MXPA98003985A - Diagnostics for and mediators of inflammatory disorders - Google Patents

Abstract

A method and kit for the diagnosis and quantification of the state of oxidation, and more specifically, the level of lipid peroxidation, of a host is provided that includes contacting a host biological sample with an antibody to an antigen formed by the reaction of a lipid hydroperoxide with a primary amine. This method assesses the risk of, or existence of, oxidative damage in the host. The invention also includes monoclonal and polyclonal antibodies, as well as antibody fragments, optionally in purified or isolated form, which are useful in this method and kit.

Description

DIAGNOSIS OF INFLAMMATORY DISORDERS AND MEDIATORS FOR THEMSELVES This application is related to the area of methods for the diagnosis and treatment of inflammatory disorders, including cardiovascular disorders. Oxidative stress is involved in a number of diseases. One product of the oxidative stress that appears to mediate the disease induced by oxidation is the peroxidated lipid. The formation of lipid peroxides in biological systems is a complex process that can be originated by a variety of means, including enzymes (such as lipoxygenases, cyclooxygenases, peroxidases, and other oxygenases) as well as by non-enzymatic means through generation of reactive, intracellular, extracellular, or cell surface oxygen species. Oxidized lipids of the cell membrane and the extracellular environment can induce profound alterations in cellular behavior. The harmful effects of peroxidized lipids and their degradation products have. been well recognized in the pathogenesis of .aterosclerosis (Herttuala, et al., Journal of Clinical Investigation, 84: 1086-1095 (1989); Gonen, et al., Atherosclerosis, 65: 265-272 (1987); and Jurgnes, et al., Biochim. Biophys P1288 / 98MX V, Acta, 875: 104-114 (1986)). Oxidation has now been considered as a potential risk factor for cardiovascular diseases (Palinski, et al., Arteriesclerosis, 10: 325-335 (1990)). 5 Since the oxidation of lipids is associated with inflammatory and cardiovascular disease states, it is an important medical objective to have a reliable test to quantify and assess the degree of lipid oxidation in a host, and in particular, a human The quantification of lipid hydroperoxides in a host can be based on a direct measurement of the hydroperoxide or on a product of degradation or reaction of the hydroperoxide. Polyunsaturated fatty acids ("PUFA") have at least two double bonds, and 15-20% have three or more double bonds. The unsaturated fatty acids that occur naturally, are never conjugated, the double bonds are separated by at least one methylene group. During the oxidation processes in the lipido hydroperoxides, PUFA double bonds can be conjugated, which is one of the oxidation routes, harmful, major. The initial products of PUFA oxidation appear to be lipid hydroperoxides (LOOH) and free radicals of hydroperoxides of lipid (LOOH- or LOO-), most of which has been P1288 / 98MX altered to contain conjugated double bonds. These products, or their additional reaction or degradation products, have been used to assess the degree of lipid oxidation in a biological sample. For example, LOOH has been reduced in vivo to an alcohol, LOH, which is inert. LOH can be detected by any method that assesses the alcohols, and if the LOH is conjugated, by any method that measures the conjugation of the double bonds. However, none of these tests are very specific, because in a biological sample, there can be any number of alcohols or molecules that contain conjugated double bonds. Alternatively, LOOH can be oxidized by a metal in vivo to form a radical in the double bond (LOOH-), which is a very reactive species. This molecule often breaks down at the location of the oxidation to form two fragments that contain aldehyde, or if it does not decompose, it frequently forms a ketone. The molecule can break down into non-symmetrical parts, and if the LOOH has more than two double bonds, it can form a variety of products. If the LOOH has three or more double bonds, malondialdehyde (MDA) is one of the decomposition products. The presence of malondialdehyde can be assessed with thiobarbituric acid (TBA), which reacts with MDA to produce a compound P1288 / 98MX fluorescent that can be easily measured and quantified. The substances reactive to TBA are referred to as "TBARS". The TBA test is not suitable for use as a diagnostic test for the state of lipid oxidation in the host, because it is prone to erroneous positive indications with other aldehydes, sugars, and amino acids. It is useful only in a research facility that uses carefully controlled samples. In addition, it only measures the amount of lipid peroxides that have three or more double bonds. Another means of measuring the LOOH in a sample is to react it directly with potassium iodide to form a colored complex, (I2) KI, which can be easily measured and quantified. A refinement of this test uses leucomethylene blue, which reacts with LOOH to form a colored product. This assay is sold commercially by Kamiya Biomedical Company. Like the TBA method, this method is useful only for analyzing laboratory samples and can not be used to analyze biological fluids or cell cultures, which may contain numerous cross-reactive substances. The aldehydes generated during the oxidation of lipids react with body fluids and tissues. Aldehydes easily modify protein thiols, lysine, histidine and other residues (Jurgens, et al.

P1288 / 98MX al., Biochemical Journal 265: 605-608 (1990); Jessup, Biochemical Journal 234: 245-248 (1986); Steinbrecher, et al., J. Lipid Res. 25: 1109-1116 (1984)). These proteins modified by the aldehydes are antigenic. Antibodies to these epitopes have provided a useful tool for the detection and localization of aldehyde-modified proteins in the atherosclerotic artery (Boyd, Am. J. Pathol., 135: 815-825 (1989); Haberland, et al., Science. 241: 215-218 (1988), Jurgens, Atheroscler, Thro 13: 1689-1699 (1993)). However, in normal or even atherosclerotic patients, the antigens of the aldehyde-modified protein can typically be found in plasma, but only at low levels in the tissues. Since lipid hydroperoxides do not exist in vivo for a long time because they are inherently unstable, and the resulting aldehydes are one of a number of metabolic pathways of LOOH decomposition and take time to produce, the reaction products of the transient LOOH, alone, with nearby compounds may be present in higher amounts than aldehydes and may represent an attractive diagnostic target. For example, lipid peroxides are highly reactive with the amino groups of proteins, lipids and lipophilic molecules. Its proximity P1288 / 98MX close to membrane proteins and lipoprotein aproproteins may suggest that these modifications may be more relevant than alterations induced by aldehydes in biological systems. The generation of LOOH in a cell membrane or lipoprotein is thus more likely to generate, at least in the initial stages, proteins that are directly modified by LOOH, than proteins that are modified by their extensive degradation products, such as aldehydes . Fruebis, Parthasarathy, and Steinberg, in 1992, published evidence suggesting that a concerted reaction occurs between the lipid peroxide radicals and the free amino groups of the polypeptides or phosphatidylethanolamine to produce fluorescent adducts of unknown structure. Proc. Nati Acad. Sci. USA, 89 ,: 10588-10592 (1992). Fruebis, et al., Incubated linoleoyl hydroperoxide with polypeptides, or an unsaturated phosphatidylethanolamine, alone, the absence of metal ions. The generation of the resulting fluorescent product was many times greater than that generated by the incubation of the polypeptides or phosphatidylethanolamine with an aldehyde, 4-hydroxynonenal (which produces fluorescent Schiff bases). Fruebis, et al, suggested two possible reaction routes, both inhibited by the interaction of a free amino group with the peroxy radical. Reaction routes The theoretical P1288 / 98MX were speculated to produce ring structures of five, six or seven members. The article does not propose an activity for these hypothetical structures, nor does it really identify this proposed structure in vivo. PCT / US95 / 05880, presented by Emory University, describes that polyunsaturated fatty acids ("PUFA") and their hydroperoxides ("ox-PUFA") induce the expression of the endothelial cell surface adhesion molecule, VCAM-1, but not the intracellular adhesion molecule (ICAM-). 1) or E-selectin in endothelial, aortic, human cells, through a mechanism that is mediated by cytokines or other non-cytokine signals. Specifically, it was described that linoleic acid, arachidonic acid, linoleyl hydroxyperoxide, and arachidonic hydroperoxide induce cell surface gene expression of VCAM-1, but not ICAM-1 or E-selectin. Saturated fatty acids (such as stearic acid) and monounsaturated fatty acids (such as oleic acid) do not induce the expression of VCAM-1, ICAM-1 or E-selectin. It was also reported that the induction of VCAM-1, by PUFA and its hydroperoxides of fatty acids is suppressed by dithiocarbamates, including pyrrolidine-dithiocarbamate (PDTC), supporting a conclusion that induction is mediated by a molecule of rusty signal, and that the P1288 / 98MX s Induction is prevented when the oxidation of the molecule is blocked, reversed, or when the signal modified by redox is otherwise prevented from interacting with its regulatory target. PCT / US93 / 10496, also filed by Emory University, discloses that dithiocarboxylates, and in particular, dithiocarbamates, block the induced expression of the endothelial cell surface adhesion molecule, VCAM-1, and are therefore useful in the treatment of cardiovascular disease, which includes atherosclerosis, restenosis after angioplasty, coronary artery disease, and angina, as well as non-cardiovascular inflammatory diseases mediated by VCAM-1. It is an object of the present invention to provide a method and kit for the assessment of the state of lipid peroxidation of a host, a means for evaluating the host-induced risk of oxidation-induced disease. It is also an object of the present invention to provide a commercially viable method and kit for the assessment of lipid peroxidation status of a host. It is another object of the present invention to provide a method and kit for the valuation of P1288 / 98MX status of lipid peroxidation of a host, as a means to evaluate the therapeutic efficiency of medical treatment for oxidation-induced disease. It is yet another object of the present invention to provide a kit for the diagnosis and quantification of the state of lipid peroxidation of a host. It is yet another object of the present invention to provide materials that are useful in the diagnosis and quantification of the state of lipid peroxidation of a host. It is yet another object of the present invention to provide a method for evaluating the ability of a drug candidate to decrease the state of lipid peroxidation of a host. It is another object of the present invention to identify and provide novel mediators of cellular responses. It is still another object of the present invention to provide new therapeutic agents and methods for the mediation of inflammatory responses. It is another object of the present invention to provide imaging agents and methods for the identification and quantification of inflammatory disorders. It is still another object of the present invention P1288 / 98MX provide a method and kit for detecting autoantibodies in the plasma of patients with diseases that cause autoantibodies, including endometriosis.

