of m/z ratio about 19.5, wherein detects two or more albumen and combines with HDL and show that described HDL is short scorching HDL.In certain embodiments, mammal is non-human mammal or people's (for example be diagnosed as and suffer from atherosclerotic people, or face the people of atherosclerotic risk, or suffer from the inflammatory reaction patient or the risk population of other illness that is feature).

[0015] in certain embodiments, be provided at the method that detects or quantitatively urge scorching HDL in the mammal.This method generally include detect combine with HDL in the mammal, contain protoheme and/or in conjunction with the level of the albumen of protoheme, wherein with normal anti-inflammatory HDL in exist contain protoheme and/or compare to contain protoheme and/or improve the described HDL of expression and be short scorching HDL in conjunction with the protein level of protoheme in conjunction with the protein level of protoheme.In certain embodiments, contain protoheme and/or comprise that in conjunction with the albumen of protoheme one or more are selected from following albumen: haemoglobin, hoptoglobin, Hemopexin, transferrins, solubility CD163 and myeloperoxidase.In certain embodiments, this method comprises the amount that detects the haemoglobin combine with HDL, and the amount of the hoptoglobin that combines with HDL of detection.In certain embodiments, this method comprises and calculates the haemoglobin combine with HDL and the product of hoptoglobin.In certain embodiments, this method comprises detecting and describedly contains protoheme and/or in conjunction with the level of albumen in the non-lipoprotein fraction of blood plasma of protoheme, wherein in HDL contain protoheme and/or in conjunction with the albumen of protoheme to increasing expression this mammiferous HDL for urging scorching HDL containing protoheme and/or comparing in the non-lipoprotein fraction of blood plasma in conjunction with this ratio that exists in the ratio of the albumen of protoheme and the object with normal anti-inflammatory HDL.

[0016] also is provided at the method that detects or quantitatively urge scorching HDL in the mammal.In a plurality of embodiments, described method comprise detect that in the mammal one or more combine with HDL, contain protoheme and/or in conjunction with the level of the albumen of protoheme; Detect that in the mammalian plasma (or serum) one or more are described to contain protoheme and/or in conjunction with the level of the albumen of protoheme, wherein HDL contain protoheme and/or in conjunction with the albumen of protoheme blood plasma is contained protoheme and/or in conjunction with the ratio of the albumen of protoheme greater than 1 expression this mammiferous HDL for urging scorching HDL.In certain embodiments, contain protoheme and/or comprise in conjunction with the albumen of protoheme and to be selected from one or more following albumen: haemoglobin, hoptoglobin, Hemopexin, transferrins, solubility CD163 and myeloperoxidase.In certain embodiments, contain protoheme and/or comprise hoptoglobin and Hemopexin in conjunction with the albumen of protoheme.In certain embodiments, for more than 2 kinds, more than 3 kinds, more than 4 kinds, more than 5 kinds or for 6 kinds of albumen, HDL contain protoheme and/or in conjunction with the albumen of protoheme blood plasma is contained protoheme and/or in conjunction with the ratio of the albumen of protoheme greater than 1 expression this mammiferous HDL for urging scorching HDL.In certain embodiments, for 1,2,3,4,5 or 6 kind of albumen for, HDL contain protoheme and/or in conjunction with the albumen of protoheme blood plasma is contained protoheme and/or in conjunction with the ratio of the albumen of protoheme greater than the value that is selected from 1,0.9,0.8,0.7,0.6,0.5,0.4,0.3 and 0.2, represent that this mammiferous HDL is short scorching HDL.

[0017] also is provided at the method that detects or quantitatively urge scorching HDL in the mammal.These methods generally include the level of measuring the protoheme that combines with HDL in the mammal, and the protoheme level that wherein combines with HDL raises this HDL of expression for urging scorching HDL than the protoheme level that combines with protectiveness HDL.In certain embodiments, the level of rising is significant on the statistics in the confidence level that is equal to or greater than 90%, 95%, 98% or 99%.

[0018] in certain embodiments, be provided at the method that detects or quantitatively urge scorching HDL in the mammal, wherein these methods generally include: measure the iron content of the HDL in the mammal, wherein the iron level of HDL raises this mammiferous HDL of expression for urging scorching HDL than the iron content of normal anti-inflammatory HDL.In certain embodiments, the level of rising is significant on the statistics in the confidence level that is equal to or greater than 90%, 95%, 98% or 99%.

[0019] in a plurality of embodiments, is provided at the method that detects or quantitatively urge scorching HDL in the mammal.These methods generally include the level that contains ferritin that combines with HDL in the mammal of measuring, and the level that contains ferritin that wherein combines with HDL raises this mammiferous HDL of expression for urging scorching HDL than the level that contains ferritin that combines with normal anti-inflammatory HDL.In certain embodiments, the level of rising is significant on the statistics in the confidence level that is equal to or greater than 90%, 95%, 98% or 99%.

[0020] also provides the method for using determination of nitric oxide to detect or quantitatively urge scorching HDL.These methods are usually directed to HDL and consume nitric oxide production ability, and wherein HDL consumes nitric oxide production ability and has situation, amount or an activity than what normal anti-inflammatory HLD consumed that nitric oxide production ability strengthens the short scorching HDL of expression.In certain embodiments, nitrogen monoxide produces through chemical method.In certain embodiments, nitrogen monoxide detects by electronic signal.

[0021] also is provided at the gathering mensuration that detects in the mammal or quantitatively urge scorching HDL.These mensuration generally include mammiferous HDL are contacted with LDL, and detect the gathering of described LDL, and wherein LDL level of assembling and the LDL that contact with normal anti-inflammatory HDL assemble and comparing expression this mammiferous HDL that raises and urge scorching HDL.In certain embodiments, gathering is measured by spectrophotometric method detection LDL coalescence rate.In certain embodiments, assemble use and do not contain albuminous pillar mensuration.

[0022] in certain embodiments, be provided in the mammal that to measure with the inflammatory reaction be the method that has situation or easy ill physique of the illness of feature.This method generally includes implements any one or more mensuration as herein described, and there is described illness in wherein positive test result explanation or is easy ill physique.In a plurality of embodiments, described illness is selected from atherosclerotic, apoplexy, leprosy, pulmonary tuberculosis, systemic loupus erythematosus, polymyalgia rheumatica, polyarteritis nodosa, chorionitis, idiopathic pulmonary fibrosis, chronic obstructive pulmonary disease, asthma, Alzheimer's, chronic renal failure, diabetes, diabetic retinopathy, nephrosis, graft rejection, transplant atherosclerotic, ischemia-reperfusion, adult respiratory system syndrome, congestive heart failure, glomerulitis, metabolic syndrome, multiple sclerosis, sepsis syndrome, drepanocytosis, vascular dementia, Crohn's disease, endothelial dysfunction, the parteriole dysfunction, AIDS, polymyalgia rheumatica, polyarteritis nodosa, chorionitis, idiopathic pulmonary fibrosis, coronary artery calcification, calcification aortic valve narrow (calcific aorticstenosis), osteoporosis, bacterial infection, virus infections, fungal infection, autoimmunity disease and rheumatoid arthritis.

[0023] in certain embodiments, to implementing mensuration as herein described from non-human mammal or people's (for example be diagnosed as and suffer from atherosclerotic people, face the people of atherosclerotic risk or suffer from the inflammatory reaction people or the risk population of other illness that is feature) sample.

[0024] in certain embodiments, in mensuration described herein, detect and/or quantitative albumen uses immunoassays (for example ELISA) to detect and/or quantitatively.

[0025] also providing treatment to suffer from the inflammatory reaction is the people of illness of feature or the method for non-human mammal.Described method generally includes to be implemented as herein described any one or more and measures, and for the more positive therapy of those positive patients' prescriptions of test in mensuration (for example give statins and/or as at United States Patent (USP) 7,166,578,7,148,197,7,144,862,6,933,279,6,930,085 and 6,664, therapeutic agent described in 230, described patent all is hereby incorporated by in all cases).

Definition

[0026] this paper can use following abbreviation: APR, acute phase response; CAD, coronary artery disease; HDL-C, the HDL cholesterol; MM-LDL, slight modify/LDL of oxidation; PON, paraoxonase; ApoA1, aPoA 1; ApoE, apo E; CM10, the weak cation chip; CVD, angiocardiopathy; CHD, coronary heart disease; FPLC, FPLC; Hb, haemoglobin; Hb-α, hemoglobin alpha-chain; Hb-β, HBB; Hp, hoptoglobin; Hx, Hemopexin; HDL, high-density lipoprotein (HDL); HPLC, high performance liquid chroma-tography; LDL, low-density lipoprotein; LDLR, ldl receptor; MetHb, methemoglobin; The NP20 chip, the positive chip; Oxy Hb, oxyhemoglobin; PON, paraoxonase; PHDL, back HDL fraction; The Q10 chip, reinforcing yin essence ion-exchange chip; RBC, red blood cell; SELDI, surface-enhanced laser desorb/ionization; SELDI-TOF-MS, surface-enhanced laser desorb/ionization flight time mass spectrum; μ LC-MSMS, micro-liquid chromatography and tandem mass spectrum; VLDL, very low density lipoprotein (VLDL).

[0027] " immunoassays " are the mensuration of utilizing the antibody of specificity bound analyte (for example hoptoglobin and/or Hemopexin).Immunoassays are characterised in that the separation of specificity binding characteristic, target and/or the quantifying analytes that uses specific antibodies.

[0028] term " polypeptide ", " peptide " and " albumen " can use alternately at this paper, are meant the polymkeric substance of amino acid residue.These terms are applicable to that wherein one or more amino acid residues are the amino acid polymer of the artificial chemical analog of corresponding natural amino acid, and the natural amino acid polymkeric substance.

[0029] term " improvement " is when using at " improving atherosclerotic one or more symptoms ", is meant to alleviate, prevent or eliminate atherosclerotic and/or distinctive one or more symptoms of related conditions.This alleviating includes but not limited to alleviate or eliminates oxidized phospholipids, alleviates atherosclerotic plaque formation and break, alleviate the clinical events such as heart attack, angina or apoplexy, the biosynthesizing that reduces hypertension, minimizing inflammatory protein, minimizing plasma cholesterol etc.

[0030] term " low-density lipoprotein " or " LDL " define according to those skilled in the art's common usage.Generally speaking, LDL is meant lipid-albumen composition, and its density range with d=1.019 to d=1.063 by super centrifuging the time exists.

[0031] term " high-density lipoprotein (HDL) " or " HDL " define according to those skilled in the art's common usage.Generally speaking, " HDL " is meant lipid-albumen composition, and its density range with d=1.063 to d=1.21 by super centrifuging the time exists.

[0032] term " I organizes HDL " or " protectiveness HDL " or " normal anti-inflammatory HDL " are meant and reduce lipid oxide (for example in low-density lipoprotein) or protect lipid to avoid high-density lipoprotein (HDL) or its component (for example apo A-I, paraoxonase, platelet activating factor PAF-AH etc.) of oxygenant oxidation.

[0033] term " abnormal function HDL " is meant at the protection lipid and avoids providing the active of reduction aspect oxidation or reparation (for example reducing) lipid oxide or do not have activity and can not or can not prevent the inflammation result's of these lipid oxides HDL substantially.

[0034] term " HDL component " is meant the component (for example molecule) that contains high-density lipoprotein (HDL) (HDL).The mensuration that the protection lipid is avoided the HDL of oxidation or reparation (for example reducing the lipid of oxidation) also comprises showing the mensuration of this active HDL component (for example apo A-I, paraoxonase, platelet activating factor PAF-AH etc.).

[0035] " Hemopexin " or " hoptoglobin " is meant natural Hemopexin of total length or hoptoglobin respectively, perhaps be meant the surrogate markers of Hemopexin or hoptoglobin, for example Hemopexin or hoptoglobin fragment, isodynamic enzyme etc., wherein the detected/quantified of mark provides the detection to the amount/concentration of full-length molecule.

[0036] term " antibody " is meant basically by one or more immunoglobulin gene encoded polypeptides, or the fragment of its specificity combination and discriminance analysis thing (antigen).The immunoglobulin gene of identification comprises κ, λ, α, γ, δ, ε and μ constant region gene, and countless immune globulin variable region gene.Light chain is K or λ by somatotype.Heavy chain is γ, μ, α, δ or ε by somatotype, and these types define immunoglobulin class IgG, IgM, IgA, IgD and IgE again respectively.Exemplary immunoglobulin (Ig) (antibody) structural unit comprises the tetramer.Every kind of tetramer all is made up of two pairs of identical polypeptied chains, and every pair all has 1 " light chain " (about 25kD) and 1 " heavy chain " (about 50-70kD).The N-end of every chain all defines about 100-110 above amino acid whose variable region, and it mainly is responsible for antigen recognizing.Variable light chain (the V of term _L) and variable heavy chain (VH) be meant these light chains and heavy chain respectively.

[0037] antibody is for example as complete immunoglobulin (Ig) or as existing by the numerous fragments that fully characterize that produce with multiple peptide enzymic digestion.Therefore, for example, the antibody below the disulfide bond of pepsin digestion in hinge area, the dimer F (ab) ' of generation Fab ₂, himself be to connect V by disulfide bond _H-C _H1 light chain.F (ab) ' ₂Can under temperate condition, be reduced, with the disulfide bond in the fracture hinge area, thus with F (ab) ' ₂Dimer changes Fab ' monomer into.Fab ' monomer be essentially Fab with part hinge area (referring to Fundamental Immunology, the 3rd edition, W.E.Paul edits, Raven Press, N.Y.1993).Although the digestion according to complete antibody has defined numerous antibody fragments, the technician will appreciate that such fragment can or utilize recombinant DNA technology from the beginning synthetic by chemical method.Therefore, term antibody used herein also comprises by modifying antibody fragment that complete antibody produces, use from the beginning synthetic those fragments (for example strand Fv) of recombinant DNA technology and being present in those fragments in the display libraries (for example phage display library).

[0038] phrase " normal health contrast " is meant individual or approximately uniform age and other groups of individuals of homogeny, and it does not have symptom or negative at the test of described illness/pathology.

[0039] phrase " detect the hoptoglobin in blood or the blood fraction and/or the level of Hemopexin and/or other albumen " and be meant detection and/or quantitative blood, blood fraction derive from blood or the sample of blood fraction in hoptoglobin and/or Hemopexin and/or other albumen.Detection can comprise hoptoglobin and/or Hemopexin and/or other albumen that direct detection is complete, and/or the fragment of detection hoptoglobin and/or Hemopexin and/or other albumen, multiple other surrogate markers of detection isotype and/or detection target protein.

[0040] phrase " provides biological sample by mammal " and is meant and directly obtains sample (for example blood sample) or obtain or provide the biological sample that has been obtained from mammal by the opposing party.

The accompanying drawing summary

[0041] Fig. 1 has shown the protein-bonded protein analysis of HDL.Protein analysis shows that (C) compares with the normal diet meals, and in the C57BL/6J mouse of atherogenicity meals (A) that ingests, HDL increases more than 10 times with combining of haemoglobin (Hb), hoptoglobin (Hp) and Hemopexin (Hx).As shown in the figure, these albumen do not combine with VLDL or LDL.

[0042] Fig. 2 shows, the HDL of the invalid mouse of ApoE of the normal diet of ingesting is short scorching, changes anti-inflammatory after oral D-4F treatment into.Before treatment (the 0th day) and with behind the fate shown in apolipoprotein A-1 simulating peptide D-4F (50 μ g/mL potable water) the treatment X-axis the invalid mouse of 9 month female apoE (every group of n=4 mouse) of the normal diet of ingesting being got blood.Measure the invalid mouse HDL of apoE and suppress the ability that LDL adds the fluorescence of DCF, as the detection of its inflammatory properties.Data show, the invalid mouse HDL of apoE is short scorching ((being extra the 0th day apoE abnormal function HDL increases to fluorescence on the fluorescence that independent LDL induces), but becoming anti-inflammatory with D-4F treatment back (1-21 days) before treatment.

[0043] Fig. 3 shows, in the serum-concentration reduction of D-4F treatment back Hemopexin.The serum-concentration of the Hemopexin in the described mouse of above Fig. 2 is measured by ELISA.

[0044] Fig. 4 shows, in the serum-concentration reduction of D-4F treatment back hoptoglobin.The serum-concentration of the hoptoglobin in the described mouse of above Fig. 2 is measured by ELISA.

[0045] Fig. 5 shows, hoptoglobin (Hp) and Hemopexin (Hx) among the HDL of the invalid mouse of D-4F treatment reduction apoE.HDL separates from mouse described in Fig. 2, and measures the content of Hp, Hx and apolipoprotein A-1 (ApoA-I).

[0046] Fig. 6 shows, the non-RBC haemoglobin that D-4F treatment causes molecular weight on the non-sex change PAGE gel to be similar to haemoglobin molecule amount contained among the RBC increases.From (the 0th day) before each treatment only of 4 mouse described in Fig. 2 with treat that serum, HDL and the red blood cell (RBC) of (the 21st day) carries out electrophoresis on non-sex change PAGE (4-15%) gel after 21 days, and come analysing haemoglobin by protein analysis.Data show, before treatment, in the serum and all haemoglobins in the HDL supernatant (it contains HDL and non-lipoprotein fraction) all on non-sex change PAGE gel with the apparent molecular weight electrophoresis of the molecular weight of the haemoglobin that is significantly higher than cracking RBC.Yet, after with D-4F treatment 21 days, the haemoglobin that significant quantity arranged in serum and HDL supernatant on gel with the apparent molecular weight electrophoresis of the molecular weight of the haemoglobin that is similar to cracking RBC.

[0047] Fig. 7 shows, the α chain of haemoglobin and β chain all increase in the wild type C57BL/6J mouse of atherogenicity meals that ingests.This figure also shows, the α chain and the β chain content of haemoglobin all increase in the invalid mouse of apoE of picked-up normal diet, and two chains all descend with the D-4F treatment, as described in Fig. 2.

[0048] Fig. 8 shows, reaches the oxyhemoglobin measured in the wild type C57BL/6J mouse that 7 days (D7C) or the atherogenicity meals of ingesting reach 7 days (D7A) or 15 weeks (W15A) among the HDL and the content of methemoglobin at the normal diet of ingesting.Also in the invalid mouse of the apoE of the normal diet of ingesting (apoE), measure the content of oxyhemoglobin and methemoglobin.As shown in the figure, in the invalid mouse of apoE of the C57BL/6J mouse of the atherogenicity meals of ingesting and the normal diet of ingesting, most of haemoglobin in conjunction with HDL is an oxyhemoglobin.

[0049] Fig. 9 shows, add to feed to raise HDL that normal diet reaches 7 days wild type C57BL/6J mouse (D7C) and do not consume the nitrogen monoxide that produces and detect with electric current (pA) with chemical mode (NO donor).

[0050] Figure 10 shows, adds oxyhemoglobin (HbO ₂) make with chemical mode (NO donor) generation and to descend in the fast wink of nitric oxide production decay curve that electric current (pA) detects.Add phosphate-buffered saline (PBS) and do not change the natural decay curve.On the contrary, add the HDL (D7A HDL-Hb) that the atherogenicity meals of ingesting reach 7 days wild type C57BL/6J mouse decay curve is sharply descended fast, show that this short scorching HDL consumes nitrogen monoxide fast.

[0051] Figure 11 shows, short scorching HDL (reaches the HDL of 7 days wild type C57BL/6J mouse from the atherogenicity meals of ingesting; D7AHDL) the nitrogen monoxide decay curve is sharply descended fast, show that short scorching HDL consumes nitrogen monoxide fast.Yet, using the oxyhemoglobin (HDL-HbO among the HDL ₂) change the K of methemoglobin (HDL-metHb) into ₃Fe (CN) ₆After handling this HDL, adding this HDL can not significantly change decay curve, shows that nitrogen monoxide is not consumed.

[0052] Figure 12 has shown that using the normal diet of ingesting to reach 7 days (the picture left above) or 15 weeks (top right plot) or the atherogenicity meals of ingesting reaches the protein analysis of serum of wild type C57BL/6J mouse in 7 days (lower-left figure) or 15 weeks (bottom-right graph) to the two-way gel of haemoglobin dyeing.The haemoglobin position of the splitting erythrocyte (RBC) of every kind of condition of the most left hurdle representative of every width of cloth figure.

[0053] Figure 13 has shown the below figure of Figure 12, and wherein the wild type C57BL/6J mouse atherogenicity meals of ingesting reach 7 days (D7A) or 15 weeks (W15A), and haemoglobin dyeing is with light blue demonstration (duplicating is grey).

[0054] Figure 14. peel off the Western blotting shown in Figure 13, and survey again with the antibody of hoptoglobin.The image (for magenta) that produces is covered on the image of Figure 13.Mazarine Regional Representative is haemoglobin and these two common positioned area of hoptoglobin wherein.

[0055] Figure 15. peel off the Western blotting shown in Figure 14, and survey again with the antibody of Hemopexin.The image (yellow) that produces is covered on the image of Figure 14.Very dark graphical representation wherein haemoglobin, hoptoglobin and Hemopexin is total to positioned area.

