BE1013881A6 - OXIDIZED LDL FRACTIONS, PROCESS FOR OBTAINING AND USE FOR DIAGNOSTIC OR THERAPEUTIC PURPOSE. - Google Patents

Abstract

The invention relates to compositions consisting of one or more fractions of low density lipoproteins (LDL) obtained by in vitro oxidation of native LDL by hydrogen peroxide or the glucose / glucose oxidase couple, in the presence of myeloperoxidase. these compositions are obtained by chromatography, in particular by Fast Protein Liquid Chromatography (FPLC) type chromatography and are purified by immunoaffinity. Monoclonal antibodies directed against the B and C / D fractions of oxidized LDL have been isolated and used for diagnostic purposes, for example for the assay of oxidized LDL or the corresponding autoantibodies, or for the study of the oxidation sensitivity of Native LDL in patients. These antibodies can also be used to treat the blood or blood fractions of a patient and to withdraw a fraction of oxidized LDL, for example on an immunoabsorption column, before being returned to the patient.

Description

Fractions of oxidized LDL, process for obtaining and use for diagnostic or therapeutic purposes.

The present invention relates to the use of myeloperoxidase for obtaining oxidized lipoproteins and corresponding monoclonal antibodies, the new monoclonal antibodies thus obtained, the lines producing these antibodies and the use of these antibodies and lines, a diagnostic test in particular for determination of cardiovascular risk as well as diagnostic application kits.

Myeloperoxidase (MPO) is an enzyme found in neutrophils and monocytes, capable of catalyzing oxidation reactions in the subendothelial space. It is well known that this oxidation involves, from hydrogen peroxide and chloride, hypochlorous acid produced in the presence of MPO.

Furthermore, it is known that low density lipoproteins (Low Density Lipoprotein, LDL), in oxidized form, are involved in certain cardiovascular diseases, in particular atherosclerosis. The presence of LDL modified by hypochlorous acid has thus been reported in atheromatous lesions.

The test based on these lipoproteins currently used to define the propensity of a given patient to develop cardiovascular diseases uses a very powerful and non-physiological pro-oxidizing agent, copper sulphate (CuSCu). However, this test is not very precise and takes place under conditions far removed from the conditions in vivo.

Patent document PCT / BE98 / 59248 discloses a method of immunodetection of LDL oxidized by malondialdehyde (MDA) and associated antibodies. Oxidation does not, however, optimally simulate the natural process.

One of the aims of the present invention is to propose a diagnostic test capable of identifying criteria allowing a more precise and earlier detection of patients at risk, by referring to an oxidative process of LDL occurring in vivo.

The development of this test involved a myeloperoxidase. In particular, it is possible to use a recombinant human myeloperoxidase with properties identical to those of natural myeloperoxidase.

The present invention also provides monoclonal antibodies, with the corresponding hybridomas, capable of recognizing specific fractions of lipoproteins oxidized by myeloperoxidase, and therefore of being used for immunodetection and the assay of these oxidized lipoproteins. Such a dosage can advantageously reflect a cardiovascular risk factor.

According to one aspect of the invention, a test is generally proposed for determining the cardiovascular risk of a patient by measuring the plasma level of oxidized LDL comprising the following steps which will be explained later: monoclonal antibody against fraction B on a plate - extraction of oxidized LDL from the plasma by adsorption on the monoclonal antibody

- recognition of LDL fixed using a polyclonal anti-apoB antibody

revelation by a marker which can for example be a detection antibody, eg if the polyclonal antibody anti.apo-B is a rabbit antibody, a goat anti rabbit IgG (Fc) antibody coupled to phosphatase alkaline - determination of LDLox concentration.