SUMMARY OF THE INVENTION A method and kit for the diagnosis and quantification of the oxidation state is provided, and more specifically, the level of peroxidation of lipids, from a host, which includes contacting a biological sample of the host with an antibody to an antigen formed by the reaction of a lipid hydroperoxide with a primary amine. This method assesses the risk of oxidative damage, or the existence thereof, in the host. The invention also includes monoclonal and polyclonal antibodies as well as antibody fragments, optionally in purified or isolated form which are useful in this method and kit. This method is based on the discovery that the modification of proteins and other compounds containing primary amines by lipid hydroperoxides results in the generation of antigenic epitopes. Now it has been discovered that these antibodies are present even in normal plasma, and can be raised above the normal levels in the plasma of patients, who suffer from disease states P1288 / 98MX _-. induced by oxidation. This test for the level of lipid peroxides in a biological sample of the patient is superior to those currently available, since it analyzes an antigen that is present in significant amounts in the plasma of the host, and can be performed reliably on samples in alive. In contrast, no proteins modified by aldehyde, antigenic in plasma by antibodies to proteins modified by aldehydes, were detected, except in cases specific such as unstable angina. In addition, antibodies to the modification of proteins and other compounds containing primary amines by lipid hydroperoxides are not cross-reactive with aldehyde-modified proteins. As an illustration of the findings underpinning the present invention, rabbit serum albumin was modified with lipid peroxides to generate a polyclonal antibody. This polyclonal antibody recognizes proteins modified by lipid peroxides, but fails to recognize unmodified proteins. Using immunohistochemistry, it was determined that this antibody recognizes epitopes present in atherosclerotic lesions in humans, the atherosclerotic arteries of monkeys fed cholesterol, and RAW macrophage cells. pre-incubated with lipid peroxide. The antibody was P1288 / 98MX is effective in Western blot analysis and can be used to detect the presence of modified epitopes even in normal plasma. The invention includes methods and kits for analyzing the state of peroxidation of lipids in a host that includes a suitable amount of an antibody that can be immobilized on a solid support and labeled or preferentially labeled with a detectable agent. The antibodies can be immobilized on a variety of solid substrates by known methods. Suitable solid substrate substrates include materials that have a membrane or coating supported by, or bonded to, bars, beads, cups, flat packs, or other solid support. Other solid substrates include cell culture plates, ELISA plates, tubes, and polymer membranes. The antibodies can be labeled or labeled with a detectable agent such as fluorochrome, a radioactive label, biotin, or other enzyme, such as horseradish peroxidase, alkaline phosphatase, and 2-galactosidase. If the detectable agent is an enzyme, it can be supplied with a kit to detect the detectable agent. A preferred means for the detection of a detectable agent employs an enzyme as a detectable agent and an enzyme substrate that changes color upon contact with the enzyme. The case can also P1288 / 98MX contain a means to evaluate the test product, for example, a color diagram, or a numerical reference diagram. The case can be designed to be quantitative or qualitative. It can be used in a scientific laboratory, a medical laboratory, or in the field. In addition, it has been discovered that certain reaction products of lipid hydroperoxides and primary amines exhibit independent biological activity as mediators of cellular responses. The term "oxycin" is used herein to refer to a fluorescent protein or lipid, which is generated by the reaction of a lipid hydroperoxide and a primary amine and which can produce a response from a target cell. Oxicins can be generated extracellularly or can be formed in the cell membrane or even intracellularly to mediate a cellular response. A wide variety of biologically active molecules having primary amines can be converted to oxycins that have biological activity. A non-limiting example of an oxycin is the stable fluorescent product of the reaction between linole hydroperoxide (13-HPODE) and an appropriate amino acid group, such as lysine, in albumin or polylysine, or a compound of small molecular weight such as P1288 / 98 X phosphatidylethanolamine. These oxycins act as potent inflammatory signals that induce gene expression of endothelial VCAM-1 through a mechanism that can be suppressed by selective antioxidants. Other cellular responses that can be produced by oxycycins include, but are not limited to, the generation or activation of MCP-1, IL-1, TNF-α, ICAM, MCSF, and E-selectin. While all the primary amines will react with the lipid hydroperoxides to form an antigenic species that can be used to generate antibodies, to determine the oxidation state of the host, not all biologically occurring peptides or primary amines will form oxycins. This is because what is required to produce an antibody response may differ from what is required to mediate a cellular response. In another embodiment, a method for assessing oxidative damage in a biological sample is provided, which includes the steps of: (i) isolating an antigen formed by the reaction in lipid hydroperoxide with a primary amine; and then (ii) identify the primary amine. The nature of the amine can provide information, in certain circumstances, with respect to the location of the oxidant disorder.

P1288 / 98 X BRIEF DESCRIPTION OF THE FIGURES Figure 1 is a bar graph graph of the recognition of rabbit serum albumin and rabbit serum albumin modified by lipid hydroperoxide by the antibody of Example 4, as measured protein in nanograms against optical density at 405 nm. Figure 2 is a bar graph plot of the LDL recognition oxidized by the antibody of Example 4, as a concentration measured in micrograms against units of optical density. Figure 3 is bar graph plot of the dose-dependent induction of VCAM-1 (A) and ICAM-1 (B) by oxidized bovine serum albumin, expressed as a percent of the maximum signal induced by TNF -to. Figure 4 is a schematic diagram of a large scale synthesis of oxycin modified by 13-HpODE employing a lipid peroxide generation system. The target protein was placed in a 10 kDa molecular weight cut-off membrane and the 13-HpoDE generation system was changed every 8-16 hours. Figure 5 is a Western blot diagram showing that sLO and linoleic acid are minimum requirements for the formation of oxSLO. Figure 6 is a Western blot diagram showing the detection of samples treated with 13- P1288 / 98MX HpODE immunoreactive after 96 hours in a large-scale reaction. Figure 7 is a graph of the induction of ICAM-1 by sLO treated with 13-HpODE as a percent of the induction by TNF over time. Figure 8 is an HPLC chromatogram indicating that the sLO modified by 13-HpODE is more hydrophobic than the untreated sLO. Figure 9 is a graph of the induction of ICAM-1 by the oxSLO fractions collected by gel filtration chromatography. The graph indicates that fraction ten induces ICAM-1 more strongly. Figure 10 is a graph of absorbance (280 nm) against the oxSLO fractions collected by gel filtration chromatography indicating that the biologically active fractions 9 and 10 comprise a portion of the total protein decomposed by gel filtration chromatography. Figure 11 is a bar graph graph of the induction of ICAM-1 by soy lipoxygenase, linoleic acid, and oxidized soy lipoxygenase as a percentage of ICAM induction by TNF. Figure 12 is a bar graph plot of ICAM-1 induction (as a function of percent TNF induction) by soy lipoxygenase and P1288 / 98MX oxidized soy lipoxygenase, synthesized by the large-scale method in human aortic endothelial cells (HAEC). Figure 13 is a bar chart graph of the ICAM-1 induction (as a function of the induction of percent TNF induction) by soy lipoxygenase and oxidized soy lipoxygenase indicates that oxSLO activates cell surface ICAM expression in a dose-dependent manner. Figure 14 is a bar graph plot of ICAM-1 induction (was a function of percentage of induction by TNF) by oxidized soy lipoxygenase. The graph indicates that oxSLO induced ICAM to a degree greater than that induced VCAM. Figure 15 is a Northern blot indicating that oxSLO, but only only induces the accumulation of VCAM, ICAM, and MCP-1 mRNA in human aortic endothelial cells (HAEC). Figure 16 is a Northern blot indicating the rapid accumulation of VCAM, ICAM and MCP-1 in endothelial, aortic, human cells.

DETAILED DESCRIPTION OF THE INVENTION I. Method for the Assessment of the Host State Oxidant.

P1288 / 98MX A. Generation of Antibody. (i) Lipido hydroperoxide. A lipid is an organic substance, oily or greasy, insoluble to water that can be extracted from cells and tissues by non-polar solvents such as chloroform and ether. The most abundant forms of the lipid is a triacylglycerol. Fatty acids are building blocks characteristic of lipids. A fatty acid is a long chain organic carboxylic acid typically having from four to twenty carbon atoms. Fatty acids are typically not present free or uncombined in cells or tissue, but are actually bound in a larger molecule such as a lipoprotein, albumin, triacylglycerol, a wax, phosphoglyceride (for example, a phosphatidylethanolamine, phosphatidylcholine, phosphatidylserine, phosphatidylnositol or cardiolipin), a sphingolipid (e.g., sphingomyelin, cerebrosides, or gangliosides), or sterol and its fatty acid esters. Materials generically referred to as ceroids and lipofuscin include fatty acids. As used herein, the term polyunsaturated fatty acid (a PUFA) refers to a fatty acid (typically of 8 to 24 carbon atoms) having at least two alkenyl bonds, and includes but is not limited to P1288 / 98 X a, linoleic acid, (C18?), Linoleic (C18?), Arachidonic (C2O?) And eicosatrienoic (C20?) • A lipido hydroperoxide, as the term is used herein, refers to: 1. (i) a polyunsaturated fatty acid; or (ii) a molecule containing a polyunsaturated fatty acid residue; 2. in which at least one of the alkenyl bonds has been converted to a hydroperoxide. The non-limiting examples of lipid hydroperoxides are: -HPETE 13-HPODE Lipid hydroperoxides can be formed in vivo enzymatically from polyunsaturated fatty acids or lipids containing a PUFA residue P1288 / 98MX by lipoxygenase, cyclooxygenase, peroxidase, or other oxygenase enzymes as well as by a non-enzymatic means through the generation of oxygen species, reactive intracellular, extracellular or cell surface. The lipid peroxides can be generated chemically by oxidation of a polyunsaturated fatty acid or lipid containing a PUFA residue using standard methods. It appears that a wide variety of lipid hydroperoxides can be used to generate an antibody that reacts with the selected amine to produce an epitope. It seems that this reaction is completely non-selective; virtually any lipid hydroperoxide will react antigenically with a primary amine. (ii) Primary Amina. Any primary amine can be used to react with the lipid hydroperoxide which forms an antigenic material. It has been found that the more hydrophobic and nucleophilic the amine, the easier the reaction is. For example, benzylamine reacts more rapidly with a lipid hydroperoxide than ethylamine. With this base, all amines such as alkyl amines of 1 to 3 carbon atoms, and ammonia are not preferred. For administration to a host, a preferred amine is one that exhibits little toxicity either alone or after administration.