[0056] Figure 16 shows, short scorching HDL does not suppress LDL and assembles, and anti-inflammatory HDL suppresses the LDL gathering.Test is from the ability of 4 LDL gatherings that have short scorching HDL and coronary artery disease or induce according to the patient who is equal to disease (CHD patient) of NCEP ATP III standard and 4 healthy volunteers' (normally) HDL inhibition phospholipase C (PLC).The value that does not add the positive control (LDL+PLC) of HDL shows with red line.The value of negative control (independent LDL) shows with the blue line of below.Data show that 4 CHD patients' HDL can not suppress the LDL gathering that PLC induces, and 4 healthy volunteers' (normally) HDL significantly suppresses the LDL gathering that PLC induces.

[0057] Figure 17 shows, before treatment, short scorching apoE abnormal function HDL does not suppress the LDL that phospholipase C (PLC) induces and assembles, but with oral D-4F treatment after 21 days, and HDL becomes anti-inflammatory, and suppresses the LDL gathering that PLC induces.As described in Fig. 2 with oral D-4F treatment before and afterwards, test apoE abnormal function HDL suppresses the ability that LDL that PLC induces assembles.As shown in FIG. 2, HDL is short scorching before treatment.As shown in Figure 17, before treatment, this apoE abnormal function HDL can not suppress the LDL gathering that PLC induces.Yet after treatment 21 days, HDL becomes (Fig. 2) of anti-inflammatory, and as shown in Figure 17, it significantly suppresses the LDL gathering that PLC induces.The value (LDL+PLC, no HDL) that has shown positive control has also shown the value of following negative control: i) LDL+PLC is together with the HDL (normal mouse HDL) of the normal C57BL/6J mouse of the normal diet of ingesting with ii) there is not the LDL (independent LDL) of PLC.

[0058] Figure 18 has shown the cholesterol spectrum of C57BL/6J mice serum sample after the FPLC fractionated.By the combining anteserum of FPLC fractionated 500 μ l from mouse (n=8).Preceding 10 1mL fractions are discarded, analyze the cholesterol level of each 1mL subsequently.The C-normal diet is fed and was raised 7 days, A-atherogenicity meals, and 7 days, the CC-normal diet was ingested 21 days, and AC-atherogenicity meals are fed and were raised 7 days, and normal diet is fed and was raised 14 days afterwards.

[0059] Figure 19 has shown the inflammatory properties from the HDL of C57BL/6J mice serum.HDL separates with HDL reagent, and analyzes its active o content (fluorescence intensity), paraoxonase (PON) determination of activity, cholesterol outflow mensuration (outflow of % cholesterol) and cholesterol level (HDL-cholesterol).The C-normal diet is fed and was raised 7 days, A-atherogenicity meals, and 7 days, AC-atherogenicity meals 7 days, were fed afterwards and were raised normal diet 14 days.* represent p＜0.01.

[0060] Figure 20 shows, has the m/z 14.9k (hemoglobin alpha-chain) and the m/z15.6k (HBB) of elevated levels in 4 atherosclerotic mouse models.The blood serum sample (n=8) of invalid mouse of LDLR (LDLR) that the atherogenicity meals of ingesting (D7=7 days, W15=15 week) or the C57BL/6J mouse of western-style food 10 days (WD) or the western-style food of ingesting was reached for 8 weeks with Q10 (pI＜7) ProteinChip array or the 12 week invalid mouse of apoE in age (apoE) of the normal diet of ingesting carries out SELDI and analyzes.Compared with the peak of the serum of the corresponding mouse control group of the normal diet of ingesting in two target SELDI peaks (m/z 14.9k and m/z 15.6k) in atherogenicity serum, or with regard to the invalid mouse of apoE, compare, and with intensity that statistical analysis is obtained with the C57BL/6J mouse of the age-matched of the normal diet of ingesting.The data that provide are the average increase multiple at each peak.The data that provide all have the statistically-significant difference meaning, p value＜0.05.It is to be noted that it is the hydrophobicity albumen and the compound sedimentation of pI＜7.0 again that the albumen of this dissecting needle to catching, this chip make on the Q10 protein chip.Haemoglobin representative by this methods analyst is in conjunction with the haemoglobin of HDL.

[0061] Figure 21 illustrates the pI mensuration at the peak of representing m/z 14.9k (hemoglobin alpha-chain) and m/z 15.6k (HBB).The damping fluid of different pH shown in being adopted as, utilize the centrifugal post of anion exchange, raise the individual blood serum sample that normal diet (C) or atherogenicity meals (A) reach the C57BL/6J mouse (n=8) in 7 days (D7) or 15 weeks (W15) and carry out desalination and fractionated feeding.With cation exchange (CM10:pI〉4) or anion exchange (Q10:pI＜4) ProteinChip array the fraction of wash-out being carried out SELDI analyzes.Shown in the relative density of 14.9k and 15.6k.

[0062] Figure 22,22B and 22C have compared non-RBC haemoglobin and RBC haemoglobin, and have compared the distribution of the lipoprotein and the non-RBC haemoglobin in the non-lipoprotein fraction of serum.Serum and lipoprotein are loaded on the 15%SDS-PAGE, and to the hemoglobin immune trace.The RBC lysate that derives from the mouse of the normal diet of ingesting loads as haemoglobin standard product.Figure 22 A: the blood serum sample (n=8) that reaches the C57BL/6J mouse in 7 days (D7) or 15 weeks (W15) from ingest normal feed (C) or atherogenicity meals (A); Figure 22 B: from VLDL, LDL, HDL and back HDL (pHDL) the FPLC fraction of the combining anteserum sample of D7 and W15 group.Figure 22 C: the cholesterol (OD490) and the protoheme (OD410) of the HDL of analysis covering D7-C and D7-A blood serum sample and the single FPLC fraction of pHDL zone (fraction 25-40).Figure 22 A shows, raises in the mouse that normal diet or atherogenicity meals reach 7 or 15 days feeding, and the amount of non-RBC total hemoglobin does not have significant difference.This figure shows that also on SDS PAGE gel, non-RBC haemoglobin is similar with the molecular weight of the haemoglobin of cracking RBC.Figure 22 B shows, feeds to raise the atherogenicity meals and reach 7 days or non-RBC haemoglobin is moved to the HDL fraction by non-lipoprotein fraction (pHDL).Figure 22 C has confirmed the data among Figure 24 B, shows ingesting normal diet after 7 days, almost the haemoglobin that does not combine with HDL, and is ingesting the atherogenicity meals after 7 days, and a lot of haemoglobins are arranged in the HDL fraction.

[0063] Figure 23 A and 23B show, have different qualities at the atherogenicity serum of the atherogenicity meals of ingesting and the non-RBC haemoglobin in the HDL fraction.Figure 23 A: the combining anteserum sample of feeding the C57BL/6J mouse (n=8) raise normal diet (C) or atherogenicity meals (A) by the FPLC fractionated, and with positive (NP20) or anion exchange (Q10; PI＜7) the ProteinChip array carries out the SELDI analysis.Shown in the relative intensity of 14.9k (Hb-α) and 15.6k (Hb-β).Figure 23 B: representative pH7.0 that will describe under Fig. 2 and the anion-exchange column fraction of pH4.0 are loaded on the 15%SDS-PAGE, and carry out Western blotting at haemoglobin.The characteristic of the change of the non-RBC haemoglobin of the atherogenicity meals of in these systems, measuring of ingesting and haemoglobin and HDL and other HDL albumen in conjunction with consistent.

[0064] Figure 24 shows, the haemoglobin in atherogenicity serum has different pI values.Merge the blood serum sample that reaches the C57BL/6J mouse (n=8) in 7 days (D7) or 15 weeks (W15) from the normal diet of ingesting (C) or atherogenicity meals (A) before use.RBC from the D7 mouse passes through the serum gradient separations, washing and cracking.Blood serum sample merges the HDL fraction from the D7 mouse by the FPLC fractionated.The serum, HDL and the RBC lysate that merge are loaded on the IEF gel (pH3-10), and carry out Western blotting at haemoglobin.RBC lysate (left swimming lane) from the mouse of the normal diet of ingesting loads as haemoglobin standard product.This figure shows that the characteristic of RBC haemoglobin does not have difference between the mouse of normal diet or atherogenicity meals that ingests.This figure also shows, less than the haemoglobin that combines with the HDL that feeds the mouse of raising normal diet, but has the haemoglobin that combines with the HDL that feeds the mouse of raising the atherogenicity meals, and its pI and RBC haemoglobin are completely different.

[0065] Figure 25 shows, non-RBC haemoglobin combines (left figure) with the HDL fraction of the atherogenicity meals of ingesting, and the non-RBC haemoglobin that is similar to the migration of RBC haemoglobin is in the atherogenicity meals 15 week back disappearances (right figure) of ingesting.To be loaded on non-sex change PAGE from the blood serum sample (right figure) of feeding the merging raise the C57BL/6J mouse (n=8) that normal diet (C) or atherogenicity meals (A) reach 7 days (D7) or 15 weeks (W15) or from the merging HDL fraction (left figure) of D7, and carry out Western blotting at haemoglobin.To be loaded on (leftmost side of left figure) on the gel from the RBC lysate of the mouse of the normal diet of ingesting again.

[0066] Figure 26 A and 26B have shown the spectrophotometric determination result of Hb among the HDL.Use Beckman DU 640 spectrophotometers, determine amount and the form of Hb by the merging FPLC fraction that contains HDL.Scanning is by all samples of 380nm to 700nm and the spectrum of pure material (Figure 26 A).Spermine nitrogen oxidation adduct (spermine NONOate) is a kind of time slowly to discharge the NO donor, and it is added sample, to observe the transformation (Figure 26 B, synoptic diagram) of oxyHb to metHb.Utilize linear regression with " the basic spectrum " of one group of pure material to detecting spectrum simulation, the concentration of deconvolute oxyHb and metHb (Vaughn etc., (2000) J.Biol.Chem., 275:2342-2348).

[0067] Figure 27 has compared 10 healthy volunteers and 10 coronary heart disease (CHD) patients or by the haemoglobin among the patient's who is equal to disease of NCEP ATPIII guide definition the HDL.Left figure has shown the non-sex change PAGE gel of the HDL fraction of the blood plasma that is merged by 10 healthy volunteers (left swimming lane) or 10 CHD patients (right swimming lane), and this gel carries out Western blotting at haemoglobin.Right figure has shown the same analysis behind the cracking RBC, and lysate is added blood plasma, separates the HDL fraction then.The result shows, the haemoglobin that combines with HDL among the patient is far more than healthy volunteer (left figure).This figure also confirms, adds excessive RBC haemoglobin to blood plasma and all produce extra haemoglobin in healthy volunteer and CHD patient's HDL fraction, but still find significantly more haemoglobin in patient's HDL.

[0068] Figure 28 A, 28B and 28C confirm, in juvenile diabetes and CHD patient's HDL, non-RBC haemoglobin and hoptoglobin all increase.By 12 healthy volunteers, 14 juvenile diabetes patient (8 adults; 6 children) or 8 CHD that take statins or CHD be equal to the disease experimenter and collect serum.Will be on 96 orifice plates at the antibody sandwich of people apoA-I.Adding is from experimenter's serum, and in 4 ℃ of incubations that spend the night.Thorough wash plate, and resist in 4 ℃ of incubations that spend the night with the goat one of antihuman hemoglobin or people's hoptoglobin.Thorough wash plate, and with put together resisting in room temperature incubation 2 hours of horseradish peroxidase (HRP) at two of goat IgG.Thoroughly wash plate adds the HRP substrate, detects optical density (OD).Figure 28 A shows non-RBC haemoglobin with μ gHRP/mL.Figure 28 B shows hoptoglobin with μ gHRP/mL.Figure 28 C has shown the non-RBC haemoglobin product with the hoptoglobin value on duty.The result shows, the value that back one method obtains makes a distinction healthy volunteer and diabetic and CHD patient, without any overlapping.

[0069] Figure 29 confirms, (+the transferrin content that D-4F) reaches in the HDL supernatant that caused mouse in 2 months significantly descends than the mouse of the potable water (no D-4F) of accepting not have D-4F to add D-4F to the potable water of the invalid mouse of the apoE at 4 monthly ages (every group of n=8).

[0070] Figure 30 confirms, (+myeloperoxidase the content that D-4F) reaches in the HDL supernatant that caused mouse in 2 months descends very significantly than the mouse of the potable water (no D-4F) of accepting not have D-4F to add D-4F to the potable water of the invalid mouse of the apoE at 4 monthly ages (every group of n=8).

Detailed Description Of The Invention

[0071] the present invention relates to new survey for abnormal function HDL (HDL) Fixed. Abnormal function HDL (for example short scorching HDL) participates in heart disease and take inflammatory reaction as feature The genesis of other illness. Therefore, be used for the mensuration of abnormal function HDL to artery congee The detection of sample sclerosis and/or the Other diseases take inflammatory reaction as feature and/or prediction prognosis have Important value, described Other diseases for example rheumatoid arthritis, lupus erythematosus, nodositas are many Arteritis, osteoporosis, atherosclerotic, apoplexy, leprosy, pulmonary tuberculosis, systemic red Yabbi sore, polymyalgia rheumatica, PAN, chorionitis, idiopathic pulmonary fibrosis, Chronic obstructive pulmonary disease, asthma, Alzheimer's, chronic renal failure, diabetes, sugar Urine characteristic of disease retinopathy, nephrosis, graft rejection, transplanted abdominal is atherosis, lack Blood pours into again, adult respiratory system syndrome, congestive heart failure, glomerulitis, metabolism Syndrome, multiple sclerosis, sepsis syndrome, drepanocytosis, vascular dementia, gram Engler sieve disease, endothelial dysfunction, parteriole dysfunction, AIDS, polymyalgia rheumatica, PAN, chorionitis, idiopathic pulmonary fibrosis, CAC, Calcified Aortic valve is narrow, osteoporosis, bacterium infection, fungal infection, autoimmunity disease and class Rheumatic arthritis, congestive heart failure, endothelial dysfunction, parteriole dysfunction, Virosis, multiple sclerosis etc.

[0072] increasing evidence shows that HDL-inflammation/anti-inflammatory property is that HDL is all Such as deciding factor important in the atherosclerotic disease, do not have with the HDL-cholesterol levels Close. The present invention relates to detect the new mensuration of the inflammatory properties of HDL. Mensuration described herein is common Can be classified as two primary categories:

[0073] the protein-bonded detection of I.HDL, it has reflected the inflammation character of HDL; With

[0074] II.HDL suppresses the detection of the ability of LDL gathering.

The protein-bonded detection of I.HDL, it has reflected the inflammation character of HDL

8 kinds of albumen of A) in atherosclerotic/hyperlipemia in mice model, being combined with HDL

[0075] we before reported, and in mouse and the mankind, the inflammatory properties of HDL is equal Coronary heart disease (CHD) index more responsive than HDL-cholesterol levels. In some embodiment In, the present invention partly relates to differentiation I group/protectiveness HDL with " abnormal function " HDL's The evaluation of specific proteins finger-print. Although in embodiment 2 and 3 for mouse HDL Set forth, but it is generally acknowledged in people and other mammal and moving identical albumen / protein fingerprint spectrum and physiological mechanism. Specifically, distinguishing normal mouse HDL causes with ingesting The specific proteins finger-print of the mouse HDL of atherogenic diet passes through The laser desorption that ProteinChip technology coupling surface strengthens/ionization flight time mass spectrum (SELDI-TOF-MS) identify. Feed and to raise C57BL/6J mouse atherogenicity meals and reach Cause the reduction of HDL-cholesterol levels, the active decline of paraoxonase, reactive oxygen species content 1 week Increase and HDL promote cholesterol to be descended by the ability of macrophage outflow. When mouse is turned to Gain normal diet and reached again for 2 whens week, the short atherogenicity feature of HDL is returned to normally Phenotype (referring to for example Figure 19). We identify the SELDI peak of 88 p＜0.05 altogether, They in the short scorching HDL that feeds the mouse raise the atherogenicity meals exist situation with The normal HDL that feeds the mouse of raising normal diet compares difference. In 88 serum peaks 74 Be returned to normal level during the meals counter-rotating. further analyze with eliminate by the short-term meals change and After the artefact that deactivated pulse atherosclerosis factor produces/change, we identify 24 The SELDI m/z peak of the albumen that representative is combined with short scorching HDL difference. Find 24 protein peaks In 14 be following in addition 3 kinds of widely used atherosclerotic/hyperlipemia animal moulds The short scorching HDL of type is common: the C57BL/6J mouse of the western-style food of ingesting, LDLR are invalid little The invalid mouse of mouse and apoE. In addition, the proteinogram of the blood serum sample of whole 4 kinds of animal models Analysis and Identification goes out a kind of 8 protein core labels (subgroups at above-mentioned 14 SELDI m/z peaks), its Can be used as serum biomarker group and identify short scorching HDL in mouse, similarly label can be used for the people With other mammal. These 8 core label proteins describe in detail in embodiment 2.

[0076] this albumen label can be easy to detect (for example use mass spectrography as herein described and/ Or protein chip), and therefore be used for for example identifying short scorching HDL the patient. Therefore, egg White label exist situation or degree can as atherosclerotic and/or take inflammatory reaction as The diagnosis of the Other diseases of feature and/or prediction prognosis.

[0077] in some cases, can use other standard method, include but not limited to electricity Swimming, chromatogram and/or immunoassays detect one or more albumen, identify or quantitative suffering to provide Fast detecting/the quantitative approach of person's albumen label intensity.

B) wonderful discovery: non-RBC blood in atherosclerotic/hyperlipemia in mice model Lactoferrin is combined with HDL

[0078] as mentioned above, we use reinforcing yin essence ion-exchange SELDI ProteinChip Technical appraisement goes out 8 species specificity albumen, and this technology can be independently or is used in combination distinguishing normal / anti-inflammatory HDL and short scorching HDL. Also in mouse model, identify 2 with short scorching HDL Significantly the biomarker peak of combination (m/z14,900 and m/z15,600). We use little liquid phase layer Analyse-tandem mass spectrum identifies the SELDI peak that represents respectively m/z 14,900 and m/z 15,600, It is respectively mouse hemoglobin alpha-chain (Hb-α, 14.9kDa) and mouse HBB (Hb-β, 15.9kDa). Western blot analysis confirms, compares Hb and short scorching HDL with normal HDL The difference combination. The biochemistry of the Hb of being combined with HDL characterizes and further shows, with short scorching The Hb of HDL combination has distinct physics and chemistry characteristic, comprises that pI reduces (pI4.0 More than the pI7.5 of pI7.0 to free Hb), with the high score that exists in the fraction that contains HDL The combination of son amount compound.

[0079] macromethod of this non-RBC hemoglobin is disclosed, amino acid sequence with RBC hemoglobin difference. The non-RBC hemoglobin of in fact, being combined with short scorching HDL The physical characteristic that changes as if owing to this hemoglobin and other albumen of being combined with HDL as Combining closely of hoptoglobin.

[0080] wonderful the present invention's discovery is, in mouse and people, always at blood plasma And have a small amount of non-RBC hemoglobin in the serum. The concentration of this non-RBC hemoglobin is about 10 micromoles. By contrast, the HC in the whole blood is greater than 1 mole. Therefore, exist 0.001% the hemoglobin of only having an appointment in the whole blood does not belong to RBC.

[0081] also wonderful the present invention's discovery is, in normal mouse and normal person, This a small amount of non-RBC hemoglobin is arranged in the non-lipoprotein fraction of blood plasma or serum. Also order The present invention that the people is surprised finds is, in the mouse of atherogenicity meals that ingests, perhaps Occur in the atherosclerotic mouse carrying out genetic manipulation, perhaps diabetes or Among the patient of CHD, perhaps in the people with other short scorching HDL cause, this non-RBC blood Lactoferrin is present among the HDL.

[0082] the main hemoglobin form that is found to be combined with HDL is the oxygenated blood red eggs In vain (oxyHb). Based on the prooxidant character of oxyHb, our Notes of Key Data is activating Under the condition of pulse atherosclerosis, Hb may help the short inflammatory matter of HDL. And, no Be limited to concrete theory, we think, in conjunction with the Hb of HDL can as atherosclerotic or The neoformation mark of other patient's condition take inflammatory reaction as feature. Some details of these researchs is retouched Be set forth in embodiment 3.

The acute phase protein of C) in atherosclerotic/hyperlipemia in mice model, being combined with HDL

[0083] as mentioned above, we find to have distinct thing with red blood cell Hb Reason and the hemoglobin (Hb) of chemical characteristic in atherosclerosis animal model in conjunction with HDL, And under atherogenic condition, help the short inflammatory matter of HDL. Therefore, for example, Compare with feeding the C57BL/6J mouse of raising normal diet, feed and raise C57BL/6J mouse (every group of n=12) the atherogenicity meals reach and caused in 1 week: i) the HDL-cholesterol levels reduces, ii) Paraoxonase activity decreased, and iii) reactive oxygen species increases, and iv) HDL promote cholesterol by The ability decline (referring to for example Figure 19, embodiment 2) of macrophage outflow.

[0084] Hemopexin and hoptoglobin are that two kinds of well-known Hb are clear Except agent. Known hoptoglobin (Hp) is combined with HDL, except its concentration in blood plasma with molten Blood and beyond increasing, it also increases in the middle of acute phase response. Wonderful the present invention finds Be, Hemopexin (Hx) also is combined with short scorching HDL, and along with acute phase response is similar Ground increases.