According to another aspect of the invention, it has been found that the rate of LDL oxidation constitutes a promising method for defining the propensity of a given patient to develop cardiovascular diseases. As already mentioned, one of the main factors intervening at an early stage in the formation of the atheromatosis lesion is the peroxidation of LDL. As oxidized LDL (oxLDL) is very quickly captured by macrophages of the arterial wall, the measurement of their concentration alone may not be representative of their production in vivo. On the other hand, the susceptibility of LDL, of a given subject to undergo peroxidative damage can be evaluated by an in-vitro test.

According to yet another aspect of the invention, a test is proposed based on the detection of natural autoantibodies in the plasma, directed against LDLs naturally oxidized by myeloperoxidase.

DETAILED DESCRIPTION OF THE INVENTION

The invention is described in more detail with reference to particular embodiments described below.

One of the aims of the invention is to provide antibodies specifically directed against certain LDL subfractions oxidized by a myeloperoxidase.

The following describes the production of myeloperoxidase, the oxidation of LDL, the separation of the fractions obtained, the production of hybridomas and the production of antibodies specific to the oxidized fractions, as well as the use of these antibodies for detection. and the dosage of oxidized LDL for diagnostic purposes.

1, Production of myeloperoxidase

The myeloperoxidase can be produced from the CHO clone producing the recombinant myeloperoxidase MPOr. The clone which was used in the context of the present invention is clone 24-1-7-57 and was cultured in Opticell (Charles River). In this system, the cells adhere to a ceramic support (Opticore) with a surface area of 4,500 cm2 and the culture medium is continuously perfused. Several parameters are constantly monitored: pH, temperature, oxygen, CO2 content, and glucose concentration.

Each day, the concentrations of lactate and glucose in the medium are measured as well as the peroxidase activity due to the myeloperoxidase synthesized and secreted in the medium.

The culture can be maintained for 2 months. The maximum production is around 30 days. About every 3 days, the entire culture medium is harvested, centrifuged and stored.

Alternatively, the cells can be kept in culture in a "cell factory", the culture supernatant also being harvested every 3 days, and the culture can be kept for 2 months.

Before each purification, it is necessary to purify the culture supernatants in order to remove cell debris therefrom. To this end, a filtration system composed of a high-flow peristaltic pump (Watson Marlow 505 s) and a tangential filter (Ultrasette, Filtron) were used.

This apparatus allows simultaneous filtration and concentration of culture supernatant before depositing on the column. After a concentration of 10 times, the supernatant is diluted approximately 2 times until reaching the resistivity of 4 mS / cm, equivalent to that of the column equilibration buffer. No loss in MPO was detected in the filtrate analyzed by Western blot, and on the other hand by ELISA, a loss of approximately 0.7% was noted there.

This tangential filtration system therefore proves to be extremely useful and reliable for the treatment of large volumes of culture supernatant to be purified.

At each purification, approximately 10 1 of filtered culture supernatant, concentrated and balanced in terms of pH and ionic strength are placed on a column of Q Sepharose Fast Flow (10 cm x 20 cm, 1570 ml of gel) balanced in buffer 20 mM phosphate pH 7.5. This anion exchange column, retaining the contaminants - mainly the serum proteins, is directly connected to a CM-Sepharose column (3.5 cm x 15 cm), cation exchange on which the MPOr is retained and is eluted at l using a NaCl gradient from 0 to 0.6 M (900 ml). The MPOr is released at a concentration of approximately 0.28 M NaCl and exits as a single protein peak. The peroxidase activity is perfectly superimposable on the peak measured at 280 nm.

The analysis of the purity of the eluted fractions was carried out by gel electrophoresis under non-reducing conditions and in the presence of SDS. When stained with Coomassie blue, 2 bands of 84 and 94 kDa appear. This second 94 kDa band is immunoreactive like that of 84 kDa and corresponds to a minor fraction of MPO.

The fractions were assayed for protein concentration (T.owry assay) and peroxidase activity (o-dianisidine as substrate), and chlorination (with monochlorodimedon as substrate). The most active fraction corresponds to the top of the elution peak.