P1288 / 98MX reaction with the lipid hydroperoxide. The amine can be attached to a larger molecule, for example, a hapten, if desired, to increase an antigenic response. Synthetic versions of molecules that contain primary amines, which occur naturally, such as peptides and proteins, are preferred. Examples include peptides or proteins having a terminal amino group, a lysine residue (e.g., albumin and polylysine) or a histidine residue. Also suitable are phosphatidylethanolamine, phosphatidylserine, and amine terminated hormones. That the reaction of the lipid hydroperoxide and the proteins occurs naturally in vivo is demonstrated by the fact that commercially available proteins such as bovine serum albumin contain the epitope lipido / amine hydroperoxide. Because of this, it is preferred to use a synthetic peptide or protein for the preparation of the antigen, and finally, an antibody, for purposes of quality control. (iii) Reaction of Lipido Hydroperoxide with the primary amine. The ability of a lipid hydroperoxide to react with a protein was demonstrated by Fruebis, Parthasarathy and Steinberg in Proc. Nati Acad. Sci. USA, P1288 / 98MX Volume 89, pp.10588-10592, November 1992 Medical Sciences. In this article, a lipid hydroperoxide was prepared by reacting linoleic acid or a linoleic acid phospholipid with soy lipoxygenase and then the hydroperoxide was incubated with polypeptides in the absence of metal ions. The general approach and experimental conditions set forth in the article can be used to prepare a wide variety of lipid / amine hydroperoxide adducts. The chemical reaction between the lipid hydroperoxide and the primary amine can be carried out in a laboratory or manufacturing plant at room temperature, pH neutral or almost neutral, simulating in vivo conditions. However, aqueous conditions are preferred, the reaction can be run in an organic solvent if the reactants and products are sufficiently soluble in the organic solvent. LOOH may be less stable in an organic solvent than in an aqueous solvent. The reaction temperature can be raised as desired, up to the boiling point of the solvent. The progress of the reaction can be inspected by fluorescence spectrophotometry. The excitation of the product is typically between 330 and 360 and the emission typically between 420 and 450. The reaction is terminated when there is no further increase in the fluorescence of the sample. In a typical reaction, the P1288 / 98MX lipid hydroperoxide and the amine are present in a ratio of approximately 1.5: 1, however, other ratios may be used as desired to optimize performance or for other purposes. (iv) Generation and Use of Antibodies. The method and kit for assessing the state of lipid peroxidation of a host can include either monoclonal or polyclonal antibodies or antibody fragments. The term "antibody", as used herein, includes monoclonal and polyclonal antibodies as well as antibody fragments that bind specifically, but reversibly to the described epitope. It is preferred that the antibody or antibody fragment be derived from a monoclonal antibody or antibody fragment. The preparation of monoclonal and polyclonal antibodies to an antigen formed by the reaction of a lipid hydroperoxide with a primary amine can be achieved using any known method, for example, those described in Zola, H. (1988) "Monoclonal Antibodies - A manual of techniques "CRC Press, and Antibodies: A Laboratory Manual, Harlow & Lane, Cold Spring Harbor (1988), incorporated herein by reference.

P1288 / 98 X In one embodiment, mainly for laboratory use, the animals are immunized with the antigen, and preferably an adjuvant. The booster immunizations are optionally continued with the antigen in PBS and mixed with the adjuvant at periodic intervals. The animals are then bled after immunizations. After the removal of clots and waste, the serum can be analyzed by ELISA. Monthly, or other periodic titles can be obtained after the initial immunization. Alternatively, the spleens are harvested from animals immunized with the antigen, and preferably an adjuvant. Spleen cells are separated and fused with immortal myeloma cells using polyethylene glycol. The fused hybridoma cells are selected and cultured in vitro. Fluids from the cell culture of hybridoma cells are analyzed for the presence of hybridoma antibodies having the desired specificity. The selection technique for identifying the appropriate monoclonal or polyclonal antibody is an important aspect in obtaining the desired immunospecificity. Hybridoma cells can be analyzed for the presence of antibodies specific for the antigen, for example, with an ELISA carried out by normal methods. In general, if the hydroperoxide product of P1288 / 98MX selected lipid and the selected amine contains an antigen with an epitope, will cause the production of a monoclonal antibody or a polyclonal antibody. If multiple antigens or an antigen with multiple epitopes are used, a polyclonal antibody will be obtained. For example, when LOOH is reacted with a serum albumin, an antigen with multiple epitopes is produced, because the albumin has a number of lysine residues at different locations in the molecule. This antigen will cause the production of polyclonal antibodies. In contrast, if the reaction product of a simple amine with LOOH is used as the antigen, that antigen can induce the formation of monoclonal or polyclonal antibodies. For example, if a phosphatidylethanolamine containing an unsaturated lipid moiety is selected, it can act as both the lipid component and the amine component. This molecule can cause the production of monoclonal or polyclonal antibodies. It has been observed that proteins from natural sources may contain a small amount of the LOOH / amine antigen. Because of this, for quality control purposes, it is preferred to react a synthetic peptide or a synthetic amine containing molecule with LOOH to form an antigen for the production of antibodies.

P1288 / 98 X The heavy variable (VH) and light variable (VL) domains of the antibody are included in the recognition of the antigen, a fact recognized first by the previous experiments with protease digestion. Additional confirmation was found by the "humanization" of rodent antibodies. The variable domains of rodent origin can be fused to constant domains of human origin such that the resulting antibody retains the antigenic specificity of the antibody of rodent origin (Morrison et al., (1984) Proc, Nat. Acad. Sci. USA 81 , 6851-6855). The "CDR graft" can be used to humanize the rodent's antibodies. Additionally or alternatively, the recombinant monoclonal antibodies can be "primatized", ie, the antibodies formed in which the variable regions are derived from two different species of primates, preferably the variable regions of the antibody from the macaque monkey. , and the constant regions of the human. The advantages of these antibodies include high homology to human immunoglobulin, in the presence of human effector functions, reduced immunogenicity and longer serum half-life (Newman et al (19929 Biotechnology 10, 1455) .The specific region for the antigen can be express as part of a bacteriophage, for example, using P1288 / 98MX the technique of McCafferty et al. (1990) Nature 348: 552-554. The antibody-like molecules of the invention can be selected from the phage display libraries using the methods described in Griffiths et al. (1993) EMBO J. 12, 725-734, in which the antigens are immobilized and used to select phages. Also, appropriate cells cultured in monolayers and either fixed with formaldehyde or glutaraldehyde or unfixed can be used to bind the phages. The irrelevant phages are washed and the bound phages are recovered by breaking their binding to the antigen and reamplifying in bacteria. This selection process by amplification is done several times to enrich the phage population for those molecules that are the antibody-like molecules of the invention. Antibody fragments include Fab-like molecules (Better et al. (1988) Science 240, 1041) Fv molecules (Sketra et al (1988) Science 240, 1038); single chain Fv molecules (ScFv) where the partner domains VH and VL are linked via a flexible oligopeptide (Bird et al. (1988) Science 242, 243; Huston et al. (1988) Proc Nati. Acad. Sci. USA 85, 5879) and single domain antibodies (dAbs) comprising isolated V domains (Ward et al (1988) Nature 341, 544). A general review of the techniques included in the P1288 / 98MX synthesis of the antibody fragments that maintain their specific binding sites is found in Winter & Milstein (1991) Nature 349, 293-299). Antibody fragments, including, but not limited to, Fab, (Fab) 2, Fv, ScFv and dAb fragments are included in this invention. Antibody-like molecules can be prepared using the recombinant DNA techniques of WO 84/03712. There may be advantages to using antibody fragments, instead of whole antibodies. The Fab, Fv, ScFv and dAb antibody fragments can all be expressed and secreted from E. coli, thus allowing the easy production of large amounts of the fragments. Complete antibodies, and fragments F (ab ') 2 are "bivalent". By "bivalent" it is meant that antibodies and F (ab ') 2 fragments have two antigen combining sites. In contrast, Fab, Fv, ScFv and dAb fragments are monovalent, having only one antigen combination site. The "antibody engineering" technique is advancing rapidly, as described in Tan L.K. and Morrison, S.L. (1988) Adv. Drug Deliv. Rev. 2: 129-142, Williams, G. (1988) Tibtech 6: 36-42 and Neuberger, M.S. et al (1988) 8th International Biotechnology Symposium Part 2, P1288 / 98MX 792-799 (all of which are incorporated herein by reference), and is well suited for preparing antibody-like molecules derived from the antibodies of the invention. Antibodies can be used for a variety of purposes that are related to the study, localization, isolation, purification of the antigen to which they bind in a specific manner. In particular, the antibody can be used in the imaging and treatment of cells that exhibit the antigen. The antibody of the invention can be coupled to a scintigraphic radiolabel, an isotope radio, a cardiovascular, anti-inflammatory drug, or another drug, an enzyme for converting a prodrug into a cardiovascular drug, an anti-inflammatory drug, or another desired drug, together with that prodrug , or another cellular process that mediates the compound. These conjugates have a "binding portion", which consists of the antibody of the invention, and a "functional portion", which consists of the radiolabel, drug or enzyme, etc. The binding portion and the functional portion can be separated by a linking portion. The binding portion and the functional portion of the conjugate (if there is also a peptide or polypeptide) can be linked together by any of the conventional ways of the crosslinking peptides, such as P1288 / 98MX those generally described in 0 'Sullivan et al. (1979) Anal. Biochem. 100, 100-108. For example, one portion can be enriched with thiol groups and the other portion reacted with a bifunctional agent capable of reacting with those thiol groups, for example, an N-hydroxysuccinimide ester of iodoacetic acid (NHIA) or N-succinimidyl- 3- (2-pyridylthio) propionate (SPDP). Amide and thioether bonds, for example achieved with the ester of m-maleimidobenzoyl-N-hydroxysuccinimide, are generally more stable in vivo than disulfide bonds. Alternatively, if the binding portion contains carbohydrates, as would be the case for an antibody or some antibody fragments, the functional portion can be linked via the carbohydrate moiety using link technology in EP 0 088 695. functional portion of the conjugate can be an enzyme for converting a prodrug into a drug, using technology similar to that described by Bagshawe and colleagues (Bagshawe (8987) Br. J. Cancer 56, 531; Bagshawe et al (1988) Br. J Cancer 58, 700; WO 88/07378). It may not be necessary for the entire enzyme to be present in the conjugate, but, of course, it must be present in the catalytic portion. You can use the so-called "abzymes", in which a monoclonal antibody is promoted to a compound included in the reaction that P1288 / 98MX one wants to catalyze, usually the reactive intermediate state. The resulting antibody can then function as an enzyme for the reaction. The conjugate can be purified by size exclusion or affinity chromatography, and tested for dual biological activities. Antigen immunoreactivity can be measured using an enzyme-linked immunosorbent assay (ELISA) with the immobilized antigen and in a live cell radioimmunoassay. An enzyme assay for β-glucosidase can be used using a substrate that changes in absorbance when glucose residues are hydrolyzed, such as oNPG (o-nitrophenyl-β-D-glucopyranoside), which releases 2-nitrophenol which is measured spectrophotometric at 405 nm. The stability of the in vitro conjugate can be tested initially by incubating at 37 ° C in serum, followed by FPLC analysis of size exclusion. In vivo stability can be tested in the same way in mice by analyzing the serum at various times after injection of the conjugate. In addition, it is possible to radiolabel the antibody with 125 I, and the enzyme with 131 I before conjugation, and to determine the biodistribution of the conjugate, the free antibody and the free enzyme in mice. Alternatively, the conjugate can be P1288 / 98 X produced as a fusion compound by recombinant DNA techniques, whereby one DNA length comprises respective regions coding for the two portions of the conjugate either adjacent to each other or separated with a region encoding a linker peptide that does not destroy the desired properties of the conjugate. Conceivably, the two functional portions of the compound can be completely or partially translatable. The DNA is then expressed in a suitable host in known ways. The antibodies or their conjugates can be administered in any suitable manner, usually parenterally, for example intravenously intraperitoneally, in non-pyrogenic, sterile, normal diluent and carrier formulations, for example isotonic saline (when administered intravenously ), once the conjugate has bound to the target cells and has been cleared from the bloodstream (if necessary), which typically takes a day or so, if desired, a prodrug can usually be administered as a dose individual by infusion, or the objective area is formed into an image. If needed, because the conjugate may be immunogenic, cyclosporin or some other immunosuppressant may be administered to provide a longer period for treatment, but this P1288 / 98 X will usually not be necessary. The synchronization between the administrations of the conjugate and a prodrug can be optimized in a non-inventive manner since the ratios of the diseased tissue / normal tissue of the conjugate (at least after the intravenous distribution) are higher after several days, whereas in At this time, the absolute amount of the conjugate bound to the target tissue, in terms of the percent of dose injected per gram, is lower than in previous moments. Therefore, the optimal interval between the administration of the conjugate and a prodrug will be a compromise between the maximum tissue concentration of the antibody / enzyme and the best distribution ratio between the affected and normal tissues. The dose of the conjugate will be chosen by the doctor according to the usual criteria. The dose of any conjugate will be chosen according to the normal criteria, particularly with reference to the type, condition and location of the target tissue and the weight of the patient. The duration of the treatment will depend in part on the speed and degree of any immunoreaction to the conjugate. The functional portion of the conjugate, when the conjugate is used for the diagnosis of inflammation, usually comprises and may consist of a radioactive atom for scintigraphic studies, for example, P1288 / 98 X technetium 99m (99m.Te) or iodine-123 (1 _? "3I), or a spinmark for nuclear magnetic resonance imaging (nmr) (also known as resonance imaging) magnetic, mri), such as iodine-123 again, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese or iron.When used in a conjugate for selective destruction of tissue, the functional portion may comprise a highly radioactive atom, such as iodine-131, rhenium-186, rhenium-188, yttrium-90 or lead-212, which emit enough energy to destroy nearby cells. other labels or labels can be incorporated into the conjugate in known ways, for example, the label can be used by synthesizing suitable reagents containing, for example, fluorine-19 instead of hydrogen.The t, al-, marks as ?? mrTpC - ,, 123-I-, 186, R -., H, 188R -, and llltIn can be bound via peptides of cysteine residues The yttrium-90 can be bound via a lysine residue. The IODOGEN method (Fraker er al. (1978) Biochem. Biophys Res. Commun. 80: 49-57) can be used to incorporate iodine-123. "Monoclonal Antibodies in Immunoscintigraphy" (Chatal, CRC Press 1989) describes other methods in detail.