[0085] as shown in FIG. 1, protein analysis shows, Hb, Hx and Hp egg White compound with derive from the HDL that feeds the C57BL/6J mouse of raising atherogenicity meals (A) The combination of fraction is raised normal diet (C) and compares 10 times of increases (〉 with feeding).

[0086] also find Hb, Hx and Hp albumen composition with from the normal diet of ingesting The HDL combination of the invalid mouse of apoE. For determining these risings relevant with the ferroheme approach HDL whether be the effectively new mark that consists of atherosclerotic inflammation in conjunction with protein level, Treat these apoE with apolipoprotein A-I mimetic peptide D-4F (the drinking water solution of 50 μ g/ml) Invalid mouse reaches 21 days, and analyze they HDL inflammatory properties and with the ferroheme approach Relevant HDL is in conjunction with albumen.

[0087] D-4F treatment i) change HDL into anti-inflammatory (referring to for example Fig. 2) by short inflammation, Ii) detect according to ELISA, significantly reduce Hx (referring to for example Fig. 3) in the serum and Hp (referring to Fig. 4 for example) level iii) reduces Hx in the HDL fraction and Hp albumen composition (referring to example Such as Fig. 5), and iv) Hb section in the invalid serum of apoE and the invalid HDL supernatant of apoE Divide the physics and chemistry characteristic that returns to normal (namely visible in red blood cell) (referring to for example figure 6). Therefore, in certain embodiments, the invention provides detect be combined with HDL, for urging The method of the ferroheme GAP-associated protein GAP of scorching HDL component.

[0088] as shown in FIG. 7, feeding the wild type of raising the atherogenicity meals In the C57BL/6J mouse, α chain and the β chain of hemoglobin all increase. α chain and the β of hemoglobin The content of chain all increases in the invalid mouse of the apoE of the normal diet of ingesting, two chains all with D-4F treats and reduces.

[0089] as shown in FIG. 8, cause feeding to raise normal diet 7 days (D7C) or to feed to raise The wild type C57BL/6J that atherogenic diet reaches 7 days (D7A) or 15 weeks (W15A) is little Oxyhemoglobin in the mouse among definite HDL and the content of ferrihemoglobin. Also raise feeding Determine oxyhemoglobin and siderosis red eggs in the invalid mouse of the apoE of normal diet (apoE) White content.

[0090] oxyhemoglobin consumes nitric oxide, and ferrihemoglobin does not consume one Nitrogen oxide. As shown in FIG. 9, adding normal mouse HDL does not change with chemical mode and produces The nitric oxide production natural decay of giving birth to. On the contrary, as shown in Figure 10, when to chemistry side Formula produces (NO donor) and adds oxyhemoglobin as the nitric oxide that electric current (pA) detects (HbO₂) time, the nitric oxide production decay that produces with chemical mode has unexpected and rapid changing Become, show that it is consumed by oxyhemoglobin. And for example shown in Figure 10, add phosphate-buffered salt Water (PBS) does not change decay curve, show do not exist owing to add that solvent (PBS) takes place one Nitrogen oxide consumes. By contrast, add to feed and to raise the atherogenicity meals and reach 7 days wild The HDL of type C57BL/6J mouse (D7AHDL-Hb) causes decay curve sharply to descend, table Bright this short scorching HDL consume nitric oxide fast.

[0091] as shown in Figure 11, use the material [K that oxyhemoglobin is changed into ferrihemoglobin₃Fe(CN) ₆] process from feeding the mouse of raising the atherogenicity meals Short atherogenic HDL reverses short scorching HDL and consumes fast nitric oxide production ability.

[0092] Figure 12 illustrates from feeding and raises normal diet and reach 7 days (the picture left above) or 15 weeks (top right plot) or feed and to raise the atherogenicity meals and reach 7 days (lower-left figure) or 15 weeks (bottom-right graph) Red blood cell (RBC) hemoglobin of wild type C57BL/6J mouse and serum hemoglobin Two-way gel. Figure 13 has copied the below figure of Figure 12, wherein wild type C57BL/6J mouse Feed and to raise the atherogenicity meals and reach 7 days (D7A) or 15 weeks (W15A), just hemoglobin Dyeing is with light blue demonstration. Peel off the Western blotting that is shown in Figure 13, and use hoptoglobin Antibody survey again (Figure 14). Peel off the Western blotting that is shown in Figure 14, and use the ferroheme combination The antibody of albumen is surveyed (Figure 15) again.

[0093] experiment that is illustrated in Figure 12-15 shows, hemoglobin, hoptoglobin and Hemopexin can be arranged on the identical particle of atherogenicity serum.

[0094] as shown in FIG. 1, for the atherogenicity meals, these particles Mainly be combined with HDL. Feed raise hoptoglobin and/or Hemopexin invalid C57BL/6J mouse atherogenicity meals reach 7 days and have confirmed that hoptoglobin is short scorching Important function during HDL forms. In brief, to wild type (WT) or ferroheme in conjunction with egg (Hx) invalid or hoptoglobin (Hp) invalid or Hemopexin and hoptoglobin in vain (Hp/Hx) all invalid C57BL/6J mouse feeds and raises normal diet (C) or atherogenicity Meals (A) reach 7 days. Mouse is got blood, measure the cholesterol in the FPLC fraction of their blood plasma Content and content of hemachrome. In fact wild type HDL does not have the ferroheme of combination. Raise when feeding When these mouse atherogenicity meals reach 7 days, there is the blood of being combined with the HDL fraction Red pigment (yellow line of below figure). When not having Hemopexin, even if ingest normal diet Also there is the ferroheme be combined with the HDL fraction, when these mouse atherogenicity of ingesting When meals reached 7 days, the content of hemachrome among the HDL of these mouse increased. By contrast, The mouse atherogenicity meals, that do not have hoptoglobin that ingests does not have in conjunction with HDL Ferroheme (bottom-right graph green line; The bottom-right graph red line). Therefore, hoptoglobin is the ferroheme knot It is necessary to close HDL.

[0095] in another experiment, to wild type (WT) or Hemopexin (Hx) Invalid or hoptoglobin (Hp) is invalid or Hemopexin and hoptoglobin (Hp/Hx) All invalid C57BL/6J mouse feeds and to raise normal diet or the atherogenicity meals reach 7 days. Mouse is got blood, and measure cholesterol level and active oxygen in the FPLC fraction of their blood plasma Material (ROS) content. In fact HDL does not have the ROS of combination. Raise these mouse actuatings when feeding When the pulse atherosclerosis meals reach 7 days, there is the ROS of being combined with the HDL fraction. There is not blood Red pigment is during in conjunction with albumen, even if also there is the ROS of being combined with the HDL fraction in the picked-up normal diet, When these mouse ingest the atherogenicity meals when reaching 7 days, among the HDL of these mouse ROS content increase. By contrast, ingest the atherogenicity meals, do not have a combination The mouse of globin does not have the ROS in conjunction with HDL. Therefore, hoptoglobin is the ROS knot It is necessary to close HDL.

[0096] data acknowledgement provided herein wonderful discovery: short scorching HDL can By the label protein of being combined with HDL or by containing the protein-bonded ferroheme level of HDL Detect. The present invention has also obtained unexpected discovery: be combined with HDL, contain ferroheme And/or in conjunction with the level of the albumen of ferroheme than the non-fat egg of these albumen in plasma/serum The ratio of the level in the white fraction is the short scorching HDL of predictability. The present invention has also obtained exceeding meaning The discovery of material: be not combined with HDL, contain ferroheme and/or in conjunction with the albumen of ferroheme Situation under, HDL can not show short scorching feature (the ROS content that for example increases).

[0097] based on these unexpected discoveries, provide numerous evaluations short scorching (abnormal function) The mensuration of HDL. These mensuration include but not limited to detect be combined with HDL 1,2,3,4, 5,6,7 or 8 kind of albumen, wherein albumen have about 9.3,14.9,15.6,15.8,16.2, 16.5,18.6,19.5 m/z ratio, wherein the combination of these albumen and HDL shows described HDL is short scorching HDL.

[0098] as mentioned above, wonderful the present invention finds it is that the m/z ratio is 14.9k Be respectively hemoglobin alpha and β chain with the albumen of 15.6k. Also wonderful the present invention's discovery is, The m/z ratio is that the albumen of 19.5k is XII group PLA₂。

[0099] in another embodiment; this mensuration relates to detecting and contains protoheme and/or in conjunction with the level of the albumen (for example haemoglobin, hoptoglobin, Hemopexin, myeloperoxidase etc.) of protoheme; or in conjunction with the level of the albumen (for example solubility CD163) of Hemopexin; these protein combination HDL, wherein said in conjunction with in the albumen of HDL more than a kind, more than 2 kinds, more than 3 kinds or 4 kinds level raise (for example comparing) with the level of in protectiveness HDL, finding show that this HDL is short scorching HDL.

[0100] in certain embodiments; mensuration comprises detect combine with HDL, contain protoheme and/or in conjunction with the level of the albumen (for example haemoglobin, hoptoglobin, Hemopexin, myeloperoxidase) of protoheme; and with the non-lipoprotein fraction of HDL in these contain protoheme and/or compare in conjunction with the level of the albumen of protoheme, wherein the level of Sheng Gaoing (for example comparing with the level of finding in protectiveness HDL) shows that this HDL is short scorching HDL.In a plurality of embodiments, mensuration comprises that the level of measuring albumen that combine with HDL, that contain protoheme (for example haemoglobin, hoptoglobin, Hemopexin, myeloperoxidase) contains protoheme in the non-lipoprotein fraction of blood plasma and/or serum these and/or in conjunction with the ratio of the level of the albumen of protoheme, the ratio of wherein at least a kind, at least 2 kinds, at least 3 kinds or 4 kinds albumen more than or equal to about this HDL of 0.2,0.3,0.4,0.5,0.6,0.7,0.8,0.9 or 1 value representation for urging scorching HDL.

[0101] in a plurality of embodiments, the predictive value of mensuration two or more contain protoheme and/or improves in conjunction with the albumen of protoheme by detecting.In a plurality of embodiments, two or more contain protoheme and/or can make up and/or other factor of weighting in conjunction with the detection of the albumen of protoheme.Therefore, for example, can use discriminant function analysis or cluster analysis to determine every kind of weight factor that detects albumen.In one exemplary embodiment, people can calculate and two or more contain protoheme and/or in conjunction with the product of the content of the albumen of protoheme (for example determine obtain by the value that multiply by each value), for example be shown in the embodiment that provides among Figure 28 C.Use is shown in the embodiment of Figure 28 C, with regard in the process of getting blood by the experimenter or after getting blood with regard to the situation of external generation haemolysis, this analysis can improve the predictive value of mensuration, and is indicated among the embodiment as shown in Figure 27.Therefore, for example, people can calculate the product that the haemoglobin that combines with HDL multiply by the value of the hoptoglobin that combines with HDL, and the two all provides the mensuration yardstick with μ gHRP/mL.

[0102] in a plurality of embodiment; mensuration comprises the level of measuring the protoheme that combines with HDL; and/or the iron content of mensuration HDL; and/or the level that contains ferritin that combines with HDL of mensuration, wherein the level of the rising of these detections (for example comparing with the level of finding in protectiveness HDL) shows that this HDL is short scorching HDL.

[0103] also provide evaluation/quantitating HDL to consume the mensuration of nitrogen monoxide ability.

II.HDL suppresses the detection of the ability of LDL gathering

[0104] in a plurality of embodiments, the invention still further relates to wonderful discovery: short scorching HDL does not prevent that LDL from assembling, and anti-inflammatory HDL prevents that LDL from assembling.Therefore, LDL assembles to measure provides the facilitated method of measuring and detect short scorching HDL.The known multiple LDL of those skilled in the art assembles mensuration, and the present invention is not intended to be limited to any concrete gathering mensuration or form.An exemplary gathering mensuration scheme is provided in embodiment 4.

[0105] as shown in Figure 16, this mensuration makes and can be easy to detect the short scorching HDL that CHD patient or CHD are equal to the patient of disease that value is with different fully with the value of the HDL acquisition of taking from healthy volunteer normally.As shown in Figure 17, this mensuration makes and is worth by the short scorching HDL that can be easy to detect the invalid mouse of apoE (atherosclerotic mouse model) different fully with the value that obtains with normal mouse HDL.And this mensuration provides a kind of simple method to confirm the validity for the treatment of the invalid mouse of apoE with apoA-I simulating peptide D-4F.

III. the purposes of Ce Dinging

[0106] therefore the present invention provides numerous mensuration that are used for the HDL of detection and/or quantitative protectiveness and/or abnormal function (short scorching); these mensuration provide and have been used to detect atherosclerotic or are the diagnosis of other illness of feature and/or the method for predict prognosis with the inflammatory reaction, and described other illness is atherosclerotic for example; apoplexy; leprosy; pulmonary tuberculosis; systemic loupus erythematosus; polymyalgia rheumatica; polyarteritis nodosa; chorionitis; idiopathic pulmonary fibrosis; chronic obstructive pulmonary disease; Alzheimer's; AIDS; polymyalgia rheumatica; polyarteritis nodosa; chorionitis; idiopathic pulmonary fibrosis; coronary artery calcification; the calcification aortic valve is narrow; osteoporosis; bacterial infection; virus infections; fungal infection; autoimmunity disease and rheumatoid arthritis; Crohn's disease etc.These measure also to detection face atherosclerotic and other inflammation disease (for example the above) people of risk and the validity of multiple treatment of monitoring and therapeutic scheme extremely useful.

[0107] in a plurality of embodiment, these mensuration comprise and detect protoheme correlativity HDL in conjunction with albumen (haemoglobin for example, and/or hoptoglobin, and/or Hemopexin, and/or myeloperoxidase etc.), detect the protein-bonded relative distribution of HDL between the non-lipoprotein fraction of HDL and plasma, detect short scorching HDL and consume nitric oxide production ability and detect HDL and suppress the ability that LDL assembles.

[0108] in this, it is to be noted,, also never it is considered as the prediction thing of atherosclerotic or abnormal function HDL in the past although known Hemopexin is weak acute phase reactant.In fact, just our understanding did not determine also in the past that it combined with HDL.This albumen is considered to main always and hoptoglobin one is used from by removing excessive protoheme in the circulation.Therefore, wonderful discovery is, Hemopexin combines with HDL, and it is in the concentration of blood plasma, the content of the Hemopexin that combines with HDL especially, but high predicted atherosclerotic and abnormal function HDL.

[0109] as implied above, in certain embodiments, the present invention relates to be used to detect atherosclerotic or be the diagnosis and/or the predict prognosis method of other illness of feature with the inflammatory reaction.Diagnostic method as herein described also can be used for treating multiple object.When destination object (for example patient) diagnose when having abnormal function HDL, they are at one or more medicines of recovery or rising protectiveness HDL level and/or multiple statins (Atorvastatin (Lipitor for example

, Pfizer), Simvastatin (Zocor

, Merck), Pravastatin (Pravachol

, Bristol-Myers Squibb), Fluvastatin (Lescol

, Novartis), Lovastatin (Mevacor

, Merck), relax and to cut down his spit of fland (Crestor

, Astra Zeneca) and Pitavastatin (Sankyo) etc.) good candidate.Recover or this class medicine (active agents) of rising protectiveness HDL level includes but not limited to that D4F is (referring to for example Li etc.; (2004) Circulation; 110:1701-1705), at PCT/US2001/26497 and/or PCT/US2001/26497 and/or PCT/US2004/026288 and/or PCT/US2005/028294 and/or PCT/US2003/09988 and/or USSN10/273; the active agents of listing in 386 etc., all these documents all are attached to herein by reference.

What [0110] will recognize is, in certain embodiments, mensuration of the present invention is carried out under different diagnosis backgrounds usually, and it is one or more particular disorder of feature that described diagnosis allows the technician to identify in the destination object with the inflammatory reaction.

The protein-bonded detected/quantified of IV.HDL

[0111] in a plurality of embodiment, mensuration of the present invention comprises and detecting and/or quantitative one or more albumen (for example HDL includes but not limited to haemoglobin, hoptoglobin, Hemopexin, myeloperoxidase, transferrins, solubility CD163 etc. in conjunction with albumen).These albumen are albumen well-known and that fully characterize.For example, hoptoglobin (referring to for example GenBank NP_005134) is for having general relatively pantomorphic positive acute phase protein (Vlierberghe etc., (2004) Clinica Chimica Acta., 345:35-42.), it is usually in conjunction with the free hemoglobin in the blood and promote its adjusting.CD163 is a kind of monocyte-macrophage specificity scavenger acceptor, absorption and removing (Aristoeli etc., (2006) Atherosclerosis 184 of mediation hoptoglobin-haemoglobin compound; 342-347).Hemopexin is plasma glycoprotein (Delanghe (2001) the Clinica Chimica Acta of a kind of 60KDa, 312:13-23) (referring to for example GenBank NP_000604, PFAM Pfam protein families database and HMM accession number PF00045).When formation dissociates protoheme in blood plasma in the decomposable process of the haemoglobin that is containing enzyme such as catalyzing enzyme, myoglobins or protoheme, its with the 1:1 ratio in conjunction with Hemopexin (Delanghe and Langlois (2001) Clinica ChimicaActa, 312:13-23; Shipulina etc., (2000) J.Protein Chem., 19:239-248; Solar etc., (1989) FEBS Lett., 256:225-229; Kuzelova etc., (1997) Biochim.Biophys.Acta, 1336:497-501).Hemopexin is the important relation thing between protoheme and the iron metabolism, with other iron transfer body hoptoglobin and transferrins by liver be used to keep the iron stable state (Delanghe and Langlois (2001) Clinica Chimica Acta, 312:13-23).

[0112] method of detected/quantified haemoglobin, hoptoglobin, Hemopexin, myeloperoxidase etc. is that those skilled in the art are well-known.For example, in (hoptoglobin of rising) or chronic liver diseases such as rheumatic fever, biliary obstruction, gastric ulcer, ulcerative colitis, erythroblastosis fetalis, hemotoncus, hemolytic anemia, owing to implement the medical science hoptoglobin in the differential diagnosis of the hemolytic anemia that the G6PD defective causes, idiopathic autoimmune hemolytic anemia, immune hemolytic anemia, drug-induced immune hemolytic anemia, primary hepatopathy and transfusion reaction (low hoptoglobin level) and measure.

[0113] any method that is used for the detected/quantified specific proteins basically all can be used for method of the present invention.Such method includes but not limited to Capillary Electrophoresis, Western blotting, mass spectrum, chromatogram (for example HPLC), Western blotting etc.

A) collection of sample and processing

[0114] biological sample of quantitative haemoglobin, hoptoglobin, Hemopexin, myeloperoxidase etc. is preferably from mammal (for example whole blood, blood plasma etc.), more preferably from human patients.Biological sample used herein is the sample of biological tissue or liquid, it contains one or more mensuration albumen as described herein (for example hoptoglobin and/or Hemopexin), and the concentration of these albumen can be associated with exist situation and/or the level of abnormal function HDL.Some preferred biological sample includes but not limited to whole blood or multiple blood fraction (for example blood plasma, serum etc.).In certain embodiments, biological sample contains HDL.In certain embodiments, biological sample comprises serum or blood plasma, and the extra sample that comprises serum or blood plasma perhaps is provided.

[0115] biological sample can perhaps can concentrate as required with suitable buffer solution dilution pre-service if needed.Can use in numerous standard aqueous buffer solution any, it uses a kind of in numerous buffers, and for example phosphate, Tris etc. are physiological pH.

[0116] as implied above, in certain embodiments, use whole blood, serum or blood plasma to measure.Acquisition and storing blood and/or blood product are that those skilled in the art are well-known.Blood obtains by venipuncture usually.Blood can add the well-known damping fluid of those skilled in the art or other reagent dilutes, and can reach 24 hours in 2-8 ℃ of storage, perhaps in storing the longer time below-20 ℃, detects then.In an especially preferred embodiment, do not having under the situation of antiseptic blood or blood product (for example serum) in-70 ℃ of standing storages.

[0117] in a plurality of embodiments, as mentioned above, sample contains HDL and/or HDL separates from sample.The method of separating HDL is that those skilled in the art are well-known, sets forth in the embodiment of this paper.

B) immunity is in conjunction with measuring.

[0118] in a preferred embodiment, use immunity that numerous processes fully verify in conjunction with any detection in measuring and/or quantitatively the albumen in the biological sample (referring to for example United States Patent (USP) 4,366,241,4,376,110,4,517,288,4,837,168,6,974,704,6,964,872,6,887,362,6,878,558,6,855,562,6,849,457,6,835,543,6,830,731,6,818,456,6,818,455,6,770,489,6,737,277,6,723,524,6,689,317,6,682,648,6,673,562,6,632,603 etc.).About the summary of general immunoassays, in addition referring to Methods in Cell Biology Volume 37:Antibodies in CellBiology, Asai edits, Academic Press, Inc.New York (1993); Basic andClinical Immunology the 7th edition, Stites and Terr edit, (1991).