According to the sugar analyzes already carried out, the two bands of 84 and 94 kDa correspond to forms of proMPO exhibiting different degrees of glycosylation. The 84 kDa band would mainly correspond to the protein exhibiting glysosylated chains rich in mannose, while the 94 kDa form would have complex glycosylation chains, up to sialic acid.

From 80 l of culture supernatant, 370 mg of pure MPO were obtained, ie approximately 4.5 mg / 1. Recombinant MPO appears in the form of 2 bands of 84 and 94 kDa, having the same protein sequence, but differing in the nature of their glycosylations.

Production of LDL subfractions oxidized by myeloperoxidase

The oxidative alterations that can occur at the LDL level are mainly of two types, on the one hand lipid peroxidation and on the other hand oxidative modifications at the apoB level. One of the most commonly used models for triggering LDL oxidation in vitro is based on the use of copper (Cu ++) as an oxidizing agent (Esterbauer et al.). This model evaluates over time the increase in degradation products of the lipid components of LDL and mainly provides information on the antioxidant status of these lipoproteins. However, the use of Cu ++ as a pro-oxidizing agent remains very controversial since its involvement in cardiovascular pathologies has not been formally demonstrated. On the other hand, other oxidizing agents such as hypochlorous acid, generated by MPO, seems to figure well among the main initiators of the formation of foam cells and therefore in the development of atheroma plaques. Indeed, the involvement of MPO in in vivo oxidation and in the pathogenesis of cardiovascular diseases is strongly suggested by the presence of MPO and oxidized derivatives of tyrosine, in particular dityrosine or 3-chlorotyrosine, in atheromatous lesions. MPO actually uses hydrogen peroxide (H202) as a substrate to oxidize halogens such as chlorine, thus allowing the formation of hypochlorous acid, a very toxic and microbicide agent. It is important to note that, unlike the case of metal ions, the oxidative damage caused by MPO mainly concerns proteins and particularly apoprotein B in the case of LDL.

2. LDL oxidation conditions

In order to illustrate the aspect of the invention based on LDL alterations generated by MPO, the supply of the hydrogen peroxide substrate was carried out in two ways: progressive production of hydrogen peroxide derived from the transformation of the glucose by glucose oxidase on the one hand, direct addition of hydrogen peroxide in the oxidation medium on the other hand.

The oxidation conditions at MPO are as follows:

- MPO / Glucose oxidase 27 system 8 mg p-rot. LDL

40 U MPO (peroxidasic units, with o-dianisine as substrate)

4U glucose oxidase 90 min at 30 ° C

- MP0 / H202 system 1.6 mg prot. LDL

8 U MPO 1 mM H202 5 min at 37 ° C

The separation of the oxidized LDL subfractions can be carried out using a Fast Protein Liquid Chromatography (FPLC) type chromatography with an Mono Q anion exchange column. The different oxidation subfractions indeed have electronegative charges depending on their degree of alteration.

It has been found that fractions (A, B, C, etc.) can thus be eluted from the column using buffers containing increasing concentrations of NaCl.

After optimizing the elution conditions, the coefficients of variation obtained for the different fractions of oxidized LDL showed good reproducibility of the technique used (CV <5% for majority peaks).

Fig. 1 illustrates the excellent separation of the oxidized fractions B, C and D obtained with elongation of the salt plates corresponding to the passage of the fractions.

FPLC results clearly show that oxidative damage linked to MPO occurs within the first 5 minutes regardless of the oxidative system (glucose / glucose oxidase or H202).

Figs. 2a and 2b illustrate the kinetics of appearance of the fractions oxidized with hydrogen peroxide (MP0 / H202) for H202 concentrations of 179 and 40 μΜ respectively.

Characterization of oxidized LDL subfractions Gel electrophoresis

Electrophoresis performed after oxidative stress shows that the damage caused by the MPO / Glucose oxidase system mainly consists of fragments of the apoB protein while the MP0 / H202 system gives rise mainly to aggregations.