B. Method for Detecting the Antigen The invention includes a method for detecting P1288 / 98 X antigens formed by the reaction of the lipid hydroperoxide with a primary amine. This test can be done on a laboratory sample or on a biological sample. As used herein, the term "biological sample" refers to a sample of tissue or fluid isolated from a host, typically a human, including, but not limited to, for example, plasma, serum, spinal fluid, lymphatic fluid, ocular fluid, and skin tissue, peritoneal fluid, urine, bile, tracts of the cardiovascular system, respiratory, intestinal, genital, uteral, or central nervous system, tears, placenta, umbilical cord, milk, endothelial cells, cells endometrial cells, epithelial cells, tumors and organs and also samples of cell culture constituents in vitro including, but not limited to, the conditioned medium resulting from the growth of cells in the cell culture medium. According to the present invention, the oxidation state, and in particular, the state of lipid peroxidation, of a host is evaluated by testing a biological sample for the presence of the level of either: (I) antigens that bind to an antibody formed as described in Section IA; or (ii) antibodies that are cross-reactive by antibodies formed as described in Section I. A. It has been determined that each P1288 / 98MX host, even healthy people, have both (I) and (ii) in their tissue and fluid in varying amounts. Actually, commercially available bovine serum albumin containing (I) and in this way will exhibit a positive response to exposure to antibodies formed in accordance with Section IA Therefore, the simple existence of (I) or (ii) in a host is not in itself an indication of a disease state induced by oxidation. Actually, the level of (I) or (ii) in a host should be considered in view of the population and individual standards. In this way, the diagnosis is completely similar to that for cholesterol, where the level of a host is compared to statistical standards. In addition, as with cholesterol tests, in certain cases, changes at individual levels can provide diagnostic information more important than the individual level itself. Typically, if a cholesterol test indicates that the patient may be at risk for heart disease, a second-level test group is performed to obtain more diagnostic information, including HDH, LDL, and triacylglycerol, in serum, absolute. At the same cholesterol level, different ratios of each component provide different diagnostic and prognostic implications. The present P1288 / 98MX test is similar to this, since if the level of lipid hydroperoxides indicates that the patient is at risk for a disorder, or has an inflammatory disorder, including a cardiovascular disorder, it may be appropriate to carry out a series of second level tests both to confirm the diagnosis and to obtain more information regarding the disorder. These second level tests can evaluate the presence and / or quantity of other substitute markers of the suspected disease. Substitute markers include, but are not limited to, LDL, HDL, triacylglycerides, L (p) A (formed by the reaction of LDL with protein a), circulating, soluble VCAM 1 levels are elevated in the serum of patients with inflammatory and systemic diseases), ICAM, E-selectin, MCSF, G-CSF, TNF-a, IL-1, and MCP-1. Substitute markers of other specific diseases are known and commercially available in assays for these markers, or are available from a medical laboratory. Alternatively, antibodies specific for the target proteins and the oxidized substitute labels can be used to analyze specific components of the modified sample by lipid peroxide, alone, to obtain more information with respect to which primary amines are included in the P1288 / 98MX oxidation process. This provides additional specificity and sensitivity for the diagnosis of atherosclerosis and other inflammatory diseases, and can provide information for the determination of which lipid peroxidation materials, if any, may be acting as oxycins. Using normal double antibody detection techniques (eg, immunoprecipitation and Western blotting), amines modified by lipid peroxide, including LDL, HDL, triacylglycerides, L (p) A, VCAM, ICAM, E-selectin, MCSF, G-CSF, TNF-a, IL-1, and MCP-1 can be identified and quantified from the tissue or fluid. This component in the complete lipid peroxide state may represent a sensitive and specific marker for inflammatory, vascular cases mediated by lipid peroxide, characteristic of atherosclerosis. Similarly, lipid peroxide modification of apo-B can be detected using both an apo-B antibody and an LOOH / amine antibody. Any test that measures the binding of an antigen to an antibody can be used to evaluate the level of the antigen or antibody in the biological sample of the host according to the present invention. A number of these tests are commonly known and commonly used. ] $ a immunocytochemistry and immunohistochemistry are P1288 / 98MX techniques that use antibodies to identify antigens on the surface of cells in solution, or on sections of tissue, respectively. The immunocytochemistry is used to quantify the formations of individual cells according to the surface markers. Immunohistochemistry is used to locate populations of particular cells or antigens. These techniques are also used for the identification of antibodies, using tissues or cells that contain the presumed autoantigen as a substrate. The antibodies are usually identified using antibodies conjugated with enzymes to the original antibody, followed by a chromogen, which deposits a colored terminal product, insoluble in the cell or tissue. Another common method of evaluation is a radioimmunoassay, in which radiolabeled reagents are used to detect the antigen or antibody. The antibody can be detected using plates sensitized with antigen. The test antigen is applied and detected by the addition of a radiolabelled ligand specific for that antibody. The amount of ligand bound to the plate gave the amount of the test antibody. This test can be reversed to test the antigen. The variations of the radioimmunoassays are competition RIA, RIA direct binding, RIA capture, RIA intercalation, and assay Immunoradiometric P1288 / 98MX (RMA). Enzyme-linked immunosorbent assays (ELISA) are a group widely used in techniques for detecting antigens and antibodies. The main ones are analogous to those of radioimmunoassays except that an enzyme is conjugated to the detection system instead of a radioactive molecule. The typical enzymes used are peroxidase, alkaline phosphatase, and 2-galactosidase. These can be used to generate colored reaction products from the colorless substrates. The color density proportional to the amount of the reagent under investigation. These tests are more convenient than the RIA, but less sensitive. The Western blotting method (immunoblotting) is used to characterize unknown antigens. The components of the biological sample are separated by gel electrophoresis. The SDS gels are separated according to the molecular weight and the IEF gels separate the samples according to the loading characteristics. The separated proteins are transferred to membranes (transferred) and identified by immunocytochemistry. Frequently used but more suitable methods of evaluation include Farr assay (in which the radiolabeled ligands bind to the specific antibody, and detect it, in solution, which is P1288 / 98MX precipitate and quantify), precipitin reactions (in which antibodies and antigens are cross-linked in large lattices to form insoluble immunocomplexes, only works if the antigen and the antibody are present in sufficient quantities, or almost equivalent, and where there are enough available epitopes to form a lattice or grid); nephelometry (measures immunocomplexes formed in solution by their ability to disperse light); immunodiffusion (detects antigens and antibodies in agar gels); Countercurrent electrophoresis (similar to immunodiffusion, except that it is used in an electric current to boost the antibody and the antigen together, useful for low concentrations of antigen antibody); radial, individual immunodiffusion (SRID) (quantifies antigens by allowing them to diffuse out of a well into a gel containing antibody, technique that can be reversed by spreading unknown antibody solutions in an antigen-containing well), rocket electrophoresis (similar to SRID, except that the test antigen is moved in the gel by an electric field); and immunofluorescence (similar to immunochemistry except that it uses fluorescence instead of enzyme conjugates). The antibody used to contact the body fluid sample is preferably immobilized on a solid substrate. The antibody is P1288 / 98MX can mobilize using a variety of media, as described in Antibodies: A Laboratory Manual, supra. Suitable solid substrates include those having a membrane or coating supported by, or attached to, bars, synthetic glasses, agarose beads, cups, flat packs, or other solid supports. Other solid substrates include cell culture plates, ELISA plates, tubes and polymer membranes. Means for labeling antibodies with detectable agents are also described in Antibodies: A Laboratory Manual, cited supra. The amount of antigen in the biological sample of the host can be determined by any means associated with the selected assay. For example, the selected immunoassay can be carried out with increasing, known amounts of antigen to produce a normal curve or color graph and then the amount of the test antigen can be determined by comparing the result of the test to the normal curve or graph that correlates the amount of the antigen-antibody complex with known amounts of antigen. The amount of antigen determined to be present in the biological sample of the host can be used to evaluate the condition of the patient in a number of ways. First, the level of the antigen can be compared to a standard of exploration based on statistical data. Second, the P1288 / 98MX antigen level, can be considered in view of the patient's own history of the level of antigen.