[0119] immunity uses " catches " to come specificity in conjunction with frequent fixedly analyte also (be haemoglobin, hoptoglobin, Hemopexin, myeloperoxidase etc. in this case, and/or its fragment) in conjunction with measuring (or immunoassays) usually.Catches is the part of specificity bound analyte.In a preferred embodiment, catches be in this case specificity in conjunction with the antibody of haemoglobin, hoptoglobin, Hemopexin, myeloperoxidase etc. and/or its fragment or isotype.

[0120] in this, it is to be noted, detect with quantitatively the monoclonal antibody and the polyclonal antibody of haemoglobin, hoptoglobin, Hemopexin and myeloperoxidase etc. are commercially available (closing antibody ab8968 (sheep polyclone), ab4248 (chicken polyclone) and ab13429 (mouse monoclonal), anti-Hemopexin antibody ab27710 (mouse monoclonal) and the ab27711 (from Abcam, the mouse monoclonal of Inc.) etc. of globin referring to for example resistive connection).

[0121] immunoassays also often utilize combination of label specificity and mark by catches and analyte form in conjunction with compound.Perhaps, label can be the 3rd part, for example another kind of antibody, its specificity binding antibody/hoptoglobin and/or antibody/Hemopexin.

[0122] in certain embodiments, label comprises the resistive connection that carries mark and closes globin and/or anti-Hemopexin and/or anti-haemoglobin and/or anti-myeloperoxidase antibody.Perhaps, antibody can not have mark, but its three resistive connections that can be labeled again close, and this three anti-antibody to species that this antibody is originated is specific.Therefore, for example, close globin antibody with detectable part such as biotin modification resistive connection, this detectable part can be by the streptavidin specificity combination of the third labeled molecule such as enzyme labeling.

[0123] other albumen that can specificity binding domain-immunoglobulin constant region, for example A albumen or G albumen also can be used as label.These albumen are normal components of streptococcus cell membrane.The constant region for immunoglobulin of they and a plurality of species has strong non-immunogenic reactivity (referring to for example Kronval etc., (1973) J.Immunol., 111:1401-1406; Akerstrom etc., (1985) J.Immunol., 135:2589-2542 etc.).

[0124] in whole mensuration, incubation and/or washing step can carry out after each reagent mix.Incubation step can be by about 5 seconds to a few hours variation, preferred about 5 minutes to about 24 hours.Yet the incubation time is depended on mensuration form, analyte, liquor capacity, concentration etc.Usually, measure in environment temperature and carry out, but they can carry out for example 4 ℃-40 ℃ in certain temperature range.

1) noncompetitive is measured form

[0125] in a plurality of embodiments, the immunoassays of detection or quantitative haemoglobin, hoptoglobin, Hemopexin, myeloperoxidase etc. can be emulative or noncompetitive.Noncompetitive immunoassays are by wherein directly detecting the mensuration of the amount of being caught analyte (be haemoglobin, and/or hoptoglobin, and/or Hemopexin, and/or myeloperoxidase etc.) in this case.In one preferred " sandwich " measured, for example, catches (for example resistive connection closes globin antibody, and/or anti-hemoglobin antibodies, and/or anti-Hemopexin antibody, and/or anti-myeloperoxidase antibody) can be directly in conjunction with the solid matrix of fixing them.Then, these immobilized antibodies are caught haemoglobin, hoptoglobin, Hemopexin and/or the myeloperoxidase that exists in the specimen.Then, the immobilized thus albumen of catching is labeled the thing combination, and described label is for example anti-for the hoptoglobin and/or the Hemopexin two that carry mark.

[0126] or, two anti-can not have mark, but its three resistive connections that can be labeled again close, this three anti-antibody to these two anti-species of originating is specific.But two anti-can use test section such as biotin modification, but this test section can be by the streptavidin specificity combination of the molecule of the third mark such as enzyme labeling.

2) competitive assay format

[0127] in competitive assay, the analyte that (external) analyte (for example hoptoglobin, Hemopexin, haemoglobin, myeloperoxidase etc.) that the amount of the analyte that exists in sample (haemoglobin, hoptoglobin, Hemopexin, myeloperoxidase etc.) adds by detection is existed in the sample is indirect detection by the amount that replaces (or competition is gone down) in the catches (for example anti-haemoglobin, hoptoglobin, Hemopexin, myeloperoxidase antibody).In a competitive assay, haemoglobin in this case of known quantity, hoptoglobin, Hemopexin, myeloperoxidase etc. are added sample, sample is contacted with catches, and catches is the antibody of specificity in conjunction with haemoglobin, hoptoglobin, Hemopexin, myeloperoxidase etc. in this case.The concentration of the analyte that exists in the amount of the haemoglobin of binding antibody, hoptoglobin, Hemopexin, myeloperoxidase etc. and the sample is inversely proportional to.

[0128] in certain embodiments, antibody is fixed on the solid matrix.The amount of the haemoglobin of binding antibody, hoptoglobin, Hemopexin, myeloperoxidase etc. can followingly be measured: detect the amount of the haemoglobin exist, hoptoglobin, Hemopexin, myeloperoxidase etc. in antibody/analyte complex, perhaps detect the amount of remaining not compound haemoglobin, hoptoglobin, Hemopexin, myeloperoxidase etc.The amount of haemoglobin, hoptoglobin, Hemopexin, myeloperoxidase etc. can wait by haemoglobin, hoptoglobin, Hemopexin, the myeloperoxidase that mark is provided and detect.

[0129] haptens inhibition mensuration is another suitable competitive assay.In this is measured, known analyte (being haemoglobin, hoptoglobin, Hemopexin, myeloperoxidase etc. in this case) is fixed on the solid matrix.The antibody of the anti-haemoglobin of known quantity, hoptoglobin, Hemopexin, myeloperoxidase etc. is added sample, make sample contact immobilized haemoglobin, hoptoglobin, Hemopexin, myeloperoxidase etc. then.In the case, the amount of the analyte that exists in amount of the immobilized haemoglobin of antibodies of anti-haemoglobin, hoptoglobin, Hemopexin, myeloperoxidase etc., hoptoglobin, Hemopexin, myeloperoxidase etc. and the sample is inversely proportional to.Moreover the amount of immobilized antibody can remaining antibody moiety detects in antibody immobilization part or the solution by detecting.

[0130] under the situation that antibody is labeled, detects and can be directly, perhaps carry out indirectly in conjunction with the mark part of aforesaid antibody by adding specificity subsequently.

3) detect albumen by RIA

[0131] in certain embodiments, use the haemoglobin of the quantitative sample of radioimmunoassay (RIA) and/or the content of hoptoglobin and/or Hemopexin and/or myeloperoxidase.The detailed protocol of radioimmunoassay is found in for example Sambrook etc., (1989) Molecular Cloning-A Laboratory Manual (the 2nd edition) 1-3 volume, Cold SpringHarbor Laboratory, Cold Spring Harbor Press, NY, etc.

4) other mensuration form

[0132] in another embodiment, use Western blotting (Western blotting) analyzing and testing and quantitatively the analyzing proteins in the sample have a situation.This technology generally comprises by the gel electrophoresis sample separation albumen based on molecular weight, the albumen that separates is transferred to suitable solid support (for example nitrocellulose filter, nylon leaching film or derivatization nylon leaching film), and sample is combined the antibody incubation of target analyte (for example haemoglobin, hoptoglobin, Hemopexin, myeloperoxidase etc.) with specificity.Specific antibody specificity combination is present in the analyte on the solid support to target analytes.These antibody can perhaps can be used specificity to detect in conjunction with the labelled antibody (for example sheep anti mouse antibodies of mark) that resistive connection closes globin and/or Hemopexin by direct mark subsequently.

[0133] other mensuration form includes but not limited to liposome immunoassays (LIA), and its use is designed for the liposome in conjunction with specific molecular (for example antibody), and discharges capsule reagent or mark.The chemical substance that detect to discharge according to standard technique (referring to Monroe etc., (1986) Amer.Clin.Prod.Rev.5:34-41) then.

[0134] aforementioned mensuration is intended to set forth and unrestricted.Use instruction provided herein, other mensuration form will be conspicuous to those skilled in the art.It is to be noted that this paper also provides concrete scheme in an embodiment.

C. evaluating and measuring

[0135] writes down mensuration of the present invention according to the well-known standard method of those skilled in the art.Under the detectable situation of level difference of measuring albumen, mensuration of the present invention is registered as the positive usually.In certain embodiments, change on the statistics significantly and change, (for example t-check, variance analysis (ANOVA), half parameter technology, nonparametric technique (for example Wilcoxon Mann-Whitney check, Wilcoxon signed rank test, Sign check, Kruskal-Wallis check etc.) are next definite for example to use any statistical test that the data set that provided is provided.On the statistics significant change preferably to the confidence level that is less than 85%, more preferably to be less than 90% confidence level, more more preferably to be less than 95% confidence level, most preferably to being less than 98% or 99% confidence level for significant.In certain embodiments, change at least 10% change, be preferably at least 20% change, at least 50% change more preferably most preferably is at least 90% change.

V. determination of nitric oxide

[0136] another discovery of the present invention is, short scorching HDL consumes nitrogen monoxide (generation nitrate), and protectiveness HDL does not consume nitrogen monoxide basically.Therefore, the detection of nitrogen monoxide consumption/loss provide fast and easily protectiveness HDL measure.

[0137] method of detection nitrogen monoxide consumption is that those skilled in the art are well-known.These methods generally include provides nitrogen monoxide or nitric oxide production chemical donor, and uses absorption spectroscopy or electrochemical method to detect nitric oxide production consumption.

VI.LDL assembles mensuration

[0138] in certain embodiments, prevent the ability that LDL assembles based on protectiveness HDL and short scorching HDL does not prevent that LDL from assembling the mensuration that is provided for non-protective HDL.Mensuration generally comprises LDL (for example LDL of Fen Liing) is contacted with described HDL, and detects aggregate amount or the speed of LDL.The method that detects the LDL gathering is that those skilled in the art are well-known.

[0139] in a simple embodiment; simply the LDL vortex is gone in the solution; through spectrophotometric method detection of aggregation speed; for example in 680nm read change (for example per 10 seconds) in time with respect to suitable contrast (for example blank solution and/or the identical experiment implemented with protectiveness HDL) absorbance (for exemplary gathering mensuration; referring to for example Khoo etc., (1988) Arteriosclerosis 8:348-358).In certain embodiments, for example use and not contain albuminous pillar detection of aggregation described in the embodiment 4.

VII. measure and optimize

[0140] mensuration of the present invention can be optimized at the purposes in specific background, for example depends on the source of biological sample and/or character and/or concrete tester and/or available analytical equipment.Therefore, for example, optimization can comprise to be determined best antibody condition in conjunction with condition determination, best sample preparation condition (for example preferred separation condition), maximization signal to noise ratio (S/N ratio), improves the method for flux etc.In addition, can select and/or optimization mensuration form according to the availability of equipment and/or reagent.

[0141] the routine selection and the optimization of survey form are that persons skilled in the art are well-known.

VIII. kit

[0142] in certain embodiments, the present invention relates to implement the kit of one or more mensuration described herein.Usually, such kit comprises that one or more are used to detect the reagent of haemoglobin and/or myeloperoxidase and/or hoptoglobin and/or Hemopexin.These reagent can include but not limited to the specific antibody of multiple protein.In certain embodiments, kit comprises one or more and is used to implement the reagent that LDL assembles mensuration or nitrogen monoxide consumption mensuration.

[0143] kit can randomly comprise the additional materials that is used to collect blood and/or separates HDL and/or LDL etc.

[0144] in addition, kit can randomly comprise the expository material that contains the guide (being scheme) of implementing the inventive method.Preferred expository material provides and has used the kit content to detect the scheme of hoptoglobin and/or Hemopexin level.Write or printing material although expository material generally includes, they are not limited thereto.The present invention includes and to store these explanations and they are conveyed to any medium of terminal user.These mediums include but not limited to electron storage medium (for example disk, tape, magnetic tape cassette, chip), optical medium (for example CD ROM) etc.These mediums can comprise the address that arrives the internet sites that these illustrative material are provided.

Embodiment

[0145] providing following examples is in order to clearly demonstrate the present invention, rather than for the invention of requirement for restriction protection.

Embodiment 1

The detection of haemoglobin, hoptoglobin and Hemopexin

[0146] shown the ELISA method that is used for hoptoglobin and Hemopexin below, and be used to prepare the HDL supernatant and in conjunction with the method for the immunoadsorption albumen of apoA-I.

Human hemoglobin ELISA

[0147] table 1. is used for the material of Hb E LISA

Remarks: make all reagent reach room temperature before use.

Adopting magnetic bead reagent to carry out HDL separates

[0148] 1. use green push pipe (green top tube) or serum separator tube and in 5 ℃ with 2300rpm centrifugal 20 minutes by blood sample in separated plasma or serum.

[0149] 2. takes out supernatant (serum or blood plasma).Attention: if sample is frozen, then melt sample, and in hydro-extractor in room temperature with 12, centrifugal 5 minutes of 000rpm.This should make any particles settling that may influence mensuration that exists in serum or the blood plasma.

[0150] 3. adds supernatant (maximum 250 μ L/ holes) to transparent round bottom 96 orifice plates.

[0151] 4. adds the magnetic bead reagent supernatant (maximum 50 μ L/ holes) of 1/5 cumulative volume and mix to each sample.It was left standstill 5 minutes.

[0152] 5. plate was placed 5 minutes at magnetic-particle concentrator top.

[0153] 6. takes out supernatant, and add micro-centrifuge tube.Attention: supernatant contains HDL.The particle that contains ApoB is removed.

[0154] 7. in 5 ℃ with 12, centrifugal 5 minutes of 000rpm is to remove any pearl.

[0155] 8. takes out supernatant.

The preparation of damping fluid

[0156] 1. the preparation bag is cushioned liquid: 25mL ddH ₂O+8mL buffer A+17mL buffer B (buffer A: 0.2M Na ₂CO ₃Buffer B: 0.2M NaHCO ₃)

[0157] 2. preparing washing damping fluid: 0.75mL polysorbas20+150mL 10X PBS+1350mL ddH ₂O.

[0158] 3. prepares sealing/dilution buffer liquid: 20mL 10X PBS+10mL polysorbas20+0.5g BSA+170mL ddH ₂O.

[0159] 4. prepares stop bath: 1.38mL H ₂SO ₄+ 48.62mL H ₂O.

The preparation of sample and typical curve

[0160] 1. sample (serum or HDL supernatant) is diluted to bag with 1:200 and is cushioned (2.5 μ LHDL are diluted to 497.5 μ L bag and are cushioned in the liquid) in the liquid.

[0161] bag that 2. is prepared as the haemoglobin of 1000ng/mL concentration is cushioned solution (the 2mL bag of 2 μ L haemoglobins is cushioned solution).

[0162] 3. by be cushioned the continuous 1:2 of liquid dilution 1000ng/mL solution with bag, obtain 500,250,125,62.5,31.25, the solution of 15.63ng/mL, prepare in triplicate standard point.

[0163] 4. include in only contain the bag be cushioned the hole of liquid as blank.

Add sample and standard items to plate

[0164] 1. to add every kind of standard items and every kind of sample of 110 μ L in triplicate to round bottom 96 hole polypropylene boards.

[0165] 2. uses the hyperchannel pipettor to take out 100 μ L and add the Immulon plate by each hole.

[0166] 3. in 4 ℃ of incubation that spend the night.

[0167] 4. antigen is rapped in the biohazard waste canister.

[0168] 5. with 300 μ L lavation buffer solutions washing 3 times.Counter-rotary-ing plate also inclines and content, with its touching 4-5 time on the suction paper handkerchief, to remove liquid fully in per step.

Sealing and antibody originally

[0169] 1. to the porose adding 200 μ L of an institute sealing damping fluid, and in room temperature incubation 1 hour.

[0170] 2. with 300 μ L lavation buffer solutions washing 3 times.Counter-rotary-ing plate also inclines and content, with its touching 4-5 time on the suction paper handkerchief, to remove liquid fully in per step.

[0171] 3. dilutes the goat antihuman hemoglobin (1 μ L antibody is at 10mL dilution buffer liquid) of puting together HRP with 1:10000.

[0172] 4. adds the 1:20000 dilution of 50 μ L to each hole.

[0173] 5. with 300 μ L lavation buffer solutions washing 3 times.Counter-rotary-ing plate also inclines and content, with its touching 4-5 time on the suction paper handkerchief, to remove liquid fully in per step.

Add TMB and stop bath

[0174] 1. mixes 1 part of TMBA and 1 part of TMBB.

[0175] 2. added 100 μ LTMB potpourris and incubation 20 minutes to each hole.

[0176] 3. adds 100 μ L stop baths to each hole.

With reading plate device reading

[0177] 1. with reading the plate device with the 450nm wavelength readings.

Hoptoglobin ELISA

[0178] table 2. is used for the material of hoptoglobin ELISA

Remarks: make all reagent reach room temperature before use.

Carrying out HDL with magnetic bead reagent separates

[0179] 1. use green push pipe (green top tube) or serum separator tube and in 5 ℃ with 2300rpm centrifugal 20 minutes by blood sample in separated plasma or serum.

[0180] 2. takes out supernatant (serum or blood plasma).Attention: if sample is frozen, then melt sample, and in hydro-extractor in room temperature with 12, centrifugal 5 minutes of 000rpm.This should make any particles settling that may influence mensuration that exists in serum or the blood plasma.

[0181] 3. adds supernatant (maximum 250 μ L/ holes) to transparent round bottom 96 orifice plates.

[0182] 4. adds the magnetic bead reagent supernatant (maximum 50 μ L/ holes) of 1/5 cumulative volume and mix to each sample.It was left standstill 5 minutes.

[0183] 5. plate was placed 5 minutes at magnetic-particle concentrator top.

[0184] 6. takes out supernatant, and add micro-centrifuge tube.Attention: supernatant contains HDL.The particle that contains ApoB is removed.

[0185] 7. in 5 ℃ with 12, centrifugal 5 minutes of 000rpm is to remove any pearl.

[0186] 8. takes out supernatant.

The preparation of damping fluid

[0187] 1. dilutes lavation buffer solution concentrate (in kit, providing) with 1:10 with SILVER REAGENT water.

[0188] 2. dilutes concentrate (in kit, providing) with 1:10 dilution EIA with SILVER REAGENT water.

Preparation plasma or HDL supernatant

[0189] 1. plasma or HDL supernatant are diluted in the EIA dilution (providing in kit) with 1:4000.

[0190] 2. with 1:100 dilution beginning (5 μ LHDL dilute in the 495 μ L dilutions).

[0191] 3. the preparation 1:100 40 times of dilutions (1:100 of 12.5 μ L dilutes in the 487.5 μ L dilutions).

The preparation standard curve

[0192] 1. with 2mL EIA dilution reprovision 100 μ g hoptoglobin standard items (in kit, providing), to produce the concentration of 50 μ g/mL.

[0193] standard items were left standstill 10 minutes, gentle agitation before the preparation dilution.

[0194] 3. by using the EIA dilution, obtains 12.5,3.13,0.78,0.195 and 0.049 μ g/mL solution, prepare in triplicate standard point with 1:4 serial dilution standard solution (50 μ g/mL).

Sample is added entering plate

[0195] 1. with 4mL EIA diluted biotinylation hoptoglobin (in kit, providing).

[0196] 2. to add every kind of standard items and every kind of sample of 35 μ L in triplicate to round bottom 96 hole polypropylene boards.

[0197] 3. uses the hyperchannel transfer pipet to take out 25 μ L, people's hoptoglobin microplate (providing in kit) is provided by each hole.

[0198] 4. use the hyperchannel transfer pipet that 25 μ L biotinylation hoptoglobins are added each hole.

[0199] 5. with band (in kit, providing) sealing of hole, and under gentle agitation in room temperature incubation 1 hour.

[0200] 6. with 200 μ L lavation buffer solutions washing 5 times.Counter-rotary-ing plate also inclines and content, with its touching 4-5 time on the suction paper handkerchief, to remove liquid fully in per step.

Add streptavidin-peroxidase conjugated thing

[0201] 1. of short duration centrifugal streptavidin-peroxidase conjugated thing.

[0202] 2. dilutes conjugate with 1:100 with the EIA dilution.

[0203] 3. add 50 μ L SP conjugates to each hole, and incubation 30 minutes.

[0204] 4. with 200 μ L lavation buffer solutions washing 5 times.Counter-rotary-ing plate also inclines and content, with its touching 4-5 time on the suction paper handkerchief, to remove liquid fully in per step.

Add substrate and stop bath

[0205] 50 μ L chromogen substrates (providing in kit) are provided in 1. every hole, and incubation 8 minutes.

[0206] 2. adds 50 μ L stop baths (in kit, providing) to each hole.

With reading plate device reading

[0207] 1. with reading the wavelength readings of plate device in 450nm.

Hemopexin ELISA

[0208] table 3. is used for the material that Hemopexin is measured.

Remarks: make all reagent reach room temperature before use

Carrying out HDL with magnetic bead reagent separates

[0209] 1. use green push pipe (green top tube) or serum separator tube and in 5 ℃ with 2300rpm centrifugal 20 minutes by blood sample in separated plasma or serum.