In the MPO / glucose oxidase system, there is a decrease in the proportion of polyunsaturated fatty acids in the phospholipids of fraction D compared to the native fraction.

Alteration of fatty acid composition

The oxidized fractions obtained by the MPO / H202 system do not show any significant modification of the fatty acid composition. These results indicate that the glucose-oxidase-induced oxidation causes peroxidative damage to the polyunsaturated fatty acids of LDL, which is not the case for the MPO / H202 system.

There are no differences in composition in terms of cholesterol esters or triglycerides in the 2 oxidation systems.

It has been found that the fractions resulting from oxidation by the MPO / Glucose oxidase system are the result of an attack by MPO followed by various root reactions while the fractions deriving from oxidation by the MPO / H202 system, appear to be quite specific to an attack by DFO.

The latter system will therefore be preferred for carrying out the invention.

3. Production of monoclonal antibodies

The antigens against which the monoclonal antibodies of the present invention are directed consist of the various oxidized fractions of LDL, more particularly however against fraction B.

One of the aims of the invention is to be able to differentiate very oxidized fractions (fractions C and D) from fractions not or only slightly oxidized (fractions A and B) in order to LDL of patients appearing to oxidize more quickly than those of normal subjects to be able to quantitatively determine the presence of fraction B.

The invention also relates to hybridomas which can be used for the production of antibodies.

Hybridomas are obtained, for example, by fusion of non-secreting myeloma cells P3 x 3Ag8.653 (ATCC CRL-1S80) with the spleen cells of the immunized mouse, in the presence of polyethylene glycol 4.000.

The fused cells are distributed in dishes of 96 wells preincubated with mouse peritoneal macrophages in the presence of a selection medium containing hypoxantine, aminopterin and thymidine. The medium is replaced on day 7 by a non-selective medium. Two weeks after the fusion, the supernatants of the clones are screened for the presence of specific antibodies on each of the fractions A to D of oxidized LDL, as well as on MPO.

In this test, the purified antigen (fraction of oxidized LDL) is fixed on the plate and the supernatant, alone or in competition with a purified fraction, is deposited on the plate. The fixed antibody is then revealed by a second antibody directed against the GIs of mice and coupled to alkaline phosphatase.

It was thus possible to obtain two monoclonal antibodies specifically recognizing LDL altered by MPO. One recognizes subfraction B, the other recognizes subfraction C and subfraction D.

In view of the characterization of the fractions obtained by the two oxidation systems (H2O2, glucose oxidase), the two antibodies recognize different epitopes.

Indeed, the anti-fraction B antibody would recognize an early alteration caused mainly by HOCl while the anti-fraction C / D antibody would be directed against damage caused not only by HOCl but also by other reactions and / or radical rearrangements obtained by oxidation due to the MPO-glucose oxidase system. With the MPO / H2C> 2 system, no D fraction is observed.

Production of anti-fraction B specific monoclonal antibodies

A monoclonal antibody directed against a poorly oxidized epitope of LDL (fraction B) was characterized. The mice were immunized according to the same protocol as above, with the difference that the B antigen was injected in the presence of aluminum hydroxide. This fraction B was oxidized using MPO in the presence of H2O2. Four hybridomas AG9, AE2, EB2 and EF2 secreting monoclonal antibodies directed against fraction B of LDL were obtained.

The supernatants of the subclones of AE2, AG9, EB2 and EF2 were tested not only for their antigenic specificity against the different fractions but also for their belonging to the different classes and subclasses of immunoglobulins. These clones are of class IgG and of subclass IgG1, the light chain is of type K. These hybridomas secrete monoclonal antibodies of specificity directed against fraction B oxidized by rMPO. Three secretory hybridomas were gradually adapted to culture without serum in hybridoma medium SFM (Life Technologies).