C. Cases. This invention also includes a kit for diagnosing the state of lipid peroxidation in a sample. The kit optimally includes an antibody that reacts with an epitope formed by the reaction of a lipid hydroperoxide with an amine found in a biological sample. The antibodies are present in the kit in an amount effective to bind to, and substantially detect, all of the antigen in the sample. The preferred kit contains sufficient antibody to bind substantially all of the antigen to the sample in about 10 minutes or less. The antibody can be immobilized on a solid support, and then labeled with a detectable people, as described above. The kit optionally contains a means for detecting the detectable agent. The antibody is labeled with a fluorochrome or radioactive label, no means to detect the environment will typically be provided, since the user would expect to have the appropriate spectrophotometer, the scintillation counter or the microscope. If the detectable agent is an enzyme, a means for detecting the detectable agent can be supplied with the kit, P1288 / 98MX and will typically include a substrate for the enzyme in sufficient quantity to detect the entire antigen-antibody complex. A preferred means for detecting a detectable agent is a substrate that is converted by an enzyme into a colored product. A common example is the use of horseradish peroxidase enzyme with 2,2'-azino-di- [3-ethyl-benzothiazoline] sulfonate (ABTS). The kit may optionally contain a lysing agent that uses the cells present in the body fluid sample. Suitable lysing agents include surfactants such as Tween-80, Nonidet P40, and Triton X-100. Preferably, the lysis agent is mobilized in the solid support together with the antibody. The case may also contain a buffer solution for washing the substrate between the steps. The buffer solution is typically a physiological solution such as a phosphate buffer, physiological saline solution, citrate buffer, or Tris buffer. The kit can include different concentrations of a preformed antigen to calibrate the assay. The kit may additionally contain a visual or numerical representation of amounts of the antigen in a normal assay calibrated for reference purposes. For example, if an assay that produces a product is used P1288 / 98MX colored, a sheet can be included that provides a representation of the increasing intensities, associated with different amounts of antigen. Alternatively, the kit can include an antigen formed by the reaction of a lipid hydroperoxide with an amine for evaluation of the level of the antibody in the biological sample. The kit can optionally include two antibodies in the detection system. The first antibody that is present in small amounts is specific for the antigen to be analyzed. The second antibody provided in higher amounts is used to detect the first antibody. For example, a rabbit antibody can be used to detect the LOOH / amine antigen, and then an anti-rabbit IgG antibody can be used to detect the bound rabbit antibody. Goat antibodies and anti-antibodies are also commonly used. As a non-limiting example, it is provided in a kit for detecting the state of lipid peroxidation of a patient, which includes a rabbit antibody specific for the LOOH / amine antigen, the anti-rabbit IgG antibody in sufficient amounts to detect the first bound antibody, a conjugated enzyme for the second antibody and a substrate for the P1288 / 98MX enzyme that changes color on exposure to the enzyme.

D. Use of the Diagnostic Cases. The diagnostic method and kits described herein can be used to evaluate a wide variety of medical conditions that are mediated by oxidation-induced events, and in particular, by lipid hydroperoxides. For example, the presence and high concentration of the lipid hydroperoxides, or the reaction products of a lipid hydroperoxide with a primary amine, can be used as an initial diagnostic marker for cardiovascular disease, which includes atherosclerosis, inflammatory disease, endometriosis, nephritis, glimerol, preeclampsia, central nervous system disorders mediated by lipid peroxidation, Alzheimer's disease psoriasis, asthma, atopic dermatitis, solid tumors, Kaposi's sarcoma, neurodegenerative disease, inflammatory bowel disease (Crohn's disease), rheumatoid arthritis, and reperfusion by ischemia. The biological sample can be selected based on the disease to be treated. For example, a specimen of tissue in the afflicted area may be optimal if the disease is tissue specific. If this method and kit indicates an abnormally increased level of lipid peroxidation in P1288 / 98 X the host, additional tests may be used to confirm the specific disorder.

E. Examples of the Rating of the Host State Oxidant. Rabbit serum albumin (RSA), soy lipoxygenase, linoleic acid, goat anti-rabbit IgG conjugated with alkaline phosphatase, octylglucoside, tetramethoxypropane and p-nitrophenyl phosphate were obtained from Sigma Chemical Company (St. Louis , Missouri). The nonenol was purchased from Aldrich Chemical Company (Milwaukee, Wl). Nonfat milk powder was purchased from Bio-Rad Chemical Company (Hercules, CA). 96-well microtiter plates and Tween 20 were purchased from Fisher Chemical Company (Pittsburgh, PA). A peptide of 20 amino acids of the sequence, YVTKSYNETKIKFDKYKAEKSHEDEL (where K means lysine) (SEQ ID No. 1) was obtained.

Example 1. Preparation of Modified Protein by Lipido peroxide. The linoleic acid was converted to linoleate 13-hydroperoxy by treatment with soy lipoxygenase (SLO) as described by Fruebis, Parthasarathy, and Steinberg, supra. The hydroperoxide of linoleic acid is P1288 / 98MX reacted immediately with immunoglobulin-free RSA and incubated at 37 ° C for 2 days. In a typical reaction, 100 nmol of linoleic acid were treated with 30 units of SLO in 1 ml of phosphate buffered saline (PBS) and the reaction was monitored by measuring the increase in solution at 234 nm. Usually, the reaction is finished in the space of 30 minutes. Then, the lipid hydroperoxide (13-HPODE) was treated with 100 μg of the lipid, IgG, another protein-free albumin in the presence of 50 μM EDTA for two days at 37 ° C. The formation of fluorescent products was established by measuring the fluorescence under the oxidation wavelength of 330 nm and the emission at 430 nm. The product was extracted with chloroform and methanol by the method of Bligh and Dyer (Bligh, et al., Can. J. Biochem. Physiol. 37: 911-917 (1959)) to remove the unreacted LOOH and then washed several times. sometimes with cold acetone, cooled with ice. The final product, RSA modified by LOOH (LOOH / RSA) was soluble in aqueous solution and had fluorescent characteristics similar to those of low density, oxidized lipoproteins (Ox-LDL). The generation of LOOH as well as the modification of the protein was carried out in the absence of any of the metals added to limit the formation of aldehydes.

P1288 / 98 X Example 2. Preparation of Oxidized LDL. LDL was isolated from the heparinized plasma of normal human donors using a Beckman TL-100 ultracentrifuge for mesa and a TLA-100.4 rotor (Santanam, et al., J. Clin.Invest 95: 2594-2600; nineteen ninety five). An individual spin gradient was used to purify the LDL from the albumin contamination. The isolation was completed within three hours of obtaining the plasma. Isolated LDL was dialyzed against phosphate buffered saline (PBS) at 4 ° C (200 x volumes) for 6 hours. The purity of isolated LDL was confirmed by the presence of an individual band on agarose gel electrophoresis and of intact apolipoprotein B on polyacrylamide gel electrophoresis with sodium dodecylsulfate. Isolated LDL (100 ug / ml) was incubated in phosphate buffered saline with 5 μm copper and incubated on a 50 mm plate at 37 ° C. After 24 hours of incubation, the solution was transferred to a glass tube and delipidated by the extraction of the lipids by the method of Bligh and Dyer. The delipidated LDL protein was dissolved in octylglucose (100 μl of 10 mg / ml were added to the protein, together with 5 μl of 1 N NaOH) as described by Parthasarathy et al. (Parthasarathy, et al., Proc. Nati. Acad. Sci. USA 84: 537-540; 1987).

P1288 / 98MX Example 3. Preparation of Proteins Modified by Aldehyde. The globulin-free RSA containing 1 mg of protein (or other proteins) in 100 μl of PBS was prepared, and then the total volume was increased by 1 ml with PBS. Nonoenol or hexanol (100 μl of 25 μM in ethanol) was added to the protein, mixed well and incubated at 37 ° C for 24 hours. Four ml of ketone cooled with ice were added to the solution and the tube was kept in the freezer for one hour. After centrifugation at 1500 rpm for 10 minutes at 4 ° C, the supernatant was removed. This step was repeated three more times, and then the precipitate was dried in a vacuum. One ml of PBS was added to the tube, and after mixing, the solution was used in an enzyme-linked immunosorbent assay (ELISA). Proteins modified by malondialdehyde were prepared as follows. A 100 μl sample of the tetramethoxypropane was prepared and 0.5 ml of 6 N HCl was added to the tube. The tube was heated at 60 ° C for 30 minutes. The pH was adjusted to 6.4 using 4 N NaOH and the total volume was adjusted to 2.7 ml using the PBS. Then 25 μl of the prepared solution was added to 2 mg of rabbit serum albumin or free of globulin or other proteins, and incubated at 37 ° C. After three hours of incubation, the solution was dialyzed against PBS and used in P1288 / 98MX ELISA.