[0210] 2. takes out supernatant (serum or blood plasma).Attention: if sample is frozen, then melt sample, and in hydro-extractor in room temperature with 12, centrifugal 5 minutes of 000rpm.This should make any particles settling that may influence mensuration that exists in serum or the blood plasma.

[0211] 3. adds supernatant (maximum 250 μ L/ holes) to transparent round bottom 96 orifice plates.

[0212] 4. adds the magnetic bead reagent supernatant (maximum 50 μ L/ holes) of 1/5 cumulative volume and mix to each sample.It was left standstill 5 minutes.

[0213] 5. plate was placed 5 minutes at magnetic-particle concentrator top.

[0214] 6. takes out supernatant, and add micro-centrifuge tube.Attention: supernatant contains the HDL of separation.The particle that contains ApoB is removed.

[0215] 7. in 5 ℃ with 12, centrifugal 5 minutes of 000rpm is to remove any pearl.

[0216] 8. takes out supernatant.

The preparation of damping fluid

[0217] 1. the preparation bag is cushioned liquid: 25mL ddH ₂O+8mL buffer A+17mL buffer B (buffer A: 0.2M Na ₂CO ₃Buffer B: 0.2M NaHCO ₃).

[0218] 2. preparing washing damping fluid: 0.75mL polysorbas20+150mL 10X PBS+1350mL ddH ₂O.

[0219] 3. prepares sealing/dilution buffer liquid: 20mL 10X PBS+10mL polysorbas20+0.5g BSA+170mL ddH ₂O.

[0220] 4. prepares stop bath: 1.38mL H ₂SO ₄+ 48.62mL H ₂O.

The preparation of sample and typical curve

[0221] 1. sample (blood plasma or HDL supernatant) is diluted to bag with 1:200 and is cushioned (2.5 μ LHDL are diluted to 497.5 μ L bag and are cushioned in the liquid) in the liquid.

[0222] bag that 2. is prepared as the Hemopexin of 1000ng/mL concentration is cushioned solution (the 2mL bag of 2 μ L Hemopexins is cushioned solution).

[0223] 3. by be cushioned the continuous 1:2 of liquid dilution 1000ng/mL solution with bag, obtain 500,250,125,62.5,31.25, the solution of 15.63ng/mL, prepare in triplicate standard point.

[0224] 4. include in only contain the bag be cushioned the hole of liquid as blank.

Add sample and standard items to plate

[0225] 1. to add every kind of standard items and every kind of sample of 110 μ L in triplicate to round bottom 96 hole polypropylene boards.

[0226] 2. uses the hyperchannel pipettor to take out 100 μ L and add the Immulon plate by each hole.

[0227] 3. in 4 ℃ of incubation that spend the night.

[0228] 4. antigen is rapped in the biohazard waste canister.

[0229] 5. with 300 μ L lavation buffer solutions washing 3 times.Counter-rotary-ing plate also inclines and content, with its touching 4-5 time on the suction paper handkerchief, to remove liquid fully in per step.

Sealing and antibody originally

[0230] 1. to the porose adding 200 μ L of an institute sealing damping fluid, and in room temperature incubation 1 hour.

[0231] 2. with 300 μ L lavation buffer solutions washing 3 times.Counter-rotary-ing plate also inclines and content, with its touching 4-5 time on the suction paper handkerchief, to remove liquid fully in per step.

[0232] 3. dilutes the anti-people's Hemopexin of chicken (1 μ L antibody is at 20mL dilution buffer liquid) of puting together HRP with 1:20000.

[0233] 4. adds the 1:20000 dilution of 50 μ L to each hole.

[0234] 5. with 300 μ L lavation buffer solutions washing 3 times.Counter-rotary-ing plate also inclines and content, with its touching 4-5 time on the suction paper handkerchief, to remove liquid fully in per step.

Add TMB and stop bath

[0235] 1. mixes 1 part of TMBA and 1 part of TMBB.

[0236] 2. add 100 μ L TMB potpourris to each hole, and incubation 20 minutes.

[0237] 3. adds 100 μ L stop baths to each hole.

With reading plate device reading

[0238] 1. with reading the wavelength readings of plate device in 450nm.

Immunoadsorption albumen haemoglobin ELISA in conjunction with ApoA-I

[0239] table 4. is used for the material in conjunction with the immunoadsorption albumen haemoglobin ELISA of ApoA-I.

Remarks: make all reagent reach room temperature before use.

Carrying out HDL with magnetic bead reagent separates

[0240] 1. use green push pipe (green top tube) or serum separator tube and in 5 ℃ with 2300rpm centrifugal 20 minutes by blood sample in separated plasma or serum.

[0241] 2. takes out supernatant (serum or blood plasma).Attention: if sample is frozen, then melt sample, and in hydro-extractor in room temperature with 12, centrifugal 5 minutes of 000rpm.This should make any particles settling that may influence mensuration that exists in serum or the blood plasma.

[0242] 3. adds supernatant (maximum 250 μ L/ holes) to transparent round bottom 96 orifice plates.

[0243] 4. adds the magnetic bead reagent supernatant (maximum 50 μ L/ holes) of 1/5 cumulative volume and mix to each sample.It was left standstill 5 minutes.

[0244] 5. plate was placed 5 minutes at magnetic-particle concentrator top.

[0245] 6. takes out supernatant, and add micro-centrifuge tube.Attention: supernatant contains HDL.The particle that contains ApoB is removed.

[0246] 7. in 5 ℃ with 12, centrifugal 5 minutes of 000rpm is to remove any pearl.

[0247] 8. takes out supernatant.

The preparation of damping fluid

[0248] 1. the preparation bag is cushioned liquid: 25mL ddH ₂O+8mL buffer A+17mL buffer B (buffer A: 0.2M Na ₂CO ₃Buffer B: 0.2M NaHCO ₃).

[0249] 2. preparing washing damping fluid: 0.75mL polysorbas20+150mL 10X PBS+1350mL ddH ₂O.

[0250] 3. prepares sealing/dilution buffer liquid: 20mL 10X PBS+10mL polysorbas20+0.5g BSA+170mL ddH ₂O.

[0251] 4. prepares stop bath: 1.38mL H ₂SO ₄+ 48.62mL H ₂O.

The preparation of sample and typical curve

[0252] 1. sample (serum or HDL supernatant) is diluted to bag with 1:200 and is cushioned (2.5 μ L HDL are diluted to 497.5 μ L bag and are cushioned in the liquid) in the liquid.

[0253] bag that 2. is prepared as the haemoglobin of 1000ng/mL concentration is cushioned solution (the 2mL bag of 2 μ L haemoglobins is cushioned solution).

[0254] 3. by be cushioned the continuous 1:2 of liquid dilution 1000ng/mL solution with bag, obtain 500,250,125,62.5,31.25, the solution of 15.63ng/mL, prepare in triplicate standard point.

[0255] 4. include in only contain the bag be cushioned the hole of liquid as blank.

Add sample and standard items to plate

[0256] 1. to add every kind of standard items and every kind of sample of 110 μ L in triplicate to round bottom 96 hole polypropylene boards.

[0257] 2. use the hyperchannel pipettor to take out the microwell plate (in the A1 kit, providing) that 100 μ L add anti-people ApoA1 bag quilt by each hole.

[0258] 3. in 4 ℃ of incubation that spend the night.

[0259] 4. antigen is rapped in the biohazard waste canister.

[0260] 5. with 300 μ L lavation buffer solutions washing 3 times.Counter-rotary-ing plate also inclines and content, with its touching 4-5 time on the suction paper handkerchief, to remove liquid fully in per step.

Sealing and antibody originally

[0261] 1. to the porose adding 200 μ L of an institute sealing damping fluid, and in room temperature incubation 1 hour.

[0262] 2. with 300 μ L lavation buffer solutions washing 3 times.Counter-rotary-ing plate also inclines and content, with its touching 4-5 time on the suction paper handkerchief, to remove liquid fully in per step.

[0263] 3. dilutes the goat antihuman hemoglobin (1 μ L antibody is at 10mL dilution buffer liquid) of puting together HRP with 1:10000.

[0264] 4. adds the 1:20000 dilution of 50 μ L to each hole.

[0265] 5. with 300 μ L lavation buffer solutions washing 3 times.Counter-rotary-ing plate also inclines and content, with its touching 4-5 time on the suction paper handkerchief, to remove liquid fully in per step.

Add TMB and stop bath

[0266] 1. mixes 1 part of TMBA and 1 part of TMBB.

[0267] 2. add 100 μ LTMB potpourris to each hole, and incubation 20 minutes.

[0268] 3. adds 100 μ L stop baths to each hole.

With reading plate device reading

[0269] 1. with reading the wavelength readings of plate device in 450nm.

Immunoadsorption protein combination globin ELISA in conjunction with ApoA-I

[0270] table 5. is used for the material in conjunction with the immunoadsorption protein combination globin ELISA of ApoA-I.

Remarks: make all reagent reach room temperature before use.

Carrying out HDL with magnetic bead reagent separates

[0271] 1. use green push pipe (green top tube) or serum separator tube and in 5 ℃ with 2300rpm centrifugal 20 minutes by blood sample in separated plasma or serum.

[0272] 2. takes out supernatant (serum or blood plasma).Attention: if sample is frozen, then melt sample, and in hydro-extractor in room temperature with 12, centrifugal 5 minutes of 000rpm.This should make any particles settling that may influence mensuration that exists in serum or the blood plasma.

[0273] 3. adds supernatant (maximum 250 μ L/ holes) to transparent round bottom 96 orifice plates.

[0274] 4. adds the magnetic bead reagent supernatant (maximum 50 μ L/ holes) of 1/5 cumulative volume and mix to each sample.It was left standstill 5 minutes.

[0275] 5. plate was placed 5 minutes at magnetic-particle concentrator top.

[0276] 6. takes out supernatant, and add micro-centrifuge tube.Attention: supernatant contains the HDL of separation.The particle that contains ApoB is removed.

[0277] 7. in 5 ℃ with 12, centrifugal 5 minutes of 000rpm is to remove any pearl.

[0278] 8. takes out supernatant.

The preparation of damping fluid

[0279] 1. the preparation bag is cushioned liquid: 25mL ddH ₂O+8mL buffer A+17mL buffer B (buffer A: 0.2M Na ₂CO ₃Buffer B: 0.2M NaHCO ₃).

[0280] 2. preparing washing damping fluid: 0.75mL polysorbas20+150mL 10X PBS+1350mL ddH ₂O.

[0281] 3. prepares sealing/dilution buffer liquid: 20mL 10X PBS+10mL polysorbas20+0.5g BSA+170mL ddH ₂O.

[0282] 4. prepares stop bath: 1.38mL H ₂SO ₄+ 48.62mL H ₂O.

Preparation HDL supernatant

[0283] 1. with 1:4000 the HDL supernatant being diluted bag is cushioned in the liquid.

[0284] 2. with 1:100 dilution beginning (5 μ LHDL dilute in the 495 μ L damping fluids).

[0285] 3. the preparation 1:100 (1:100 of 12.5 μ L dilutes in the 487.5 μ L damping fluids) 40 times of dilutions.

The preparation of typical curve

[0286] 1. with 2mL damping fluid reprovision 100 μ g hoptoglobin standard items (in kit, providing), to produce the concentration of 50 μ g/mL.

[0287] standard items was left standstill 10 minutes, diluting preceding gentle agitation.

[0288] 3. by using damping fluid, obtains 12.5,3.13,0.78,0.195 and 0.049 μ g/mL solution, prepare in triplicate standard point with 1:4 serial dilution standard solution (50 μ g/mL).

Add sample and standard items to plate

[0289] 1. to add every kind of standard items and every kind of sample of 110 μ L in triplicate to round bottom 96 hole polypropylene boards.

[0290] 2. use the hyperchannel pipettor to take out the microwell plate (in the A1 kit, providing) that 100 μ L add anti-people ApoA1 bag quilt by each hole.

[0291] 3. in 4 ℃ of incubation that spend the night.

[0292] 4. antigen is rapped in the biohazard waste canister.

[0293] 5. with 300 μ L lavation buffer solutions washing 3 times.Counter-rotary-ing plate also inclines and content, with its touching 4-5 time on the suction paper handkerchief, to remove liquid fully in per step.

Sealing and antibody originally

[0294] 1. to the porose adding 200 μ L of an institute sealing damping fluid, and in room temperature incubation 1 hour.

[0295] 2. with 300 μ L lavation buffer solutions washing 3 times.Counter-rotary-ing plate also inclines and content, with its touching 4-5 time on the suction paper handkerchief, to remove liquid fully in per step.

[0296] 3. dilutes anti-people's hoptoglobin (1 μ L antibody is in 20mL dilution buffer liquid) of puting together HRP with 1:20000.

[0297] 4. adds the 1:20000 dilution of 50 μ L to each hole.

[0298] 5. with 300 μ L lavation buffer solutions washing 3 times.Counter-rotary-ing plate also inclines and content, with its touching 4-5 time on the suction paper handkerchief, to remove liquid fully in per step.

Add TMB and stop bath

[0299] 1. mixes 1 part of TMBA and 1 part of TMBB.

[0300] 2. add 100 μ LTMB potpourris to each hole, and incubation 20 minutes.

[0301] 3. adds 100 μ L stop baths to each hole.

With reading plate device reading

[0302] 1. with reading the wavelength readings of plate device in 450nm.

In conjunction with the immunoadsorption proteinemina red pigment of ApoA-I in conjunction with protein ELISA

[0303] table 6. is used in conjunction with the immunoadsorption proteinemina red pigment of the ApoA-I material in conjunction with protein ELISA.

Remarks: make all reagent reach room temperature before use

Carrying out HDL with magnetic bead reagent separates

[0304] 1. use green push pipe (green top tube) or serum separator tube and in 5 ℃ with 2300rpm centrifugal 20 minutes by blood sample in separated plasma or serum.

[0305] 2. takes out supernatant (serum or blood plasma).Attention: if sample is frozen, then melt sample, and in hydro-extractor in room temperature with 12, centrifugal 5 minutes of 000rpm.This should make any particles settling that may influence mensuration that exists in serum or the blood plasma.

[0306] 3. adds supernatant (maximum 250 μ L/ holes) to transparent round bottom 96 orifice plates.

[0307] 4. adds the magnetic bead reagent supernatant (maximum 50 μ L/ holes) of 1/5 cumulative volume and mix to each sample.It was left standstill 5 minutes.

[0308] 5. plate was placed 5 minutes at magnetic-particle concentrator top.

[0309] 6. takes out supernatant, and add micro-centrifuge tube.Attention: supernatant contains the HDL of separation.The particle that contains ApoB is removed.

[0310] 7. in 5 ℃ with 12, centrifugal 5 minutes of 000rpm is to remove any pearl.

[0311] 8. takes out supernatant.

The preparation of damping fluid

[0312] 1. the preparation bag is cushioned liquid: 25mL ddH ₂O+8mL buffer A+17mL buffer B (buffer A: 0.2M Na ₂CO ₃Buffer B: 0.2M NaHCO ₃).

[0313] 2. preparing washing damping fluid: 0.75mL polysorbas20+150mL 10X PBS+1350mL ddH ₂O.

[0314] 3. prepares sealing/dilution buffer liquid: 20mL 10X PBS+10mL polysorbas20+0.5g BSA+170mL ddH ₂O.

[0315] 4. prepares stop bath: 1.38mL H ₂SO ₄+ 48.62mL H ₂O.

The preparation of sample and typical curve

[0316] 1. sample (HDL supernatant) is diluted to bag with 1:200 and is cushioned (2.5 μ LHDL are diluted to 497.5 μ L bag and are cushioned in the liquid) in the liquid.

[0317] bag that 2. is prepared as the Hemopexin of 1000ng/mL concentration is cushioned solution (the 2mL bag of 2 μ L Hemopexins is cushioned solution).

[0318] 3. by be cushioned the continuous 1:2 of liquid dilution 1000ng/mL solution with bag, obtain 500,250,125,62.5,31.25, the solution of 15.63ng/mL, prepare in triplicate standard point.

[0319] 4. include in only contain the bag be cushioned the hole of liquid as blank.

Add sample and standard items to plate

[0320] 1. to add every kind of standard items and every kind of sample of 110 μ L in triplicate to round bottom 96 hole polypropylene boards.

[0321] 2. use the hyperchannel pipettor to take out the microwell plate (in the A1 kit, providing) that 100 μ L add anti-people ApoA1 bag quilt by each hole.

[0322] 3. in 4 ℃ of incubation that spend the night.

[0323] 4. antigen is rapped in the biohazard waste canister.

[0324] 5. with 300 μ L lavation buffer solutions washing 3 times.Counter-rotary-ing plate also inclines and content, with its touching 4-5 time on the suction paper handkerchief, to remove liquid fully in per step.

Sealing and antibody originally

[0325] 1. to the porose adding 200 μ L of an institute sealing damping fluid, and in room temperature incubation 1 hour.

[0326] 2. with 300 μ L lavation buffer solutions washing 3 times.Counter-rotary-ing plate also inclines and content, with its touching 4-5 time on the suction paper handkerchief, to remove liquid fully in per step.

[0327] 3. dilutes the anti-people's Hemopexin of chicken (1 μ L antibody is in 20mL dilution buffer liquid) of puting together HRP with 1:20000.

[0328] 4. adds the 1:20000 dilution of 50 μ L to each hole.

[0329] 5. with 300 μ L lavation buffer solutions washing 3 times.Counter-rotary-ing plate also inclines and content, with its touching 4-5 time on the suction paper handkerchief, to remove liquid fully in per step.

Add TMB and stop bath

[0330] 1. mixes 1 part of TMBA and 1 part of TMBB.

[0331] 2. add 100 μ L TMB potpourris to each hole, and incubation 20 minutes.

[0332] 3. adds 100 μ L stop baths to each hole.

With reading plate device reading

[0333] 1. with reading the wavelength readings of plate device in 450nm.

It is to be noted that [0334] method as herein described is intended to set forth and unrestricted.Use instruction provided herein, those skilled in the art can be easy to obtain other mensuration and mensuration form.

The result

[0335] merges the blood plasma that 10 healthy volunteers and 10 confirmations suffer from the patient of CHD.Separate HDL, and on non-sex change PAGE gel electrophoresis, use protein analysis that haemoglobin is carried out Western blotting.As shown in the left figure of Figure 27,, in 10 healthy volunteers' HDL, there is obviously more haemoglobin than 10 CHD patients.As shown in the right figure of Figure 27, the amount of the haemoglobin that before HDL separates, in blood plasma adding RBC lysate has increased by two groups, has combined with HDL, but under significant difference between healthy volunteer and the CHD patient even the condition at these extreme haemolysis, still obviously keep.

[0336] in other experiment, collects serum by another group of 12 healthy volunteers and 14 juvenile diabetes patients (8 adults and 6 children) and 8 CHD patients or according to the patient who is equal to disease of NCEPATP III standard.Aftermentioned patient accepts statins all.Will be on 96 orifice plates at the antibody sandwich of people apoA-I.Adding is from experimenter's serum, in 4 ℃ of incubations that spend the night.Thorough wash plate, and in 4 ℃ of goats one anti-incubations that spend the night with antihuman hemoglobin or people's hoptoglobin.Thorough each plate of washing, and with put together horseradish peroxidase (HRP), resist in room temperature incubation 2 hours at two of goat IgG.Thoroughly each plate of washing, and adding HRP substrate detects optical density (OD).Figure 28 A has shown non-RBC haemoglobin with μ gHRP/mL.Figure 28 B has shown hoptoglobin with μ gHRP/mL.Figure 28 C has shown the non-RBC haemoglobin product with the hoptoglobin value on duty.The result shows that the value that the aftermentioned method obtains is distinguished healthy volunteer and diabetic and CHD patient, without any overlapping.

[0337] these results show, the new mensuration of this paper report can be used for detecting the patient such as the disease of diabetes and CHD, and are provided for distinguishing they and normal person's means.

Embodiment 2

The proteinogram of short scorching HDL

[0338] we report, and the inflammatory properties of HDL all is atherosclerotic indicator more responsive than HDL-cholesterol levels in mouse and people.In the present embodiment, we have described use ProteinChip technology coupling surface laser enhanced desorb/ionization flight time mass spectrum (SELDI-TOF-MS) and have identified the specific proteins finger-print of distinguishing the normal mouse HDL and the mouse HDL of the atherogenicity meals of ingesting.Feed raise C57BL/6J mouse atherogenicity meals reach cause that the HDL-cholesterol levels reduces by 1 week, paraoxonase actively descends, reactive oxygen species content increases and HDL promotes the ability drop of cholesterol by the macrophage outflow.Reached for 2 whens week again when mouse being changed back normal diet, the short atherogenicity feature of HDL is returned to normal phenotype.We identify the SELDI peak of 88 p＜0.05 altogether, and their situations that exists in the short scorching HDL that feeds the mouse of raising the atherogenicity meals are compared different with the normal HDL that feeds the mouse of raising normal diet.74 in 88 serum peaks are returned to normal level when meals reverse.Further analyzing with after eliminating by artefact/change that the short-term meals change and deactivated pulse atherosclerosis factor causes, we identify 24 SELDIm/z peaks, the albumen that its representative combines with short scorching HDL difference.Find that 14 short scorching HDL for following 3 kinds of widely used atherosclerotic/hyperlipemia animal models in addition in 24 protein peaks are common: the C57BL/6J mouse of the western-style food of ingesting, the invalid mouse of LDLR and the invalid mouse of apoE.In addition, the proteinogram Analysis and Identification of the blood serum sample of whole 4 kinds of animal models goes out a kind of 8 protein core labels (subgroups at above-mentioned 14 SELDI m/z peaks), and it can be used as serum biomarker group and identifies short scorching HDL.