The supernatants of these 3 secreting hybridomas were purified by passage through a column of Protein A-Sepharose CL-4B conditioned in PBS at pH 8. The elution was carried out in 0.1M citric acid at different pH, (6.5 IgGl - 4.5 IgG2a - 3 IgG2b).

Production of monoclonal IgG specific for fraction B

c ^ one mg total AG948F4A2 Fraction 1 1, 3 mg

Fraction 2 2.6 mg EB2E9G62IH2 4.53 mg EB2G3G2 2.6 mg

The recognition of the fraction B antigen oxidized by the 3 monoclonal antibodies is very similar and is illustrated in FIG. 3 (Box antigen)

The following table presents the characteristics of some monoclonal antibodies isolated in the context of the present invention.

4. ELISA test of naturally oxidized LDL circulating in plasma

ELISA tests measuring LDL oxidized by MPO in the circulation have thus been developed in the context of the present invention.

These tests do not require an ex vivo oxidation of LDL (constituting a very delicate phase), it is possible to carry out this test as a routine.

One of the tests according to an implementation of the invention can be summarized as follows: - fixation of the anti-fraction B monoclonal antibody on the plate - extraction of oxidized LDL from the plasma by adsorption on the monoclonal antibody

- recognition of LDL fixed using a polyclonal anti-apoB antibody

- revelation by a detection antibody - determination of the LDLox concentration in the sample using a standard range of fraction B of LDL oxidized by MPO and isolated by FPLC (Box fraction)

To confirm the feasibility of the test according to the invention, the standard of the fraction (Box) of oxidized LDL was applied at increasing concentrations (from 0 to 24 μg of protein per well). The 3 monoclonal antibodies (EB2E9G621H2, EB2-g3g2 and AG948F4A2) recognizing the oxidized fraction B were tested.

The results show a good response of the 3 antibodies to increasing concentrations of standards, linear in the first phase and reaching a ceiling for higher concentrations. This plateau can be explained by the upper detection limit of the test and / or by a disturbance due to the presence of NaCl in the Box fraction (greater when the Box concentration increases). Each curve has been reproduced several times with the same Box fraction and shows good reproducibility.

However, Box fractions deriving from two different oxidations do not generate identical standard curves. In fact, separation by FPLC allows separation as a function of the electric charge, but does not provide information on the chemical nature of the epitope. To

O

to obtain a pure Box fraction standard recognized specifically by the monoclonals, according to the invention, purification of the Box fraction is proposed by immunoaffinity (column composed of the monoclonal coupled to a matrix such as activated Sepharose CL-6B).

Furthermore, the conservation of the fractions can be favorably carried out in the presence of glycerol or sucrose.

The dose-response curve of native LDL (at plasma concentration) was also tested in parallel with the corresponding plasma. As expected, increasing concentrations of native LDL cause a proportional increase in the signal. However, the response obtained for plasma is zero or significantly lower than that of native LDLs. This indicates that one (or more) component (s) of the plasma can inhibit the recognition of LDLox by the monoclonals.

Among the plasma components capable of influencing the binding of LDLox with the monoclonal, two main categories can be distinguished: -lipoproteins, and in particular particles containing apoB (native LDL and VLDL) -proteins

The reliability of the test was confirmed, by verifying that apoB not modified by MPO, and present on LDL as well as on VLDL, could not compete with modified apoB, at the level of the monoclonal antibody.

The dose-response curve for native LDLs shows a signal that may correspond to the presence of a small proportion of oxidized LDLs in these native LDLs.

It has also been verified that albumin does not interfere with the antibody-antigen bond.

Several solutions are proposed to eliminate or minimize the interference observed in the plasma.

First of all, the sample can be pre-purified using semi-permeable membranes allowing the elimination of molecules up to 100,000 Da. On the other hand, IgG can be removed by immunoprecipitation techniques.