Example 4. Preparation of the Antibody. Three male rabbits with a body weight of 3-4 kilograms were purchased from Myrtle Rabbitry (Thompson Station, TN). For the primary immunization, 1.5 mg / ml of the rabbit serum albumin modified with lipid peroxide was dissolved in PBS, mixed with Freunds' complete adjuvant (Sigma, St. Louis, MO), then injected subcutaneously. Reinforcement immunizations with antigen in PBS were continued and mixed with the incomplete Freunds adjuvant at four week intervals. The rabbits were bled 10 to 14 days after the immunizations and the blood was allowed to stand for 4 hours at room temperature and at 4 ° C overnight. After removal of the clot and debris, by centrifugation for 20 minutes at 3000 rpm, the serum was analyzed by ELISA and stored at 20 ° C. Monthly titers were followed and terminal blood was removed at 6 months after the initial immunization. The antibody titer LOOH / RSA in the animals increased with time and stabilized at approximately four months.

P1288 / 98MX Example 5. ELISA assay for Proteins Modified by LOOH. Using ELISA, it was investigated whether the LOOH / RSA antibody recognized the protein modified by LOOH and the unmodified protein. RSA free unmodified IgG control was used. The wells or cavities of the ELISA plates were coated with 50 μl per well of different solutions of the RSA modified with lipid peroxide and incubated at 37 ° C for 3 hours. Plates were washed three times with Tween 20 with 0.05% PBS blocked for 3 hours with 300 μl of a fat-free milk powder, 1% together with 0.05% Tween 20 in PBS. After blocking, the plates were washed three times with 0.05% Tween 20 PBS, and the anti-LOOH / RSA sera were diluted to 1: 250. Fifty μl were added to each well and incubated at 37 ° C for 3 hours overnight. After a three-fold wash with 0.05% Tween 20 in PBS, the anti-rabbit IgG conjugated with alkaline phosphatase was diluted at 1: 38000 and 50 μl was added to each well and incubated at 37 ° for 2 hours. After washing 6 times with 0.05% Tween 20 in PBS, 50 μl of p-nitrophenyl phosphate was added to each well and incubated at 37 °. The plates were dried at 15 minute intervals for 2 hours. The curve was constructed by plotting the reading of OD against the P1288 / 98MX concentration of LOOH / RSA. The antigens (LOOH / RSA, RSA, Ox-LDL, and other modified proteins) were placed in 96-well plates overnight at room temperature. The wells or cavities were blocked by 3% BSA in PBS for 2 hours and washed three times with PBS. The anti-LOOH / RSA antibody was diluted 1: 250 with PBS containing 3% BSA added to each well. After two hours of incubation at 37 ° C, the wells were washed with PBS. The goat anti-rabbit IgG conjugated with alkaline phosphatase was diluted to 1: 38,000 and added. After 2 hours of incubation at 37 ° C, the wells were washed again and p-nitrophenol phosphate was added. The color development was determined by a plate reader (Anthos II). Figure 1 is a bar graph plot of the recognition of rabbit serum albumin and rabbit serum albumin modified by lipid hydroperoxide, as measured in nanograms of protein against optical density at 405 nm. As illustrated, the antibody at a dilution of 1: 250 selectively recognized the modified protein in a concentration-dependent manner. It was also recognized as 10 ng of RSA (less than 2 nM of modified RSA) at significantly higher levels than those of the control protein. The unmodified control protein was poorly recognized by the antibody to P1288 / 98 X at high concentrations. Controls without primary or secondary antibodies were not recognized by the antibody other modified proteins (human and bovine serum albumin, modified by LOOH, catalase and cytochrome C) were also recognized by the antibody. The Aproprotein B100 (apo B) component of LDL underwent extensive modification during LDL oxidation. A number of studies have shown that the modification includes new antigenic epitopes generated upon the covalent modification of lysine residues by aldehydes. In order to determine whether Ox-LDL also contains intact LOOH-modified plants, it was investigated whether the LOOH / RSA antibody recognizes LDL or Ox-LDL. Figure 2 is a graph of bar graphs illustrating the recognition of ox-LDL by the antibody of Example 4 as measured in microgram concentration against optical density units. As indicated in Figure 2, Ox-LDL was recognized by the antibody in a concentration-dependent manner. The antibody was also able to recognize as 0.25 μg of the Ox-LDL protein (< 0.5 nM apo B). Native LDL, prepared in the presence of butylated hydroxytoluene (BHT) was not recognized even at 2.5 μg concentrations. In separate experiments it was determined that the lipid component of Ox-LDL was also P1288 / 98MX recognized by the antibody, suggesting that the Ox-LDL aminophospholipids, such as phosphatidylethanolamine can be modified in a similar manner.

Example 6. Immunohistochemistry. The ability of the antibody to recognize modified proteins in tissues was tested under two conditions. In the first study, RAW cells, preincubated with LOOH, were used. In the second study, the atherosclerotic arteries from monkeys fed cholesterol were immunoblotted with the antibody. RAW macrophages were incubated with 100 μM 13-HPODE for 1, 2 or 3 days, as follows. Confluent cells and 6-well plates were treated with 13-HPODE for 1, 2 or 3 days in serum-free DMEM (minimal essential Dulbecco's modified Eagle medium). fresh 13-HPODE was added on the second and third day to the respective cell plates. At the end of the first, second and third day, cells were fixed with Bouins solution for 10 minutes and immunocytochemistry was performed using antibody against LOOH / RSA. After fixation, the cells were washed three times with PBS. The anti-LOOH / RSA antibody was diluted to 1: 250 with PBS containing 3% BSA was added to each well. For the negative control, no primary antibody was added. After two hours of P1288 / 98MX incubation at room temperature, the wells were washed with PBS three times and goat anti-rabbit IgG conjugated with alkaline phosphatase was diluted to 1: 100 and added. After 2 hours of incubation at room temperature, the wells were washed again and incubated with fast red. After color development, the reaction was terminated and the cells were photographed using a microscope with a camera attachment. The frozen segments of the abdominal aorta of a group of male cynomologo monkeys were evaluated by immunohistochemistry. The animals were fed a moderately high-fat diet for five years consisting of high-protein monkey feed supplemented with 8.2% dry egg yolk and 10% lard. The final diet contained 37.5% saturated fat, 44.9% monounsaturated fat, 17.5% polyunsaturated fat with 0.25% cholesterol. The average serum level of these animals was 306 mg / dL. These cholesterol levels are sufficient to produce a range of atherosclerosis lesions in monkeys. Human aortas that exhibit different degrees of atherosclerosis development were obtained from organ donors at the time of tissue collection and were collected with the approval of the committee of the Emory University Human Subjects Committee. The aortas of cynomologo monkeys, and of humans P1288 / 98MX fixed in paraformaldehyde and frozen in O.C.T. before sectioning at 5 μm in a cryostat. Tissue sections were then immunoblotted using the antibody at a dilution of 1: 500 followed by biotinylated goat anti-rabbit IgG (Fisher Scientific) used at a 1: 200 dilution and visualized by the Vector ABC-AP system using red vector as a chromagene (Vector Laboratories). The immunohistochemistry showed that the cells incubated with LOOH were immunostained with the antibody and the antigenic epitopes were present intracellularly. The control cells and controls lacking in the primary antibodies failed to show immunoreactivity. Those arteries showed intense immunoreactivity. Immunoreactivity was localized in areas rich in macrophages of foam cells as determined by the counter staining or staining by macrophages.

Example 7. Western Blot Analysis Western blot analysis was performed after separation of the proteins in 7.5% crosslinked acrylamide gels using 2.5 μg of protein. Transfer samples were detected using anti-LOOH / RSAk, diluted 1: 250 as the primary antibody and a goat anti-rabbit IgG conjugated with peroxidase as the P1288 / 98MX secondary antibody. The cross-reactivity bands were visualized by chemiluminium detection. Using the Western blot analysis of LDL and Ox-LDL, it was confirmed that the antibody of Example 4 was able to recognize Ox-LDL but not native LDL. Western blot analysis of normal human plasma showed that at least three different proteins are recognized by the antibody. Plasma samples were prepared in the presence of BHT and it is unlikely that these epitopes were generated in vitro. The identity of these proteins is unknown, although from the mobility in the gel it is suspected that these proteins can represent albumin and apo B.

EXAMPLE 8. Cross Reactivity of the Antibody of Example 7 with Proteins Modified by Aldehyde. A number of antibodies have been described for the aldehyde-modified proteins. Since the incubation of the LOOH with the protein is of long duration, it is possible that the aldehydes may have contributed to the generation of the antigenic epitopes. In order to establish whether the antibody to LOOH / RSA recognizes proteins modified by the virus, twenty amino acid peptides of the sequence YVTKSYNETKIKFDKYKAEKSHEDEL (where K means lysine) were prepared (SEQ.ID .: No. 1). This peptide was used P1288 / 98MX because plasma proteins, such as albumin, from commercial sources may already possess similar epitopes, either as a result of in vivo generation or as a result of generation during in vitro purification procedures. The data from ELISA of MDA, hexanal, nonenal, and synthetic peptide modified by LOOH is given in Table 1. The antibody failed to recognize the synthetic peptide or its aldehyde-modified derivatives to any significant degree. In contrast, the synthetic peptide modified with LOOH was avidly recognized by the antibody.

Table 1: Recognition of the peptide modified by LOOH and the peptides not modi fi ed by aldehyde by the anti-LOOH / RSA antibody.

Microtiter wells were coated with P1288 / 98MX increasing concentrations of the unmodified or modified peptide. After blocking the wells with non-fat milk powder, 100 μl of a 1: 250 dilution of the anti-LOOH / RSA antibody was added to each well. After washing, the anti-rabbit IgG conjugated with alkaline phosphatase was added to each well. After the addition of the substrate, p-nitrophenyl phosphate, the OD was measured at 405 nm using a microplate reader. The values represent averages of a triplicate conjunct of the optical density readings of the wells or cavities from one of at least two separate tests. Western blot analysis of the aldehyde-modified peptide and LOOH confirmed that none of the synthetic proteins modified by aldehyde were recognized to any significant degree, whereas the peptide modified with LOOH was recognized by the antibody.