Experimental technique

Zoopery

[0339] in these experiments, use C57BL/6J, the LDLR in 8-12 age in week invalid/and C57BL/6J and apoE be invalid/the C57BL/6J female mice.Mouse absorbs a kind of in described 3 kinds of meals in the middle of experimental period: normal diet (Ralston Purina mouse feed), or contain the atherogenicity meals (Teklad/Harlan Catalog) or the western-style food (Teklad/Harlan of 15.8% fat, 1.25% cholesterol and 0.5% cholic acid (w/w/weight), Madison WI, meals numbering 88137; 42% fat, 0.15% cholesterol, w/w).Blood serum sample separates from the overnight fast mouse, freezing as previously mentioned be kept in 10% sucrose (Navab etc., (2000) J.Lipid Res., 41:1481-1494), and in-80 ℃ of storages until use.

Lipoprotein separates

[0340] blood serum sample comes fractionated by gel infiltration FPLC (FPLC) system that is composed in series by 2 Pharmacia Superose 6 posts.Serum (0.5ml) is with the 0.5ml/ minute flow velocity wash-out of aseptic PBS to carry via nonmetal BeckmanHPLC pump, and every 1ml fraction collection.According to manufacturer's scheme, (Thermo, Louisville CO) measure the cholesterol level of each fraction, use BCA to measure that (Promega, Madison WI) measure the protein content of each fraction to use cholesterol reagent.

Being used for the SELDI analysis separates with the HDL of isolated measuring

[0341] (Polymedco, Cortland Manor is NY) according to manufacturer's scheme fresh separated HDL with LipiDirectHDL reagent.Mensuration contain HDL supernatant cholesterol level and carry out the BCA protein determination, and use in back 48 hours in separation.

Reactive oxygen species among the HDL (ROS)

[0342] uses 2,7,7 ' dichlorofluorescein diacetin (H as previously mentioned ₂DCFDA:Invitrogen, Carlsbad, CA) measure ROS content among the HDL (Navab etc., (2001) J.Lipid Res., 42:1308-1317).In brief, with HDL and H ₂The methanol solution of DCFDA (10 μ g/ml) was in 37 ℃ of incubations 30 minutes.Measure the DCF formation of fluorescence intensity detection in 485nm/525nm as the ROS indicator.

Paraoxonase (PON) is measured

[0343] measure as previously mentioned PON activity among the HDL (Van Lenten etc., (1995) J.Clin.Invest., 96:2758-2767).With HDL and paraoxon incubation, analyze the PON activity by detecting in 12 minute time period in the increase of the absorbance of 405nm.PON active unit is defined as the every ml HDL per minute that applies and forms 1nmol 4-nitrophenol.

The cholesterol outflow

[0344] carry out as previously mentioned cellular cholesterol outflow (Navab etc., (2004) Circulation, 109:3215-3220).In brief, in 24 hole tissue culturing plates, cultivate mouse RAW264.7 cell, and in containing the DMEM nutrient culture media of 10% FBS overnight growth.Use the serum free medium washed cell, and with ³H-cholesterol (0.5 μ Ci/ml) and acetylation LDL (50 μ g/ml) are loaded on the nutrient culture media of the BSA that contains 0.5% FAF together, and (Sigma, St.Louis spend the night in MO).The cell of washing mark is resuspended in the nutrient culture media that contains 0.5%BSA, with HDL in 37 ℃ of incubations 6 hours.The cholesterol outflow is represented with the percent that is discharged into the gross activity counting in the nutrient culture media.

Be used for the specimen preparation that SELDI analyzes

[0345] (Ciphergen Biosystems, Fremont CA) handle serum and HDL sample on reinforcing yin essence ion-exchange (Q10) chip according to manufacturer's scheme.In brief, (1 * PBS/0.1%Triton X-100, pH7) balance Q10 array spot is 15 minutes with binding buffer liquid in room temperature in the dampening chamber.Each sample is at first used 9M urea/2% Chaps/50mM TrisHCl, and pH9.0 dilutes with 1:5, is diluted to 1:25 with binding buffer liquid again.On the Q10 protein arrays chip that every kind of dilute sample point sample of 5 μ l is crossed to balance, and in the dampening chamber in room temperature incubation 30 minutes.Wash chip 2 times with binding buffer liquid, use HPLC H ₂O washing 1 time, air-dry then.Earlier with the 100% saturated sinapic acid solution of 0.5 μ l the sequential process chip of 50% saturated sinapic acid solution succeeded by 1 μ l.Sinapic acid solution is by using the saturated EAM solution of sinapic acid (3,5-dimethoxy-4 '-hydroxycinnamic acid) (50% acetonitrile and 0.5% trifluoroacetic acid) prepared fresh.

Ciphergen protein chip SELDI-TOF-MS analyzes

[0346] analyzes array with Ciphergen ProteinChip reader (model PB SII).Protein spectrum uses average 65 LASER SPECKLEs to produce with the laser intensity of 230-280 arbitrary unit.For the data acquisition of low molecular weight protein (LMWP), detection is measured scope and is set between the 2-18kDa, and maximum is measured and is 25kDa.For high-molecular-weight protein, detection is measured scope and is set between the 20-150kDa, and maximum is measured and is 250kDa.The mass-to-charge ratio (m/z) that is captured in every kind of albumen on the array surface is measured according to following external calibration standard items (Ciphergen Biosystems): bovine insulin (5,733.6Da), people's ubiquitin (8,564.8Da), ox cytochrome c (12,230.9Da), ox superoxide dismutase (15,591.4Da), the cow's milk globulin A (18,363.3Da), horseradish peroxidase (43,240Da), BSA (66,410Da) and the chicken conalbumin (77,490Da).

Statistical study

[0347] analyzes data with ProteinChip data analysis software 3.2 editions (CiphergenBiosystems).For each relatively, raw intensity data all uses total gas current standardization of all analytical spectras in each group.For the low-molecular-weight scope, peak strength is to 3,000-25, and the total gas current standardization of the m/z between the 000Da, for the high molecular scope, peak strength is to 4,000-250, the total gas current standardization of the m/z between the 000Da.The biomarker wizard application (calculate by nonparametric; Ciphergen Biosystems) is used to compile all spectrum and the quantitative mass peak of automatic detection.Each analytical spectra group is carried out the sample statistics, and (anti-inflammatory HDL is to short scorching HDL; Normal serum is to atherogenicity serum).Calculate the protein diversity (multiple variation) between each group.If, think that then albumen is the difference combination between two groups in the statistically-significant difference (p＜0.05) of observing its intensity when one group is compared.

The result

Model system

[0348] Hedrick etc. shows, feed for C57BL/6J LDL receptor deficiency (LDLR is invalid) mouse short-term to raise (reaching 7 days) atherogenicity meals that the active and quality of blood plasma PON is sharply descended, blood plasma and HDL lipid hydroperoxide increase (Hedrick etc., (2000) Arterioscler.Thromb.Vasc.Biol., 20:1946-1952).Interesting is, after the edible atherogenicity meals of mouse reach 7 days, changes back normal diet and reaches 3 days and cause PON quality and activity to be returned to normal level.Yet Hedrick etc. point out, change back normal diet and reach 3 days and do not cause all HDL characteristics to recover (ibid) fully.Identify that at us we have used similar mouse model system in the protein-bonded work of difference of urging among the scorching HDL.Feed the C57BL/6J mouse raise normal feed and have anti-inflammatory HDL, and feed the C57BL/6J mouse of raising the atherogenicity meals have short scorching HDL (Shih etc., (1996) J.Clin.Invest., 97:1630-1639).As shown in Figure 18 and 19, when with meals when the 7th day becomes other 14 days normal feed by the atherogenicity meals, HDL cholesterol (Figure 18 and Figure 19, the bottom right), reactive oxygen species content (Figure 19, upper left), PON activity (Figure 19, upper right) and the cholesterol outflow (Figure 19, lower-left) of HDL mediation return to normal level.We infer, use from these 3 kinds of experiment conditions (at the normal feed of ingesting of whole experimental phase, to reach 21 days in some experiments; At the atherogenicity meals of ingesting of whole experimental phase, reach 21 days in some experiments; Perhaps ingest atherogenicity meals 7 days, the normal feed 14 days of then ingesting again) HDL can be provided at the system that identifies the albumen that combines with HDL or dissociate when HDL changes short scorching state into.

Specific SELDI peak combines with short scorching HDL difference

[0349] we at first use SELDI to analyze and identify the albumen that combines with HDL when ingesting the atherogenicity meals.Female C57BL/6J mouse in 8 ages in week (n=8/group) the normal feed of ingesting reaches 7 days (C), the atherogenicity meals of ingesting reach 7 days (A), the normal feed of ingesting reaches 21 days (CC), the atherogenicity meals of perhaps ingesting reach 7 days, and the normal feed (AC) of then ingesting reaches and 14 days.When each end cycle, collect the blood serum sample (after overnight fast, obtaining) of each meals group.The HDL of each serum separates with LipiDirectHDL reagent, and carry out SELDI and analyze, identifying, and compare with serum and HDL from the mouse of accepting normal feed from atherogenic serum of the mouse of the atherogenicity meals of ingesting and the proteinogram of HDL.Obtain proteinogram by every group single blood serum sample (n=8).The proteinogram of ' C ' group is compared with the proteinogram of ' A ' group, and the proteinogram of ' CC ' group is compared with the proteinogram of ' AC ' group.In first group analysis, HDL (' C ' group) than normal serum in the HDL of atherogenicity serum (' A ' group) detects discrepant m/z peak, as previously mentioned by the further statistical analysis (Kozak etc. of Ciphergen ProteinChip software, (2003) Proc.Natl.Acad.Sci., USA, 100:12343-12348).In the atherogenicity meals of ingesting reach 21 days the HDL of mouse, detect altogether 88 and reach the remarkable different peak (p＜0.05) of HDL of 21 days mouse with the normal diet of ingesting.

Protein peak among the HDL reflects that meals change

[0350] when to these proteinograms of ' AC ' group analysis, in 88 peaks 74 are returned to when ingesting normal diet visible normal level in HDL, point out these peaks representatives with HDL by anti-inflammatory to the short scorching proteins associated spectrum that changes, vice versa (table 12).

The total proteinogram that in the short scorching HDL that short-term and long-term meals are induced, exists

[0351] also reaches the HDL that HDL and the normal feed of ingesting in the atherogenicity serum of C57BL/6J mouse (n=8) in 15 weeks (W15A) reaches the mice serum in 15 weeks (W15C), produce the SELDI spectrum by the atherogenicity meals of relatively ingesting.We have further determined at the HDL that the short-term meals are induced (74 above-mentioned peaks) and the peak that has among (15 week) HDL that meals are induced for a long time.We infer that this relatively can be eliminated because the short-term meals change the artifact/change that produces.When comparing with the HDL of the mouse of the normal diet of ingesting, we identify altogether 24 specific and remarkable different SELDI peaks (table 7) in the HDL of the mouse of accepting any cycle of atherogenicity meals (7 days atherogenicity meals and 15 all atherogenicity meals).

[0352] table 7. is in short-term or the total SELDI peak that combines with HDL after the atherogenicity meals of ingesting for a long time.

Digitized representation in the parenthesis changes the protein peak that multiple descends: the NC=no change, ingest atherogenicity meals 7 days of ingest normal diet 7 days of C=, A=, the AC=atherogenicity meals of ingesting reach 7 days, ingest normal diet 21 days of then ingest normal diet 14 days, CC=.15wA=ingests 15 weeks of atherogenicity meals, and 15wC=ingests 15 weeks of normal diet.

[0353] because the atherogenicity meals comprises 0.5% cholic acid, so we repeat above-mentioned all experiments to the HDL of the blood serum sample of the mouse (n=8) that derives from the western-style food (WD) that do not contain cholic acid of ingesting, and identifying the albumen label of forming by 21 m/z peaks, it represents the albumen (table 8) that combines with HDL difference when the higher fatty acid high cholesterol meals of ingesting (western-style food that contains the atherogenicity meals of cholic acid and do not contain cholic acid).When comparing with the HDL of the mouse of the normal diet of ingesting, in the mouse of atherogenicity meals or western-style food that ingests, have to combine/increase HDL (with the black matrix numbering) among 13 protein peaks and the HDL strongly, the albumen (in parenthesis) (table 8) of 8 kinds of depolymerization/reduction HDL is arranged in the mouse of atherogenicity meals or western-style food that ingests.

[0354] table 8. and the total SELDI peak that combines from the C57BL/6JHDL difference of western-style food of ingesting with the mouse of the atherogenicity meals of ingesting.

Digitized representation in the parenthesis changes the protein peak that multiple descends: the NC=no change, ingest atherogenicity meals 7 days of ingest normal diet 7 days of C=, A=, the AC=atherogenicity meals of ingesting reach 7 days, ingest normal diet 21 days of then ingest normal diet 14 days, CC=.15wA=ingests 15 weeks of atherogenicity meals, and 15wC=ingests 15 weeks of normal diet, ingest western-style food 7 days of WD=.

The total proteinogram that in the hyperlipemia mouse model, combines with HDL difference

[0355] is the proteinogram of the HDL of the HDL of the differentiation atherosclerotic mouse model identified of checking and control mice, we further use other well-known atherosclerotic mouse model, comprise the invalid mouse of apoE of the invalid mouse of LDLR of the western-style food of ingesting and the normal diet of ingesting.These mouse have short scorching HDL (Navab etc., (2005) Ann.Med., 37:173-178 to the atherosclerotic sensitivity; Shih etc., (1996) J.Clin.Invest., 97:1630-1639; Ridker (2002) Circulation, 105:2-4).HDL sample from these atherogenicity mouse is carried out SELDI analyze, and with these spectrums of C57BL/6J mouse model cross validation.These researchs have not only been eliminated by artifact/variation that the short-term meals change and deactivated pulse atherosclerosis factor causes, and identify total HDL biomarker in a plurality of atherogenicity models.21 peaks that combine with short scorching HDL that identify in the C57BL/6J mouse model have 14 to be common (table 9) of detected those spectrums in the HDL of invalid mouse of LDLR and the invalid mouse of apoE.

[0356] the total SELDI peak that combines with the HDL difference of 3 kinds of atherosclerotic mouse models of table 9..

Digitized representation in the parenthesis changes the m/z peak that the protein peak that multiple descends: WT=combines with short scorching HDL difference in wild type C57BL/6J mouse, the m/z peak that LDLR=combines with the short scorching HDL of the invalid mouse of LDLR, the m/z peak that ApoE=combines with the short scorching HDL of the invalid mouse of ApoE.

The potential serum biomarker that combines with short scorching HDL

[0357], the blood serum sample of whole 4 kinds of mouse Atherosclerosis Model is carried out the SELDI analysis in the mode identical with the analysis that HDL is implemented for further identifying representative in the haemocyanin spectrum of atherogenic/albumen of combining with HDL during the hyperlipemia meals of ingesting.SELDI analyzes 13 peaks is accredited as total biomarker (table 10) in the mice serum of atherogenic/hyperlipemia.8 those spectrum (14 above peaks) common (tables 11) for identifying among the HDL of the meals of the atherogenic/hyperlipemia of ingesting are arranged in 13 protein peaks that detect in the serum of atherogenic/hyperlipemia in addition.These situation hints, the potential source biomolecule mark of 8 kinds of albumen being identified for combining with short scorching HDL, it can directly detect in mice serum.

[0358] the total SELDI peak that combines with the serum difference of 3 kinds of atherosclerotic mouse models of table 10..

Digitized representation in the parenthesis changes the protein peak that multiple descends.The m/z peak that WT=combines with atherogenic serum difference in wild type C57BL/6J mouse, the m/z peak that LDLR=combines with atherogenic/hyperlipemia serum of the invalid mouse of LDLR, the m/z peak that ApoE=combines with the atherogenic serum of the invalid mouse of ApoE.

[0359] the total albumen label that in 3 kinds of C57BL/6J mouse Atherosclerosis Model, combines of table 11. with HDL and serum difference.

m/z(×10 3)

(9.3)

14.9

15.6

15.8

16.2

16.5

(18.6)

(19.5)

Discuss

[0360] in recent years, having become is clear that, the HDL function may be to have more than HDL-C optionally to treat target (Castellani etc., (1997) J.Clin.Invest., 100:464-474 to antiatherosclerosis the time; Navab etc., (2005) Ann.Med., 37:173-178; Ridker (2002) Circulation, 105:2-4; Ansell etc., (2003) Circulation, 108:2751-2756).Although many albumen are relevant with HDL with enzymatic activity, know that seldom which kind of concrete proteinogram helps to distinguish better normally/anti-inflammatory HDL and short scorching HDL.We utilize the ProteinChip technology in existing mouse Atherosclerosis Model, and identify the m/z peak of the representative albumen relevant with HDL difference in the mouse of atherogenicity meals that ingests.

[0361] single mouse sample (every group of n=8) is carried out our all experiments, each sample is with triplicate detection.In addition, only between each group, exist the m/z peak of conspicuousness (p＜0.05) just to be accepted as the candidate peak, be used for further analysis.In first group of experiment, when being become by atherogenicity meals (7 days), meals ingest normal diet again in the time of 14 days, and in HDL, always have 74 m/z peaks (table 12) and be returned to normal level.Because any artifact that the meals short term variations causes, further compare these peaks for eliminating at the proteinogram of the HDL sample of the mouse that derives from 15 weeks of atherogenicity meals of ingesting.Interesting is, 74 initial peaks only have 24 (table 7) to appear to derive among the HDL of mouse in 15 weeks of atherogenicity meals of ingesting, and 59 remaining m/z peaks of prompting may change and/or deactivated pulse atherosclerosis factor owing to the short-term meals.Yet, also might be 59 remaining m/z peaks represent still that atherogenicity at HDL transforms early stage with urge the albumen that scorching HDL combines.

[0362] the table 12. SELDI peak relevant with the short scorching HDL of C57BL/6J mouse.

Digitized representation in the parenthesis changes the protein peak that multiple descends.Ingest normal diet 21 days of ingest atherogenicity meals 7 days of ingest atherogenicity meals 7 days of ingest normal diet 7 days of NC=no change, C=, A=, AC=, and the normal diet 14 days of ingesting, CC=.

[0363] interestingly is, when comparing with the proteinogram of the mouse that derives from the western-style food of ingesting (table 8), in the HDL of the C57BL/6J mouse of the atherogenicity meals of ingesting (or short-term or long-term) following 3 kinds of albumen: m/z 5100, m/z 35300 and m/z197000 do not appear.Because cholate is the main difference between used two kinds of meals, these 3 kinds of albumen might have been represented cholate metabolism and the distinctive albumen of toxicity.The evaluation of these albumen is useful to understanding the HDL inflammatory properties relevant with the cholic acid metabolism with the sign susceptible of proof.Surprisingly, when by deriving from that two kinds of other short scorching HDL of atherosclerosis animal model (invalid mouse of the LDLR of the western-style food of ingesting and the invalid mouse of apoE of normal diet of ingesting) prepare proteinogram and during with the total proteinogram contrast of the short scorching HDL of C57BL/6J mouse, having only 14 to be these 3 kinds of models common (table 8 and table 9) in 21 peaks.

[0364] the haemocyanin analysis of spectrum provides the simpler and easy and efficient strategy of using biomarker in medical diagnosis on disease and/or drug effectiveness.We identify and compare numerous albumen (table 13) different in serum with the spectrum that derives from HDL.Evaluation is from the core sequence (table 10) at 13 peaks of all different atherosclerotics/hyperlipemia in mice models.When we relatively derived from the last several histones spectrum of serum analysis of spectrum and HDL analysis of spectrum, we identified one group of total 8 protein peak (table 10).Represent the albumen at these 8 peaks can form one group of very important mark, it not only directly measures the blood serum sample that is used to identify short scorching HDL, and is used for further understanding the research of short scorching HDL character.

[0365] table 13. and C57BL/6J mouse is atherogenic/SELDI peak that hyperlipemia serum difference combines.Digitized representation in the parenthesis changes the protein peak that multiple descends.Ingest normal diet 21 days of ingest atherogenicity meals 7 days of ingest atherogenicity meals 7 days of ingest normal diet 7 days of NC=no change, C=, A=, AC=, and the normal diet 14 days of ingesting, CC=.

[0366] C57BL/6J that induces of table 14. and short-term and long-term meals atherogenic/total SELDI peak that hyperlipemia serum difference combines.

Digitized representation in the parenthesis changes the protein peak that multiple descends.Ingest atherogenicity meals 7 days of ingest normal diet 7 days of NC=no change, C=, A=, the AC=atherogenicity meals of ingesting reach 7 days, ingest normal diet 21 days of the normal diet 14 days of then ingesting, CC=.W15A=ingests 15 weeks of atherogenicity meals, and W15C=ingests 15 weeks of normal diet.