However, it has been observed that interference can be limited by diluting the sample. In fact, an increase in interference has been observed for small dilutions of the plasma, while larger dilutions tend to limit these disturbances. On the other hand, in parallel with a dilution of the sample, various techniques known per se make it possible to amplify the signal.

By way of example, a preferred variant of the test for direct measurement of circulating naturally oxidized LDLs is described below:

Sandwich ELISA test - coating of F 96 maxisorb plates (nunc immuno plate No 4 4 2304) with monoclonal antibodies (100 μΐ / well) plate no 1 Mab AG 948 F4 A2 (anti B) at 5.3 ng / well plate no 2 Mab EB 2 A 9 G 6 21 H2 (anti B), at 5.3 ng / well plate no 3 Mab EB2 G3 G2 (anti B) at 5.3 ng / well in the 50 mM carbonate / bicarbonate buffer, pH 9.6 - Saturation of the plates in casein 0.1% after washing in TBS Tween, 60 min at 37 deg - arrangement of the samples: plasma diluted 10 times in PBS pH 7.5 (H2O HPLC), 60 min at 37 deg , then washing with TBS Tween. Ideally a negative control and an oxidized standard B are also placed on the plate.

- fixation of the second antibody: polyclonal mouse antibody against apolipoprotein B 100 at 1 pg / ml, 60 min at 37 ° C.

- fixation of the coupled antibody: goat anti rabbit IgG (Fc) antibody coupled to alkaline phosphatase (Proméga, cat no S3731), diluted 7,500 times in casein buffer.

- revelation: using the alkaline phosphatase substrate: para nitrophenyl phosphate at 1 mg / ml in diethanolamine buffer pH 9.8.

It is found that the 3 antibodies provide comparable results.

An example of results is shown in FIG. 4 for 13 patients and a negative control (50 min of revelation) using the antibody EB 2 A 9 G 6 21 H2.

By reproducing the test with 50 × diluted plasma samples the results are identical.

Conclusion: The invention therefore demonstrates the feasibility and the advantages of the LDI assay, modified by the action of MPO in the circulation.

5. ELISA type test for measuring sensitivity to ex-vivo oxidation of LDL

According to the invention, the kinetic characteristics of the oxidation of LDL by myeloperoxidase are also advantageously used for diagnostic purposes.

An LDL oxidation was thus carried out on a microplate in order to develop one of the tests according to the invention based on the kinetics of the oxidation.

This test includes h steps:

1) Fixation of plasma LDL on a polyclonal anti-apo B antibody

2) Oxidation of LDL by the MPO / hydrogen peroxide system. 12.5 mU MPO were used in the presence of 40μΜ H202 per well, these conditions having then been modified to slow down oxidation, by reducing the amount of pro-oxidizing agents by 5 times and using 2.5 mU MPO and 8μΜ H202 per well.

3) Recognition of a specific LDL epitope oxidized by a monoclonal antibody according to the invention 4) Recognition of the monoclonal antibody by IgG coupled to alkaline phosphatase, anti mouse IgG.

In order to optimize the oxidation conditions which discriminate between patients and healthy people, we used the two monoclonal antibodies obtained (anti fraction B and anti fraction C / D) at our disposal.

The assay procedure is illustrated in FIG. 5.

Several series of oxidation were carried out on plasma from healthy volunteers and patients. The oxidation of LDL was followed over time by the appearance of epitopes B and C. It quickly appeared necessary to determine a threshold oxidation value defining normal or pathological oxidation. To this end, the measurement of a C / B ratio can constitute an internal standard of the reaction.

Twenty-five samples from healthy volunteers were tested to determine a “normal” mean C / B ratio, the mean obtained corresponds to a C / B ratio of 1.26 and the standard deviation is 0.16. The established “normal” range therefore corresponds to a C / B ratio ranging from 0.94 to 1.58 (mean ± 2 SD).