II. Biological Activity of Oxycines. A. Definition of Oxycines. It has been found that in certain reaction products of the lipid hydroperoxides and primary amines independent biological activity is exhibited as mediators of cellular responses. The term "oxycin" is used herein to refer to a fluorescent protein or lipid that is generated by the reaction of a hydroperoxide of P1288 / 98MX lipid and a primary amine that can elicit a response from a target cell. Oxicins can be generated extracellularly or can be formed in the cell membrane to mediate a cellular response. Oxycins can also be generated intracellularly. It can be converted into a wide variety of biologically active molecules that have primary amines to oxycins that have a biological activity. An example of an oxycin is the stable fluorescent product of the reaction between línoleic peroxide (13-HPODE) and an appropriate amino acid group, such as lysine, in albumin or polylysine, or a compound of small molecular weight such as phosphatidylethanolamine. Certain oxycins act as potent inflammatory signals that induce endothelial VCAM-1 gene expression through a mechanism that can be suppressed by selective antioxidants. Other cellular responses that can be caused by oxycycins are the generation or activation of MCP-1, IL-1, TNF-alpha, ICAM, MCSF, and E-selectin. Oxicins can be used as mediators of cellular inflammatory responses, including cardiovascular responses since the oxidation of lipids is associated with inflammatory and cardiovascular disease. The specific disease states that are inflammatory or cardiovascular are of nature P1288 / 98 X inflammatory or cardiovascular include atherosclerosis, endometriosis, glimeral nephritis, preeclampsia, and disorders of the central nervous system, mediated by peroxidation of lipids, Alzheimer's disease, autoimmune disorders, psoriasis, asthma, atopic dermatitis, skin cancer, disease neurodegenerative, irritable bowel disease (Crohn's disease), rheumatoid arthritis, ischemic reperfusion, osteoarthritis, asthma, dermatitis, multiple scleroris, post-angioplasty restenosis, coronary artery disease, and angina. While all the primary amines will react with the lipid hydroperoxides to form an antigenic species that can be used to generate antibodies to determine the oxidation state of the host, not all naturally occurring peptides or primary amines will form oxycycins. This is because only epitopes are required to elicit an antibody response, however, in order to mediate a cellular response, the number and location of the sites reacted is critical for the complementary binding as a receptor. Non-limiting examples of oxytocin epitopes are described Proc. Nati, Acad. Sci. USA, volume 89, pp 10588-10592, November 1992 Medical Sciences, incorporated P1288 / 98MX herein by reference. In particular, epitopes can include: HC-CH R-HC CH CH. ^ N PROTEI PROTEIN R = alkyl The antibodies of the present invention can be used to block the harmful activity of antigen, particularly by physically intervening with its ability to mediate a cellular response B. In Vitro Synthesis and Characterization of Oxycin Lipid modification of soy lipoxygenase (sLO) was achieved with the incubation of lipid hydroperoxide with lipoxygenase, using a modified method of Freubis, Parthasarathy, and Steinberg (Proc. Nati. Acad. Sci. USA, 89, 10588-10592). For this synthesis, it was important P1288 / 98MX maintaining a high ratio of lipid to protein. In a small-scale reaction (1 ml), 250 μM linoleic acid was directly incubated with sLO (50 units, 80 ng) at room temperature for three days; the agitation of the mixture was important for the introduction of air into the reaction. All reactions were run in Tris 10 mM, pH 8.0, 15 mM sodium chloride. The non-purified reaction mixture was used to generate rabbit polyclonal antibodies and mouse monoclonal antibodies against oxidatively modified oxicin lipoxygenase (oxSLO). In scaling up the reaction, it became impossible to maintain the high lipid-to-protein ratio due to immiscibility problems. A method for the synthesis of lipoxygenase oxycin modified by 13-HpODE, or the modification of the "target" protein was developed (see, figure 4). The target protein, 1-3 mg in a volume of up to 1.5 ml, was sequestered within the 10 kDa molecular weight cut-off membrane. This allowed the continuous exchange of the 250 ml of the lipid peroxide generation system with the retention of the protein target. Catalytic amounts of soy lipoxygenase, 1.260 U, and 800 μM linoleic acid, were used in the peroxide generation system to catalyze the formation of 13-hydroperoxy- [S-]P1288 / 98MX (E, Z)] -9, 11-octadecadienoic. The generation system was exchanged with a fresh sLO / linoleic acid every eight to sixteen hours for a period of at least 170 hours. The reaction was performed in 10 mM Tris, pH 8.0, 15 mM sodium chloride with constant stirring. An advantage of the large-scale method was that any protein greater than 10 kDa or a mixture of proteins can be used as the target protein. The criteria for the formation of oxicins oxidized by 13-HpODE include induction of ICAM-1 in the endothelial cell-based assay (which is discussed in detail in the last sections of this report) and immunoreactivity with the antibodies of the oxycin. The minimum requirements for the formation of oxycin were evaluated using the small scale reaction. As observed by Western analysis using polyclonal antibody, sLO and linoleic acid were minimum requirements for the three day reaction (see figure 5). Shaking the reaction improved the yield. There was no cross-reactivity when the sLO was covered with a shock absorber alone. The course of time for the formation of oxycin in the large-scale reaction was followed by immunoreactivity and biological activity (induction of ICAM-1, see figures 6 and 7). Immunoreactivity activity was observed before biological activity. He P1288 / 98MX initial product observed by Western analysis was a high molecular weight band that migrated at approximately 80 kDa. The reaction products observed at the last time points formed a ladder with a predominant species at approximately 25 kDa. The course of reaction time for both immunoreactivity and biological activity was dependent on lipid concentration; the increased concentration of linoleic acid increased the reaction rate. The oxycin reaction products were examined by reversed-phase, analytical high-performance liquid chromatography (RP-HPLC) and gel filtration. HPLC analysis using a C18 column revealed that the reaction products were more hydrophobic than the starting materials, consistent with the addition of the lipid to the lipoxygenase (see Figure 8). Preparative gel filtration was used to partially purify oxycin (see figures 9 and 10). A biological activity was found in fractions 9 and 10 although the majority of the protein was found in the other fractions. The stability of oxycin was evaluated based on immunoreactivity and biological activity. Oxycin was stable to a number of acidic and basic conditions and to the presence of reducing agents, as observed by Western analysis. In the pre-incubation with 3.5 M MgCl2, P1288 / 98MX 10 mM sodium phosphate, pH 7.2; 5.0 M LiCl, 10 mM sodium phosphate, pH 7.2; 100 mM triethylamine, pH 11.5; and 100 mM glycine, pH 2.5, immunoreactivity was maintained with polyclonal and monoclonal antibodies. In contrast, incubation with 3.5 M MgCl 2, 10 mM sodium phosphate, pH 7.2 caused a loss of biological activity while the remaining storage conditions had no effect. If the loss of activity was due to a change in the conformation of the protein, as an oxidation state, or another cause remains to be determined. The biological activity was maintained after denaturation with heat and denaturation with heat in the presence of dithiothreitol. The addition of EDTA, Trolox, and probucol also had no effect on the induction of ICAM-1. After acid hydrolysis of the protein, the biological activity was lost.

Example 9. Characterization of the biological activity of oxSLO: oxSLO induced the expression of ICAM in Endothelial, Aortic, Human cells (HAEC). In a small-scale synthesis reaction, sLO converted linoleic acid 250 μm (LA) to 13-HpODE in the space of the first hour of incubation at room temperature. During the three subsequent days of P1288 / 98MX incubation, 13-HpODE presumably modified the enzyme oxidatively and the generation of epitopes in the enzyme that can cause biological activity such as the expression of cell surface ICAM in HAEC as shown in the ELISA assay depicted in Figure 11. The HAEC cultured on microtiter plates were exposed in treatment for 16 hours before the ELISA assay. The sLO or LA alone after three days of incubation did not become active, suggesting that the oxidative modification of the sLO is essential for the creation of the active, biological epitope. In the large-scale synthesis reaction, only in the presence of the 13-HpODE generation system the sLO becomes active as shown in figure 12. The negative control of sLO represented the sLO enzyme incubated with buffer only during the extended period of time. It was confirmed that the oxSLO synthesized by any method are functionally and immunologically identical. In addition, it has been determined that the formation of a biological activity is specific to the protein. The reaction of rabbit serum albumin using the large or small scale oxycynin reaction generates immunoreactive products, but not biologically active ones. ICAM expression of the cell surface, activated by oxSLO was in a manner dependent on the P1288 / 98MX dose, as shown in figure 13. 40 ng / ml of the oxSLO was sufficient for a significant signal in the standard ELISA assay. Again the equal amount of unmodified sLO failed to activate the HAEC. A similar result is observed in the Northern blot as shown in Figure 15.

Example 10. Expression of VCAM, E-selectin and MCP-1 induced by oxSLO in HAEC. OxSLO not only induced ICAM, but also two other adhesion molecules (VCAM in Figure 15, E-selectin in Figure 16) and a chemokine molecule (MCP-1 in Figure 15 and 16). Figure 14 showed induction of the cell surface VCAM expression of the HAEC, although to a lesser degree than the induction of ICAM. The ELISA data is supported by the Northern blot data as illustrated in Figure 15. The amount of VCAM mRNA accumulation after 4 hours of the oxSLO treatment was less than the ICAM mRNA. In addition, the mRNA level of MCP-1 and E-selectin was as high as TNF induction, suggesting that MCP-1 and E-selectin may be key components of the oxSLO-mediated signaling cascade. The induction of these preinflammatory genes was very fast as shown in Figure 16. In the space of 2-6 hours of stimulation, the P1288 / 98MX level of mRNA of the indicated genes all reached a constant state.

Example 11. Expression of IL-lβ by oxSLO in macrophage. In addition to having a profound effect on endothelial cells as summarized in Table 2, oxSLO induced the accumulation of another il-lß mRNA of cytokine in the macrophage cell line (RAW) as shown in the figure 14. The RAW cells were treated with oxSLO for 4 hours and the total RNA was harvested and analyzed. In the development of atherosclerosis, both endothelial cells and macrophages are shown to be extremely important. The present in vitro data strongly supports the notion that a single class of protein modified by lipid hydroperoxide, lipids or lipophilic molecules that are defined as oxycin can mediate a proinflammatory signal in the atherosclerotic lesion areas.