[0367] represent the albumen at these m/z peaks (table 11) to combine with the short scorching HDL difference of C57BL/6J mouse hyperlipidemia and Atherosclerosis Model.As initial screening, by ion exchange column fractionated normal serum and the atherogenicity serum that adopts different pH damping fluids, then carry out SELDI and analyze, to obtain the pI value (Kozak etc. at every kind of target protein peak, (2005) Proteomics, 5:4589-4596).Use the pI and the quality information of each protein peak, our searching database (TagIdent), and obtain to represent the candidate albumen at target SELDI peak.We determine, find that in the mouse of atherogenicity meals that ingests haemoglobin combines with HDL.The details of these researchs provides in embodiment 3.

[0368] in a word, we have characterized 8 m/z SELDI biomarker peaks in short scorching HDL, and confirm their differential expressions in atherogenicity/hyperlipemia serum.In a word, these marks will help to determine to participate in the molecular mechanism that normal/anti-inflammatory HDL changes short scorching HDL in mouse.The identifying of these marks and the mark in the short scorching HDL of people helps developing that clinical assays is improved atherosclerotic and be the early detection of other patient's condition of feature with the inflammatory reaction.

Embodiment 3

With in the mice serum of the atherogenicity of ingesting/hyperlipemia meals The haemoglobin of high-density lipoprotein (HDL) combination

[0369] in embodiment 2, we use the reinforcing yin essence ion-exchange SELDI ProteinChip technical appraisement of distinguishing normal/anti-inflammatory HDL and short scorching HDL in the mouse Atherosclerosis Model to go out 8 species specificity protein fingerprint spectrums.Use little liquid layer to analyse-tandem mass spectrum, we will represent the SELDI peak of m/z 14,900 and m/z 15,600 be accredited as respectively the mouse hemoglobin alpha-chain (Hb-α, 14.9kDa) and the mouse HBB (Hb-β, 15.9kDa).Western blot analysis confirms that when comparing with normal HDL, Hb combines with short scorching HDL difference.The biochemical characteristics of the Hb that combines with HDL further shows, have different physics and chemical characteristic with the Hb of short scorching HDL combination, comprise that pI reduces (more than pI4.0 and the pI7.0 pI7.5 to free Hb), combines with the high molecular weight component that exists in containing the fraction of HDL.The principal mode that is found the haemoglobin that combines with HDL is oxyhemoglobin (oxyHb).Based on the prooxidant character of oxyHb, our Notes of Key Data, Hb has the short inflammatory matter that helps HDL under atherogenic condition.In addition, we infer that the Hb that combines with HDL can be the neoformation mark of other patient's condition of feature as atherosclerotic with the inflammatory reaction.

Experimental technique

Zoopery

[0370] in the experiment of comparing 3 kinds of mouse models, uses 8-12 the wild type LDL receptor deficiency (LDLR is invalid) and the invalid C57BL/6J female mice (every group of n=8) of apoE in age in week.In these experiments, ingest a kind of in following 2 kinds of meals of mouse: normal diet (Ralston Purina mouse feed), or contain the atherogenicity meals (Teklad/HarlanCatalog) of 15.8% fat, 1.25% cholesterol and 0.5% cholic acid (w/w/weight).For short-term research, mouse (every group of n=8) the described meals of ingesting reach 7 days, and for studying for a long period of time, the mouse described meals of ingesting reached for 15 weeks.Blood serum sample separates the mouse from overnight fast, freezing being kept in 10% sucrose, and in-70 ℃ of storages until use.

Lipoprotein separates

[0371] blood serum sample of He Binging separates by the system level that is composed in series by 2 Pharmacia Superose 6 posts.Serum (0.5ml) is with the 0.5ml/ minute flow velocity wash-out of aseptic PBS to carry via nonmetal BeckmanHPLC pump, and every 1ml fraction collection.Discarded preceding 10 1mL fractions, according to manufacturer's scheme to each 1mL fraction of collecting subsequently carry out the cholesterol level analysis (Thermo, Louisville, CO) and the BCA protein determination (Promega, Madison, WI).VLDL, the LDL, HDL and the back HDL fraction that merge all experiments.For some experiment, (NY) fresh separated is from the HDL of single blood serum sample for Polymedco, Cortland Manor to use LipiDirectHDL reagent according to manufacturer's scheme.Mensuration contains the cholesterol level and the BCA protein determination of the supernatant of HDL, and uses in after separation 48 hours.

With doing the specimen preparation that SELDI analyzes

[0372] according to manufacturer's scheme (Ciphergen Biosystems, Fremont, CA) preparation and handle lipoprotein or blood serum sample on normal phase (NP-20), reinforcing yin essence ion-exchange (Q10) and weak cation exchange (CM10) ProteinChip array.In brief, (1 * PBS/0.1% Triton X-100 is pH7) in equilibrium at room temperature NP-20, Q10 and CM10 array spot 10 minutes with binding buffer liquid.Will be to array chip with sample (serum dilutes with 1:25, and the lipoprotein fraction is diluted with the 1:2) point sample of binding buffer liquid dilution, and in the dampening chamber in room temperature incubation 30 minutes.Wash chip 2 times with binding buffer liquid, use HPLC H ₂O washing 1 time, air-dry then.Earlier with the 100% saturated sinapic acid solution of 0.5 μ l the sequential process chip of 50% saturated sinapic acid solution succeeded by 1 μ l.The sinapic acid solution saturated EAM solution of sinapic acid (3,5-dimethoxy-4 '-hydroxycinnamic acid) (50% acetonitrile and 0.5% trifluoroacetic acid) prepared fresh.

Ciphergen Protein Chip SELDI-TOF-MS analyzes

[0373] as analyzing array with Ciphergen ProteinChip reader (model PB SII) described in the embodiment 2.

Statistical study

[0374] as (Ciphergen Biosystems, Fremont CA) analyze data using ProteinChip data analysis software 3.2 editions described in the embodiment 2.

Represent the evaluation of the albumen at specific m/z peak

The haemocyanin fractionated

[0375] scheme according to the manufacturer goes up desalination with serum at P-6Micro Bio-Spin chromatographic column (Bio-Rad, Hercules CA).Use the centrifugal post of Q10 anion exchange (Ciphergen Biosystems, Fremont, CA) the damping fluid series elution fractionation separation of serum sample that descends with following pH: pH8.5,7.5,7.0,6.0,5.0,4.0 and 2.0 according to manufacturer's scheme.Adopt the SELDI-TOF-MS PSII analyzing proteins fraction of Q10 or CM10 protein chip array.Two arrays are all used the binding buffer solution equilibria of 10mM HCl before use.

Haemocyanin purifying, passive wash-out and check by SELDI-TOF-MS

[0376] merga pass SELDI analyzes the fraction that confirms to contain most target peaks, and by centrifugal evaporation drying.The fraction that contains described albumen is further separated on SDS-PAGE, then with Simply Blue Safe stain (Invitrogen, Carlsbad, CA) dyeing.Cutting-out has the band corresponding to the molecular weight of target peak, gel piece cut in half, the passive as previously mentioned wash-out of half gel piece (Le Bihan etc., (2004) Proteomics, 4:2739-2753).In brief, dehydrating gel, dry in heat block, and rehydrated in organic mixture.Ultrasonic to gel, vortex afterwards.The albumen of wash-out is used for analyzing the existence that confirms target peak by SELDI ProteinChip.Remaining half gel that is used for as described below digests.

Trypsinization

[0377] carries out trypsinization in the gel (Gomez etc., (2003) Mol Cell Proteomics 2:1068-1085) as previously mentioned.In brief, contain the eluent (confirming the back) of target peak, use the iodoacetamide alkylation, and handle with trypsase (Promega) by SELDI with DTT reduction.With the saturated gel slice recovering peptide of hplc grade water, and extract with acetonitrile/trifluoroacetic acid.Extract is dry in cold SAVANT Speed Vac (Global MedicalInstrumentation), and carries out μ LC-MSMS (ibid) as previously mentioned.Use Sonar ms/ms ^TM(Genomic Solutions) and TurboSEQUEST ^TM(ThermoElectron Corp) uses data retrieval mouse database.

Electrophoresis and Western blotting

[0378] other reagent that is used for electrophoresis of IEF, Tris/HCl gel and all available from Bio-Rad (Hercules, CA).With blood serum sample (2 μ L) load sample to 15%SDS-PAGE, IEF (pH3～10), the non-sex change of Tris/HCl (4～15%) or IEF-Tris/HCl 2D gel.For the 2D gel, downcut every swimming lane by the IEF gel, and be inserted in the native gel according to manufacturer's scheme (Bio-Rad).With the blood serum sample load sample to gel, and according to manufacturer's scheme (Bio-Rad) be transferred to nitrocellulose filter (GE Healthcare, Piscataway, NJ).This film to the haemoglobin of 1:1000 (MP Biomedicals, Irvine, CA) or 1:10,000 apoA-1 (Bethyl Laboratory, Montgomery, TX) Western blotting.Two anti-(the GE Healthcare) that put together HRP use with the 1:10000 dilutability, and band manifests with ECL detectable (GEHealthcare).

The spectrophotometry of Hb

[0379] merges the FPLC fraction that contains HDL, use Beckman DU 640 spectrophotometers to detect Hb.Spectrum by 380nm-700nm scanning all samples and pure hemoglobin material.(CaymanChemical Michigan) adds the sample that contains oxyhemoglobin (oxyHb) to the adduct of time release NO donor spermine nitrogen oxidation slowly, to observe the transformation of oxyHb to methemoglobin (metHb).Utilize linear regression with " the basic spectrum " of one group of pure material to detecting spectrum simulation, the concentration of deconvolute thus oxyHb and metHb (Vaughn etc., (2000) J.Biol.Chem., 275:2342-2348).When consume oxyHb produce ratio that metHb shows as 1:1 simultaneously total hemoglobin in all samples, all during conservation, confirmed the validity of the method for deconvoluting.

The result

The serum of SELDI peak m/z 14.9k and m/z 15.6k and atherogenic/hyperlipemia With the HDL combination

[0380] use the SELDI of reinforcing yin essence ion-exchange (Q10) ProteinChip the analysis showed that, the peak of representing m/z 14.9k and m/z 15.6k is than to impinging upon the serum (Figure 20 that derives from 4 kinds of different atherosclerotics/hyperlipemia in mice models, last figure) and among the HDL (Figure 20, figure below) all be enhanced minimum several times.Carry out serum and HDL sample that the atherogenicity meals in short-term (7 days) and long-term (15 week) are fed the C57BL/6J mouse of raising for controlling oneself, carry out the experiment that the albumen of representing SELDI peak m/z 14.9k and m/z 15.6k was identified and characterized in all subsequently being used to.

Use size, pI and TagIdent to identify potential candidate albumen SELDI peak m/z 14.9 and M/z 15

[0381] be definite identity of representing the albumen at two SELDI peaks, we at first check the pI scope at two peaks.As described under, carry out the anion exchange fractionated to deriving from the single blood serum sample that ingest normal diet or atherogenicity meals reach the C57BL/6J mouse (every group of n=8) in 7 days (short-term) or 15 weeks (for a long time) in method.Fraction is with the buffer solution elution of different pH, and further analyzes (Figure 21) with CM10 and Q10SELDI chip.Most of intensity of representing two peak m/z14.9k and m/z15.6k is by (Figure 21) under the buffer solution elution in pH7.5 to pH8.0 scope.Use is analyzed the size of determining by SELDI-TOF-MS and is carried out online TagIdent (albumen database) by the corresponding pI that the anion exchange fractionated is determined and retrieve (Figure 21).Use allow 0.5% size deviation and+search criteria of 2pI scope, Hb-α and Hb-β are accredited as the potential candidate albumen of 14.9kDa and 15.6kDa respectively.Based on document, the pI of free Hb is in the scope of 7.5-8.5.Interesting is, two peaks also finding to represent m/z14.9k and m/z 15.6k with from atherogenicity serum, combine (Figure 21) with the fraction of pH7.0 and pH4.0 buffer solution elution.These Notes of Key Datas, i) Hb in atherogenic sample has different chemical characteristics, or ii) represents the albumen of m/z 14.9k and m/z 15.6k different with Hb.As follows, subsequently research discloses, and Hb is represented at these peaks in fact really, results from combining closely of the albumen that combines HDL with other such as hoptoglobin with the different physics of the Hb of HDL combination and chemical characteristic.

Fractionated and tryptic peptide fragmentation and confirm two biologies by the tandem mass spectrum analysis The identity of labelled protein is Hb-α and Hb-β

[0382] be the identity that further confirms two biomarkers, after removing albuminization and anion-exchange chromatography by the mice serum partial purification corresponding to the peak of size separately.Partially purified albumen is carried out trypsinization, then carry out μ LC-MSMS and analyze, the fragment of acquisition is retrieved people's albumen database (Sonar and SEQUEST).The result confirms that 14.9kDa albumen is α-Hb, and 15.6kDa albumen is β-Hb.

In mouse model, haemoglobin is accredited as the potential mark of short scorching HDL

[0383] for further in atherogenic serum and HDL, having a situation by non-SELDI method validation Hb, we at first test the blood serum sample from the mouse of the atherogenicity meals of ingesting on SDS-PAGE, then carry out Western blotting at Hb.Hb total amount in the blood serum sample is not remarkable different (Figure 22 A) between normal and atherogenic serum.

[0384] total concentration that should be pointed out that Hb in the serum (being non-RBC Hb) is about 10 μ mol.By contrast, the Hb concentration in the whole blood is greater than 1mol.Therefore, only have an appointment 0.001% Hb is present in the blood outside the RBC.As shown in Figure 22 A, the amount of the non-RBC Hb of this in the mice serum of the normal feed of ingesting is compared with the mouse of the atherogenicity meals of ingesting does not have difference.

[0385] yet, in the lipoprotein of the FPLC fractionated of normal serum (promptly from the mouse of the normal diet of ingesting serum), Hb combines with back HDL (pHDL) fraction, and the Hb in atherogenicity serum combines (Figure 22 B) with the HDL fraction.In addition, confirmed observed result (Figure 22 C) in Figure 22 B by the content of hemachrome that independent FPLC fraction is carried out the Hb that OD detect to obtain in 410nm.In the atherogenicity meals mouse in 15 weeks that ingests, find Hb only in the HDL fraction (Figure 22 B).These experiments show that Hb is the mark of short scorching HDL in mouse.

The haemoglobin that combines with atherogenicity serum shows distinct physics and change Learn characteristic

[0386] western blot analysis of blood serum sample does not demonstrate the significant difference aspect the Hb quality (Figure 22 A) between normal serum and the atherogenicity serum, prompting Hb and being combined in when ingesting the atherogenicity meals of HDL are changed, and the maintenance of the quality of haemoglobin is constant.Use the SELDI of Q10 array to experimentize, in order to the biomarker of finding to combine, the albumen of this experimental selection pI＜7 with atherogenic serum.In addition, in the experiment of above-mentioned anion exchange fractionated, find that two peaks of the representative m/z14.9k of significant quantity and m/z15.6k combine (Figure 21) with atherogenicity serum fraction with pH7.0 and pH4.0 buffer solution elution.These Notes of Key Datas, i) quality of Hb is not significantly changed under the atherogenicity condition, ii) the chemical characteristic of Hb is changed under the atherogenicity condition, with distinct pI value is card (Figure 21), iii) because the HDL under the atherogenicity condition changes and/or Hb changes, Hb is in conjunction with HDL, and ingesting the atherogenicity meals after 15 weeks, and Hb only combines with HDL.

[0387], use NP20 and Q10 array that blood serum sample and FPLC lipoprotein fraction from study for a long period of time (W15) are carried out the SELDI analysis for characterizing the different qualities of Hb in atherogenicity serum.The NP20 array is caught the Hb (Figure 23 A, left figure) of equivalent in conjunction with all albumen by normal and atherogenic blood serum sample.In addition, NP-20 array Hb peak combines with pHDL fraction in the deactivated pulse atherosclerosis sample, and combines (Figure 23 A, left figure) in the atherogenicity sample fully with the HDL fraction.These data are consistent with the western blot analysis after the SDS-PAGE that is shown in Figure 22 B.On the other hand, the Q10 array is caught Hb (Figure 23 A, right figure) in serum that is only obtained by the atherogenicity sample and HDL fraction, and the Hb of prompting combination in atherogenicity serum has different characteristics, combines with HDL.For further check has the situation that exists of the Hb of different pI under the atherogenicity condition, by SDS-PAGE succeeded by protein analysis analyze that SELDI verified, from the serum fraction of the anion-exchange column of Figure 21.Atherogenicity serum comprises Hb (Figure 23 B) in the anion-exchange column fraction from pH7 and pH4.

The sign of the Hb that combines with the HDL fraction

[0388] is physics and the chemical characteristic of further verifying the Hb that combines with HDL, the blood serum sample of D7 and W15 group and the HDL fraction of D7 group are carried out isoelectric focusing (IEF) and native gel electrophoresis.The IEF gel shows, the Hb in atherogenicity serum has the pI value of about 4 reduction, and the pI of normal Hb is about 7.5 (Figure 24).Normal Hb (pI7.5) to the transformation of the Hb of modified (pI4.0) in the sample of D7 group high-visible (Figure 24).These changes are not the changes because of RBC, demonstrate the Hb (Figure 24) with normal pI because separate from the RBC of same group of mouse.In addition, the Hb that combines with the HDL fraction in the D7 group demonstrates the Hb (Figure 24) of modified forms.Native gel shows that the Hb immunoreactivity combines with high molecular (HMW) particle in atherogenic blood serum sample, and 15 week of atherogenicity meals back Hb only combines (Figure 25) with HDL ingesting.In addition, the non-sex change 2D of IEF/ gel (Figure 12) confirms, has various ways (based on pI) from the Hb of atherogenicity serum, and these forms mainly combine with the HMW particle.Be the concrete form (oxyHb or metHb) of definite Hb that combines with HDL, the HDL fraction that is combined is carried out spectrophotometric analysis.Find that the main Hb form that combines with HDL is oxyHb, a small amount of metHb (Figure 26 and table 15) is arranged.

[0389] oxyHb that in D7 and W15 mouse, combines of table 15. and the concentration of metHb with HDL

Discuss

[0390] the proteinogram analysis is an effective means of determining the albumen of differential expression and/or combination in blood serum sample, allows to estimate fast important function biologically.CiphergenBiosystems (Fremont, CA) develop ProteinChip technology coupling surface laser enhanced desorb/ionization flight time mass spectrum (SELDI-TOF-MS), be beneficial to proteinogram analysis (Rubin and Merchant (2000) Am.Clin.Lab.19:28-29 of complex biological mixtures; Weinberger etc., (2002) Curr.Opin.Chem.Biol.6:86-91; Fung etc., (2001) Curr.Opin.Biotechnol.12:65-69; Issaq etc., (2002) Biochem.Biophys.Res.Commun.292:587-592).SELDI is unique with regard to its ability of being analyzed trace (low flight mole) analyte by complexity source (serum, urine, excrement, CSF, tissue culture extract, cell lysate).When the target analyte is known, utilize " bait " molecule (for example electric charge, hydrophobicity, specificity binding affinity, antibody) and chip surface covalently bound, " preactivated " surperficial ProteinChip array is also as the open platform at multiple different choice.Effectiveness (Wright etc., (1999) Prostate Cancer-Prostatic Dis.2:264-276 of the cancer protein labeling in the SELDI-TOF-MS scientific discovery serum have been confirmed in the recent period; Li etc., (2002) Clin.Cheam.48:1296-1304).We successfully utilize the SELDI-TOF-MS system, and draft report be used for evaluation (Kozak etc., (2003) Proc.Natl.Acad.Sci., USA, the 100:12343-12348 of the biomarker of oophoroma early detection; Kozak etc., (2005) Proteomics5:4589-96).

[0391] behind the pI at two peaks determining to represent m/z 14.9k and m/z 15.6k (Figure 21), database retrieval is accredited as Hb-α and Hb-β the potential source biomolecule mark that combines with short scorching HDL in the atherogenicity serum.These find to use μ LC-MSMS method to confirm.We worry this discovery at first, because mechanical treatment and/or specimen preparation can cause RBC cracking and Hb to discharge potentially.Yet, carefully repeat these experiments subsequently and determine that Hb is not the artifact, but the specificity of the short scorching HDL that in atherogenicity serum, exists and effective mark.At first, we show that total Hb quality does not have difference (Figure 22 B) between the blood serum sample of the mouse that derives from ingest normal diet and atherogenicity meals.Hb total concentration in the serum (being non-RBCHb) is about 10 μ mol.By contrast, the Hb concentration in the whole blood is greater than 1mol.Therefore, only there is about 0.001% Hb in the blood outside RBC.As shown in Figure 22 A, the amount of the non-RBC Hb of this in the mice serum of normal feed of ingesting does not have difference than the mouse of the atherogenicity meals of ingesting.