Plasmas from 33 patients were tested and compared to the "normal" range established on plasmas from "healthy" people. Eleven of them stood out from the “normal” (FIG. 6). Among the patients with a sensitivity to oxidation higher than the “normal”, it was particularly interesting to note that some did not present classic risk factors such as cholesterol and / or high triglycerides, or more recent as small LDL and dense or Lp (a), and that however they presented a severe coronary artery disease (unstable angina, infarction, ...).

This test therefore proved to be of great interest in primary prevention by highlighting a potential risk while the other parameters commonly used prove to be negative.

FIG. 6 thus illustrates a comparison of the sensitivity to LDL oxidation of 33 patients compared to a control population, the results being expressed as a percentage relative to the control.

The intra-test reproducibility, tested by carrying out several kinetics on the same plate, is entirely acceptable. On the other hand, the inter-test reproducibility, tested by comparison of the results obtained for the same sample on different plates, is poor. We will therefore introduce a control sample on each plate to be able to interpret the results obtained on different plates.

The kinetics obtained show that with the anti-fraction B antibody, the results are very reproducible while those obtained with the anti C / D present variations. It was indeed verified that the same individual taken at different times gave comparable results, by taking 3 blood samples several days apart from the same volunteer.

It can therefore be concluded that the measurement of the oxidation kinetics of LDL can be carried out under good conditions using the anti-fraction B antibody.

6. Elisa test for measuring circulating autoantibodies against oxidized LDL (fraction B)

According to yet another aspect of the invention, a test is proposed based on the detection of natural autoantibodies in the plasma, directed against LDLs oxidized by myeloperoxidase.

It has in fact been observed that the plasma contains autoantibodies directed specifically against the fractions of LDL oxidized by myeloperoxidase in the presence of H2O2, and not against the fractions obtained by oxidation with other agents such as copper sulphate, AAPH or an azoinitiator.

The level of autoantibodies recognizing these oxidized LDLs is a reflection of the oxidative status insofar as the modification of LDLs results in the appearance of new epitopes making the lipoproteins more antigenic. This assay also makes it possible to evaluate the capacity of the immune system to respond to the appearance of these modified LDLs.

According to the invention, therefore, an ELISA type test is proposed which makes it possible to quantitatively determine the presence of autoantibodies against specific subfractions in patients with cardiovascular pathology.

The following table indicates the responses observed for 5 patients, in the form of the ratios between the signal obtained with oxidized LDL and that of the corresponding blank, that is to say for native LDL. A ratio of 1 (or <1) means that the sample does not contain autoantibodies against oxidized LDL. Conversely, we can postulate that a ratio> 2 translates the presence of autoantibodies.

In 3 of the 5 subjects, the presence of autoantibodies against LDL oxidized by MPO is observed, while no subject appears to contain autoantibodies against LDL oxidized by copper or AAPH.

7, Specific immunoadsorption

According to another aspect of the invention, a radiation therapy method is proposed which consists in treating the blood of a patient in order to remove the antigens therefrom (LDL oxidized fractions, in particular fraction B). This method consists in passing the blood, or a fraction of blood, of a patient through a system where he will come into contact with immobilized oxidized LDL anti-fraction B antibodies, for example on an immunoadsorption column, before d to be returned to the patient, the blood or the fraction of blood being freed from said atherogenic antigens.

Claims (11)