P1288 / 98MX Table 2: OxSLO induced the accumulation of VCAM-1, ICAM-1 and MCP-1 mRNA in HAEC.

Example 12: Biological Activity of the Product of the Oxidant Modification of Rabbit Serum Albumin by Linoleic Hydroperoxide. We investigated whether the oxidative modification of rabbit serum albumin, RSA, by linoleic hydroperoxide alters the structural or biological property of an element (peptide or non-peptide) in this material, conferring a biological function as an inflammatory signal of vascular endothelial cell . Using the RSA P1288 / 98 X oxidatively modified (oxRSA) prepared as described in Example 1, Endothelial, aortic, human cells cultured for 12 hours were exposed with a range of oxRSA concentrations from 1 to 100 nM (nanomolar), and analyzed for expression on the cell surface of inducible adhesion molecules, VCAM-1, ICAM-1 or the constitutively expressed adhesion molecule, ICAM-2 by the ELISA assay. As shown in Figure 3, expressed as a percent of the signal induced by TNF-a, maximum, ox-RSA exhibited a dose-dependent induction of VCAM-1 at 40% and ICAM-1 at 80% of the maximum signal of TNF-a. The approximate EC50 of the oxRSA was 10-15 nM for both VCAM-1 and ICAM-1. This induction was not due to the contaminating lipopolysaccharide since polymyxin B (10 μg / ml) completely inhibited the induction of LPS but had no effect on the activation of oxRSA. In addition, the limulus test for LPS in the oxRSA preparation was below detection. ICAM-2 expressed constitutively was not affected. This result suggests that oxRSA functions in a sensitive manner in addition to the dose as a potent inflammatory factor for endothelial cells in the low nanomolar range.

P1288 / 98MX Example 13: Effect of Oxidant Modification Product of Rabbit Serum Albumin by Linole Hydroperoxide on mRNA accumulation. It was then investigated without the induction of cell surface expression of VCAM-1 and ICAM-1 by ox-RSA is due at least in part to changes in mRNA accumulation. Northern filter analysis was performed from RNA isolated from HAEC exposed for 4 hours with either TNFa (lOOU / ml) or oxRSA (25nM). Similar to that observed at the protein level, VCAM-mRNA levels are induced by oxRSA at levels of approximately 30-40% observed for TNFa. The ox-RSA ICAM-1 mRNA was also induced.

Example 14: Effect of the Modification Product Oxidant of Rabbit Serum Albumin by Linoleic hydroperoxide in the transcriptional activation of the VCAM-12 promoter through a DNA-binding complex similar to NF-kB. To determine if oxRSA modulated the nuclear regulatory cases associated with gene expression of Redox sensitive VCAM-1, human aortic endothelial cells, cultured or not (CTL) were exposed for two hours to TNFa (100OO / ml) or 25nM of oxycin P1288 / 98MX RSA-LOOH (oxRSA), prepared nuclear extracts and gel unit change assays performed using 32 P labeled kLOkR, the tandem NF-kB binding sites of the human VCAM-1 promoter. The appropriate cold competitor and superchange controls using anti-p50 and anti-p65 antisera (R &D Systems) were performed to establish the binding specificity of the DNA sequence of the NF-kB complex band. Both TNF and oxRSA induced DNA-binding activity similar to NF-kB. This suggests that oxycins may play a role in transcriptional activation pathways mediated by NF-kB in the vasculature.

Example 15. Titers of autoantibodies of plasma samples from subjects with endometriosis and control. Oxidation that occurs in the peritoneal cavity of subjects with endometriosis may result in the presence of modified autoantibodies to protein and these antibodies may be increased in patients suffering from endometriosis. To determine if these autoantibodies are present in patients diagnosed with endometriosis, the plasma samples taken from these patients were evaluated for the presence of antibodies that react with three antigens: the product P1288 / 98MX reaction of the lipid hydroperoxide with rabbit serum albumin, the reaction product of the lipid hydroperoxide with LDL, and that with MDA. ELISA assay: 10 μg of Ox-LDL, MDA-LDL or albumen modified with lipid peroxide (LOOH-RSA) (positive antigens) and LDL, human acetyl LDL albumin (Negative controls) were plated with a volume of 100 μl in a 96-microtiter plate. After incubation overnight at 4 ° C, the plates were blocked by incubation with 3% milk powder by incubation for 2 hours. After the washings, human IfF, bound, was detected using an antibody of Anti-human IgG, conjugated with peroxidase or alkaline phosphatase. The bound enzyme conjugated IgG was quantified by color detection using specific substrates. The results are given in tables 3-5. As indicated, there was a statistical ince in the levels of tive antibodies in patients with endometriosis over the controls.

Table 3: Antigen: LOOH / RSA (200 μl plasma samples). The results are expressed in OD equivalent of the p-nitrophenol formed. t-Test: Equal variations that assume two samples P1288 / 98MX Table 4: Antigen: Ox-LDL (200 μl plasma samples) Table 5: Antigen: MDA-LDL (200 μl plasma samples) t-Test: Equivalent variations that assume two samples P1288 / 98 X This invention has been described with reference to its preferred embodiments. Variations and modifications of the method, kit, and materials, including antibodies, will be obvious to those skilled in the art from the foregoing detailed description of the invention. It is proposed that all these variations and modifications be included within the scope of the appended claims.

P1288 / 98MX Table 1: oxSLO induced the accumulation of mRNA of VCAM-1, ICAM-1 and MCP-1 in HAEC Tment VCAM-1 ICAM-1 MCP-1 (arbitrary units (arbitrary units)) TNF (units of 82.06 71.92 101.65 100 / ml) sLO (0.8 μg / ml) 3.36 3.14 12.34 sLO (0.08 μg / ml) 0.12 -0.09 3.1 sLO (0.008 μg / ml) -0.14 -0.51 1.12 sL modified (1 24.17 19.74 116.62 μg / ml) sLO modified (0.2 11.51 9.41 96.28 μg / ml) sLO modified (0.04 9.3 2.06 37.35 μg / ml

Claims (25)

1. NOVELTY OF THE INVENTION Having described the present invention, it is considered as a novelty and, therefore, the content of the following CLAIMS is claimed as property: 1. A method for the assessment of lipid oxidation in a biological sample that includes contacting the biological sample with an antibody that binds to an antigen formed by the reaction of a lipid hydroperoxide with a primary amine.
2. 2. A kit for the evaluation of lipid peroxidation in a biological sample that includes (I) an antibody or antibody fragment that binds to a reaction product of a lipid hydroperoxide with a primary amine, or (ii) an antibody that binds to the antibody of (I).
3. 3. An isolated antibody that binds to an antigen formed by reacting a lipid hydroperoxide with a primary amine.
4. 4. An antibody fragment that binds an antigen formed by reacting a lipid hydroperoxide with a primary amine.
5. 5. The antibody according to claim 3 or 4, immobilized on a solid support.
6. 6. The antibody according to claim 3 or 4, P1288 / 98MX labeled or labeled with a detectable agent. The antibody according to claim 5, wherein the solid support is selected from a membrane and a coating supported by, or attached to bars, beads, cups, or flat packets. The antibody according to claim 5, wherein the solid support is selected from a cell culture plate, ELISA plate, tube, and polymer membrane. The antibody according to claim 3 or 4, which is labeled with a detectable agent selected from the group consisting of a fluorochrome, a radioactive label, biotin, horseradish peroxidase, alkaline phosphatase, 2-galactosidase, or another enzyme. 10. The antibody according to claim 3, which is a conjugate. The antibody according to claim 3 or 4, wherein the antigen is the reaction product of linoleic hydroperoxide and a primary amine. The antibody according to claim 11, wherein the appropriate amino acid group such as lysine, in albumin or polylysine, or a compound of small molecular weight such as phosphatidylethanolamine. The antibody according to claim 3 or 4, which is humanized. P1288 / 98MX 14. A method for titrating the level of lipid peroxidation in a biological sample, which includes contacting the sample with either: (I) an antigen that binds to an antibody that is immunoreactive with an antigen formed by the reaction of: a lipid hydroperoxide with a primary amine, or (ii) an antibody or antibody fragment or conjugate that is cross-reactive with an antibody that is immunoreactive with an antigen formed by the reaction of lipid hydroperoxide with an amine 15. The method according to claim 13, wherein the level of (I) or (ii) in a host is compared to a population standard. 16. A method for assessing oxidative damage in a biological sample, comprising the steps of: (I) isolating an antigen formed by the reaction of lipid hydroperoxide with a primary amine; and then (ii) identify the primary amine. 17. A method for diagnosing a disorder related to oxidation in a biological sample, comprising evaluating the level of a reaction product of a lipid hydroperoxide with a primary amine. 18. The method according to claim 16, wherein the condition related to oxidation is selected from the group consisting of disease P1288 / 98 X cardiovascular, atherosclerosis, inflammatory disease, endometriosis, glimerol nephritis, preeclapsis, central nervous system disorders mediated by lipid peroxidation, Alzheimer's disease, psoriasis, asthma, atopic dermatitis, solid tumors, Kaposi's sarcoma, neurodegenerative disease , inflammatory bowel disease (Crohn's disease), rheumatoid arthritis and reperfusion due to ischemia. 19. A method to induce the inflammatory effect in a biological sample or host, which includes contacting the sample or host with a fluorescent product of the reaction between a lipid hydroperoxide and a primary amine that induces this effect. The method according to claim 13, wherein the linoleic hydroperoxide is 13-HPODE. The method according to claim 13, wherein the amine is selected from the group consisting of amino acid, protein, peptide, or phosphatidylethanolamine. The method according to claim 13, wherein the inflammatory effect is produced by induction of a mediator selected from the group consisting of VCAM-1 MCP-1, IL-1, TNF-α, ICAM, MCSF, and E -selectina. 23. The antibody according to claim 6, in combination with a means for detecting the agent P1288 / 98MX detectable. The antibody according to claim 21, wherein the combined means for detecting a detectable agent using an enzyme as a detectable agent and an enzyme substrate that changes the color on contact with the enzyme. 25. A method for assessing the ability of a substance to decrease the state of lipid peroxidation of a biological sample, which comprises comparing the level of the reaction product of a lipid hydroperoxide and a primary amine in the host sample before and after put the sample in contact with the substance. P1288 / 98 X