[0392] secondly, at normal HDL in short scorching HDL transforming process (the D7 normal diet is to D7 atherogenicity meals), we do not find that in deriving from the RBC lysate of same mouse any Hb changes (quality and quality), and pointing out Hb to be combined under the short scorching condition with short scorching HDL again is the specificity phenomenon.The 3rd, Hb depends on the degree of urging scorching condition with combining of short scorching HDL, and this confirms (Figure 22 B and Figure 24 and 25) by the Hb that compares in D7 and the W15 lipoprotein sample.At last, comprising the invalid mouse of apoE (it has short scorching HDL when ingesting normal diet) in 4 kinds of interior different atherosclerotic/hyperlipemia model, Hb is in conjunction with HDL in discovery.In a word, these results show, are the marks of short scorching HDL in conjunction with the Hb of HDL.

Reported already [0393] that the normal pI of Hb was between pI7.0 to 8.0.We find to have at least two kinds of Hb materials (Figure 21 and Figure 24) with different pI values with the Hb of short scorching HDL combination.Ingesting the atherogenicity meals after 15 weeks, and all Hb that combine with HDL show this unusual pI.The change of discovery pI of Hb after the atherogenicity meals of ingesting is not the change because of haemoglobin, but because of the albumen of combining closely with haemoglobin among the HDL.Because find that most of under these conditions Hb is an oxyhemoglobin, so the oxidative stress of being induced by the atherogenicity meals might cause these changes.The RBC Hb of same mouse does not have these to change prompting, and free under these conditions Hb can be subjected to influence in various degree.The total serum Hb of normal diet or atherogenicity meals of ingesting does not have this true prompting of difference, and the RBC cracking of increase is not the influence factor in this process.

[0394] hoptoglobin (Hp) and Hemopexin (Hx) are respectively to haemoglobin (Hb) (K _d1pM) and protoheme (K _d＜1pM) have a plasma proteins of high binding affinity.They are mainly expressed in liver, belong to acute phase protein family, and it synthesizes in inflammatory process is induced (Bowman and Kurosky (1982) Adv Hum Genet.12:189-261; Altruda etc., (1985) NucleicAcidsRes.13:3841-3859).What fully determine is, Hb (in red blood cell the abundantest and on function important protein) is in case discharged by red blood cell, become height toxicity with regard to oxidation characteristic owing to protoheme, it participates in the Fenton reaction, generation causes the reactive oxygen species (Hoffman etc. of cellular damage, (1995) Hematology:BasicPrinciple and Practice. is the 2nd edition, New York, NY:Churchill Livingstone).The toxicity of protoheme is owing to the hydrophobicity of protoheme increases, the hydrophobicity of protoheme can make protoheme insert (Balla etc. in lipid film and other lipophilic compartment not with protein combination the time, (1993) Proc.Natl.Acad.Sci., USA, 90:9285-9289).Usually, in the process of hemocytoblast stoning and red blood cell aging disintegration, there is a spot of extravascular hemolysis, Hb is discharged in the blood plasma.In the illness relevant such as hemorrhage, hemoglobinopathy, ischemia-reperfusion or malaria, discharge a large amount of free Hb (Wagener etc., (2001) TrendsPharmacol Sci.22:52-54) with intravascular hemolysis.In case enter in the blood plasma, free Hb is a dimer by the Hp combination with regard to fast dissociating.The metabolism of blood plasma Hb is considered to the major function of tissue macrophages, and tissue macrophages can be removed thing acceptor CD163 by macrophage and absorb Hb-Hp compound (Schaer etc., (2006) Blood 107:373-380; Fabriek etc., (2005) Immunobiology 210:153-160), and internalization they (Kristiansen etc., (2001) Nature409:198-201).Interesting is that nearest research is accredited as LDH receptor related protein (LRP)/CD91 (Hvidberg etc., (2005) Blood 106:2572-2579) acceptor of being responsible for removing Hemopexin-protoheme compound.LRP/CD91 expresses in several cell types, comprises macrophage and liver cell, and these cells can pass through receptor mediated endocytosis internalization protoheme-Hx compound (Hunt etc., (1996) J Cell Physiol.168:71-80).In the experiment of this paper report, we do not see that Hb is discharged by RBC, but existing Hb changes the form (previous not report) that combines with the HDL fraction into.Might be to form the Hb-Hp-Hx compound under the oxidative stress condition, it combines with HDL, so that by removing fast in the circulation.In fact, reported already that Hp combined (Rademacher etc., (1987) Anal Biochem.160:119-126 with the major protein component apoA1 of HDL; Kunitake etc., (1994) Biochemistry 33:1988-1993; Porta etc., (1999) Zygote 7:67-77; Spagnuolo etc., (2005) J.Biol.Chem..280:1193-1198).Hp combines with apoA1 and has changed HDL function (Balestrieri etc., (2001) Mol Reprod Dev.59:186-191; Cigliano etc., (2001) Steroids 66:889-896).The Notes of Key Data among Figure 12-15, non-RBC haemoglobin, hoptoglobin and Hemopexin are under atherogenic condition all in the same compound in HDL.

[0395] Hb be with hyperglycemia, oxidative stress, hypertension, insulin resistance, obesity and

The sick relevant damage of urine and known mark (Zhang etc., (2004) Proteomics4:244-256 of disease; De Valk and Marx (1999) Arch Intern Med.159:1542-1548; Alayash etc., (2001) Antioxid Redox Signal 3:313-327).It is poisonous that Hb also is considered to, because free Hb also is potential oxygenant (Alayash (1999) Nat Biotechnol.17:545-549) because of its protoheme (Fe) with in conjunction with the reactive group of protoheme, also shown described reactive group in conjunction with protoheme oxidation LDL (Paganga etc., (1992) FEBS Lett.303:154-158 in vivo; Miller etc., (1996) ArchBiochem Biophys.326:252-260; Ziouzenkova etc., (1999) J.Biol.Chem..274:18916-18924).Our discovery shows that first in the oxidative stress environment, Hb combines with the HDL fraction in mouse.Short scorching HDL in atherogenicity serum contains lipid hydroperoxide (LOOH), does not have the paraoxonase activity, and activated monocyte can not prevent the LDL oxidation and show less cholesterol outflow.We report at this, and Hb combines with short scorching HDL specificity in the atherogenicity mouse.Be not limited to concrete theory, we think that Hb combines with HDL and relates to HDL by the transformation of anti-inflammatory to short inflammation.

[0396] in a word, Hb combines with short scorching HDL in atherosclerosis animal model.Expand to the people, we think that the Hb in conjunction with HDL can be used as the mark of short scorching HDL.

Embodiment 4

Use Qiagen not contain albuminous pillar and carry out LDL gathering method for measuring Material

[0397] uses and not contain albuminous pillar and carry out LDL and assemble and measure usually that used material is shown in table 16.

[0398] table 16. is used for LDL and assembles the material of measuring

Method:

The preparation of sample

[0399] 1. dilutes 25 μ L serum or blood plasma with 75 μ L dilution buffer liquid.

[0400] 2. with the of short duration centrifugal centrifugal post that does not contain albumin/IgG of 500 * g, so that take out resin by screw-cap.

[0401] 3. takes off screw-cap, pull down the bottom seal of centrifugal post, and discharge store buffer liquid by run by gravity.

[0402] 4. following equilibrium centrifugation post: the dilution buffer liquid of 2 * 0.5mL equal portions is pipetted on the centrifugal post, and makes every part to flow out by run by gravity.

[0403] 5. seals centrifugal post with QIAfilter Cartridge cap.

[0404] sample that 6. will prepare in step 1 adds on the post.

[0405] 7. the close lid of centrifugal post, violent jolting is to obtain homogeneous suspending liquid.In room temperature incubation 5 minutes on shaking table.

[0406] 8. takes off QIAfilter Cartridge, and centrifugal post is transferred to clean centrifuge tube.

[0407] cap 1/4 circle of 9. outwarding winding.

[0408] 10. by collecting effluent in centrifugal 10 seconds with 500 * g.

The dilution buffer liquid washing column of [0409] 11. usefulness 2 * 100 μ L equal portions is by collecting every part of washing fraction in centrifugal 10 seconds with 500 * g.

Two washing fractions of the outflow fraction of [0410] 12. combining step 10 and step 11.

Removal contains albumen and the cholesterol determination of Apo B

[0411] 1. precipitating the albumen that will contain ApoB by dextran sulfate removes by not containing in the albuminous sample.For precipitating with dextran sulfate, the SigmaHDL cholesterol reagent that will contain dextran sulfate and magnesium ion is dissolved in the distilled water.With every kind of sample mix of dextran sulfate (1.0mg/ml) Yu the 500 μ L of 50 μ l, and in room temperature incubation 5 minutes, subsequently with 3, centrifugal 10 minutes of 000g.In experiment, use the supernatant that contains HDL.

[0412] 2. uses the T-CHOL of standard cholesterol determination detection in not containing the HDL supernatant of albumin/apoB.

[0413] 3. the HDL supernatant is added to each hole with 10 μ g concentration.

The preparation of phospholipase C (PLC)

[0414] 1. adds the ddH of capacity to PLC bottle (250 unit/bottle) ₂O is to obtain 56.3 units/mL.For example, add 4.4mL ddH to the bottle that contains 250 units ₂O.

[0415] 2. of short duration vortex.

[0416] 3. 200 μ LPLC solution five equilibriums is gone in the eppendorf pipe.

[0417] freezing in-20 ℃.

Incubation LDL+ sample

[0418] 1. following to each hole adding (all contrasts, sample etc. are all to carry out in triplicate):

The component in each hole of table 17..

Remarks: each Kong Douying has 75 μ g LDL (for example, if the concentration of LDL is 8.6mg/mL, then adding 8.7 μ L).Value can change according to the concentration of LDL and sample.

[0419] 2. on nutator in 37 ℃ of incubation plates 1 hour.

[0420] 3. reads plate with the wavelength of 478nm with reading the plate device.

With the PLC incubation

[0421] 1. 20 μ L PLC solution are all accepted in each hole except that " independent LDL ".

[0422] 2. adds 200 μ L damping fluids to 200 μ L PLC solution.

[0423] 3. of short duration vortex.

[0424] 4. adds the PLC solution of the dilution of 20 μ L as shown to each hole.

[0425] 5. reads plate during 0 minute, 5 minutes, 10 minutes, 30 minutes, 45 minutes and 60 minutes in 478nm.

Embodiment 5

D-4F treatment reduces combining of haemoglobin in the mouse and scavenger thereof and high-density lipoprotein (HDL) Target

[0426] we before reported, and apolipoprotein A-1 (apoA-I) analogies D-4F changes HDL into anti-inflammatory by short inflammation in mouse and monkey.Owing to find that haemoglobin in atherosclerosis animal model (Hb) combines with HDL, and under the atherogenicity condition, helping the short inflammatory matter of HDL, we manage to determine D-4F treatment whether can reduction Hb and urge combining of scorching HDL in atherosclerosis animal model.

Method and result

[0427] data among Fig. 2 show, the HDL that takes from the invalid mouse of apoE (atherosclerotic mouse model) is short scorching.Giving oral apoA-I simulating peptide (D-4F) afterwards, HDL is transformed into anti-inflammatory.As shown in Fig. 3 and 4, consistent with the inflammatory properties change of HDL, the Hemopexin in mice serum and the density loss of hoptoglobin.

[0428] HDL that analyzes mouse shows, when will urging that scorching HDL is parallel and changing anti-inflammatory into, the hoptoglobin among the mouse HDL and the content of Hemopexin also descend, and the apoA-I content among the HDL does not change (Fig. 5).When the level of the Hemopexin of serum and HDL and hoptoglobin reduces, concentration with the non-RBC haemoglobin of the characteristic migration that is similar to normal RBC haemoglobin increases (Fig. 6), the content of hemoglobin of HDL descend (the bar figure of Fig. 7 rightmost side).As shown in Figure 29 and 30, give D-4F and also significantly reduce the transferrins in the HDL-supernatant and the content of myeloperoxidase.

Conclusion

[0429] Hb and removing thing albumen Hemopexin thereof and hoptoglobin are the components of short scorching HDL.D-4F will urge wherein a kind of mechanism that scorching HDL changes anti-inflammatory HDL into and can be by preventing and/or reverse combining of the albumen that contains enzymatic oxidation thing Hb and HDL.Obviously be accompanied by the change of haemoglobin, hoptoglobin, Hemopexin, transferrins and myeloperoxidase with urging material treatment that scorching HDL changes anti-inflammatory HDL in conjunction with HDL.These results show that mensuration described herein can be used for monitoring and improves abnormal function HDL and reduce atherosclerotic therapy.

[0430] it being understood that embodiment described herein and embodiment only are used for purposes of illustration, those skilled in the art can expect that it is included in the scope of the application's the spirit and scope and the claim of enclosing according to multiple modification of the present invention or variation.All publications, patent and patented claim that this paper quotes all are hereby incorporated by in all cases.

Claims (38)

1. one kind is detected in mammal or the quantitative method of short scorching HDL, and described method comprises:

Provide biological sample by described mammal, described sample comprises HDL; With

Detect the albumen that two or more combine with HDL, wherein said two or more albumen are selected from the albumen of m/z ratio about 9.3, the albumen of m/z ratio about 14.9, the albumen of m/z ratio about 15.6, the albumen of m/z ratio about 15.8, the albumen of m/z ratio about 16.2, the albumen of m/z ratio about 16.5, the albumen of m/z ratio about 18.6 and the albumen of m/z ratio about 19.5, wherein detect described two or more albumen that combine with HDL and show that described HDL is short scorching HDL.

2. the process of claim 1 wherein that described detection comprises the described albumen that detects more than 3 kinds.

3. the process of claim 1 wherein that described detection comprises the described albumen that detects more than 4 kinds.

4. the process of claim 1 wherein that described detection comprises the described albumen that detects more than 5 kinds.

5. the process of claim 1 wherein that described detection comprises the described albumen that detects more than 6 kinds.

6. the process of claim 1 wherein that described detection comprises the described albumen that detects more than 7 kinds.

7. the process of claim 1 wherein that described detection comprises the whole 8 kinds of described albumen of detection.

8. the process of claim 1 wherein that described mammal behaves.

9. the process of claim 1 wherein that described mammal suffers from atherosclerotic people for being diagnosed as.

10. the process of claim 1 wherein that described mammal is to be diagnosed as the people who faces the atherosclerotic risk.

11. the process of claim 1 wherein that described detection comprises immunoassays.

12. method that in mammal, detects or quantitatively urge scorching HDL, described method comprises: detect and to contain protoheme and/or in conjunction with the level of albumen protoheme, that combine with described mammiferous HDL, wherein contain protoheme and/or contain protoheme and/or compare in conjunction with the protein level of protoheme and improve the described HDL of expression for urging scorching HDL in conjunction with what exist among the protein level of protoheme and the normal anti-inflammatory HDL.

13. the method for claim 12 wherein saidly contains protoheme and/or comprises that in conjunction with the albumen of protoheme one or more are selected from following albumen: haemoglobin, hoptoglobin, Hemopexin, transferrins, solubility CD163 and myeloperoxidase.

14. the method for claim 12, wherein said method comprise the amount of the haemoglobin that detection combines with HDL and the amount of the hoptoglobin that detection combines with HDL.

15. comprising, the method for claim 14, wherein said method calculate the haemoglobin combine with HDL and the product of hoptoglobin.

16. the method for claim 12, described method comprises also that identical described in the non-lipoprotein fraction that detects blood plasma contains protoheme and/or in conjunction with the level of the albumen of protoheme, wherein in HDL contain protoheme and/or in conjunction with the albumen of protoheme to increasing the described mammiferous HDL of expression and be short scorching HDL containing protoheme and/or comparing in the non-lipoprotein fraction of blood plasma in conjunction with this ratio that exists in the ratio of the albumen of protoheme and the destination object with normal anti-inflammatory HDL.

17. the method for claim 12, wherein said detection comprises immunoassays.

18. the method for claim 12, wherein said detection comprises ELISA.

19. a method that detects or quantitatively urge scorching HDL in mammal, described method comprises:

Detect that one or more that combine with described mammiferous HDL contain protoheme and/or in conjunction with the level of the albumen of protoheme;

Detect that in the described mammiferous blood plasma one or more are described identical to contain protoheme and/or in conjunction with the level of the albumen of protoheme, wherein HDL contains protoheme and/or in conjunction with the albumen of protoheme blood plasma is contained protoheme and/or is short scorching HDL in conjunction with the ratio of the albumen of protoheme greater than the described mammiferous HDL of 1 expression.

20. the method for claim 19 wherein saidly contains protoheme and/or comprises in conjunction with the albumen of protoheme and to be selected from one or more following albumen: haemoglobin, hoptoglobin, Hemopexin, transferrins, solubility CD163 and myeloperoxidase.

21. the method for claim 19, wherein in the described albumen two or more, HDL contains protoheme and/or in conjunction with the albumen of protoheme blood plasma is contained protoheme and/or is short scorching HDL in conjunction with the ratio of the albumen of protoheme greater than the described mammiferous HDL of 1 expression.

22. the method for claim 19, wherein for three kinds in the described albumen or more kinds of for, HDL contains protoheme and/or in conjunction with the albumen of protoheme blood plasma is contained protoheme and/or is short scorching HDL in conjunction with the ratio of the albumen of protoheme greater than the described mammiferous HDL of 1 expression.

23. the method for claim 19, wherein for 1,2,3 in the described albumen or 4 kind, HDL contain protoheme and/or in conjunction with the albumen of protoheme blood plasma is contained protoheme and/or in conjunction with the ratio of the albumen of protoheme greater than the value that is selected from 1,0.9,0.8,0.7,0.6,0.5,0.4,0.3 and 0.2, represent that described mammiferous HDL is short scorching HDL.

24. a method that detects or quantitatively urge scorching HDL in mammal, described method comprises:

Measure the protoheme level that combines with described mammiferous HDL, the protoheme level that wherein combines with HDL raises the described HDL of expression for urging scorching HDL than the protoheme level that combines with protectiveness HDL.

25. the method for claim 24, the level of wherein said rising has the statistically-significant difference meaning in the confidence level that is equal to or greater than 90%, 95%, 98% or 99%.

26. a method that detects or quantitatively urge scorching HDL in mammal, described method comprises:

Measure the iron content of described mammiferous HDL, the iron level of wherein said HDL raises the described mammiferous described HDL of expression for urging scorching HDL than the iron content of normal anti-inflammatory HDL.

27. the method for claim 26, wherein the level of Sheng Gaoing has the statistically-significant difference meaning in the confidence level that is equal to or greater than 90%, 95%, 98% or 99%.

28. a method that detects or quantitatively urge scorching HDL in mammal, described method comprises:

Measure the level that contains ferritin that combines with described mammiferous HDL, wherein the level that contains ferritin that combines with described HDL raises the described mammiferous described HDL of expression for urging scorching HDL than the level that contains ferritin that combines with normal anti-inflammatory HDL.

29. the method for claim 28, wherein the level of Sheng Gaoing has the statistically-significant difference meaning in the confidence level that is equal to or greater than 90%, 95%, 98% or 99%.

30. method that detects or quantitatively urge scorching HDL, described method comprises that measuring HDL consumes nitric oxide production ability, and wherein HDL consumes nitric oxide production ability and has situation, amount or an activity than what normal anti-inflammatory HLD consumed that nitric oxide production ability increases the short scorching HDL of expression.

31. the method for claim 30, wherein said nitrogen monoxide produces with chemical mode.

32. the method for claim 30, wherein said nitrogen monoxide detects by electronic signal.

33. one kind is measured with the inflammatory reaction in mammal is the method that has situation or easy ill physique of the illness of feature, described method comprises:

Implement any one or more mensuration among the claim 1-32, there is described illness in wherein positive test result indication or is its easy ill physique.

34. the method for claim 33, wherein said illness is selected from atherosclerotic, apoplexy, leprosy, pulmonary tuberculosis, systemic loupus erythematosus, polymyalgia rheumatica, polyarteritis nodosa, chorionitis, idiopathic pulmonary fibrosis, chronic obstructive pulmonary disease, asthma, Alzheimer's, chronic renal failure, diabetes, diabetic retinopathy, nephrosis, graft rejection, transplant atherosclerotic, ischemia-reperfusion, adult respiratory system syndrome, congestive heart failure, glomerulitis, metabolic syndrome, multiple sclerosis, sepsis syndrome, drepanocytosis, vascular dementia, Crohn's disease, endothelial dysfunction, the parteriole dysfunction, AIDS, polymyalgia rheumatica, polyarteritis nodosa, chorionitis, idiopathic pulmonary fibrosis, coronary artery calcification, the calcification aortic valve is narrow, osteoporosis, bacterial infection, virus infections, fungal infection, autoimmunity disease and rheumatoid arthritis.

35. a treatment suffers from the inflammatory reaction people's the method for the illness that is feature, described method comprises:

Implement any one or more mensuration among the claim 1-32; And

Be the positive more positive therapy of those object prescriptions of test in described mensuration.

36. a method that detects or quantitatively urge scorching HDL in mammal, described method comprises:

Described mammiferous HDL is contacted with LDL, and detect the gathering of described LDL, wherein LDL gathering level is compared the described mammiferous described HDL of expression that raises for urging scorching HDL with the gathering of the LDL that contacts with normal anti-inflammatory HDL.

37. the method for claim 36, wherein said gathering detects the LDL coalescence rate by spectrophotometric method and measures.

38. using, the method for claim 36, wherein said gathering do not contain albuminous pillar mensuration.

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