1. Composition consisting of an oxidized fraction of LDL obtained by in vitro oxidation of LDL by hydrogen peroxide or the glucose / glucose-oxidase couple in the presence of myeloperoxidase. 2 Composition according to claim 1 purified by chromatography. 3. Composition according to the preceding claim obtained using a separation by chromatography of the Fast Protein Liquid Chromatography (FPLC) type. 4. Composition according to the preceding claim obtained using buffers of increasing salt concentrations. 5. Composition according to any one of the preceding claims which has also been purified by immunoaffinity. 6. Composition according to the preceding claim purified by immunoaffinity using a column composed of a monoclonal antibody coupled to a matrix and directed against said fraction. 7. Composition according to the preceding claim in which the column is an activated Sepharose column. 8. Composition according to any one of the preceding claims, characterized in that it constitutes sub-fraction B, sub-fraction of oxidized LDL closest to native LDL in Fast Protein Liquid Chromatography type chromatography with an exchange column. Mono Q anions. 9. Composition according to any one of claims 1 to 8 characterized in that it constitutes the sub-fraction C, sub-fraction of the oxidized LDL which follows the sub-fraction B in chromatography of the Fast Protein Liquid Chromatography type with a column anion exchanger Mono Q. 10. Composition consisting of an oxidized fraction of LDL obtained by in vitro oxidation of LDL by the couple glucose / glucose oxidase in the presence of myeloperoxidase characterized in that it comprises the sub-fraction D, sub-fraction of the most oxidized LDL distant from native LDL in Fast Protein Liquid Chromatography with a Mono Q anion exchange column 11. Composition according to claim 10 characterized in that it consists of a mixture of subfractions C and D. 12. Composition according to claim 1 or 2 in which the myeloperoxidase used is a recombinant human myeloperoxidase. 13. Monoclonal antibody directed against a composition according to any one of claims 1 to 12. 14. Method for obtaining oxidized fractions of LDL characterized in that the LDL are oxidized by addition of hydrogen peroxide in the presence of myeloperoxidase and the resulting oxidized fraction is separated and then fractionated by Fast Protein Liquid Chromatography (FPLC) chromatography using buffers with increasing salt concentrations. 15. Use of the oxidation fractions obtained according to the preceding claim for obtaining monoclonal antibodies. 16. Antigen constituted by an isolated oxidation fraction according to the method of claim 14. 17. Antigen according to the preceding claim constituted by fraction B. 18. The antigen according to claim 16 consisting of fraction C or fraction B, or a mixture of these LDL oxidation fractions. 19. Monoclonal antibody directed against one of the antigens of claims 16 to 18. 20. Use of an antibody according to claims 13 or 19 for an ex vivo oxidation test of LDL of a patient. 21. Use of an antibody according to one of claims 13 to 19 for a test for measuring circulating LDLox. 22. Use of an isolated oxidized LDL fraction as standard for the determination of oxidized antifraction autoantibodies in a patient. 23. Use according to claim 22 in which the oxidized fraction is fraction B. 24. Test, in particular for determining the cardiovascular risk of a patient, characterized in that it comprises the following stages: - fixing of an anti-fraction B monoclonal antibody on a solid substrate - extraction of oxidized LDL from plasma by adsorption on the monoclonal antibody - recognition of LDL fixed using a polyclonal anti-apoB antibody - quantitative revelation of the polyclonal antibody - determination of the LDLox concentration. 25. The test of claim 24 in which the quantitative revelation is carried out by the action of alkaline phosphatase coupled to an antibody directed against the anti-apo B polyclonal antibody. 26. Tests according to the preceding claim, in which the anti-apo B polyclonal antibody is a rabbit antibody and the antibody coupled to alkaline phosphatase is a goat anti-rabbit IgG (Fc) antibody. 27 Test according to claim 24, in which the anti-apoB polyclonal antibody is coupled to a peroxidase. 28. Test according to claim 24, in which the concentration of LDLox in the sample is determined by comparison with a sample or a standard range of fraction B of LDL oxidized by MPO and isolated by FPLC (Box fraction). 29. Test according to claim 28 in which the plasma is previously diluted between 5 and more than 40 times. 30. The test of claim 28 in which the IgGs from the plasma are eliminated by immunoprecipitation techniques. 31. A method for determining the circulating level of oxidized LDL, characterized in that it uses monoclonal antibodies obtained from at least a fraction of the LDL oxidized ex-vivo by myeloperoxidase. 32. Method for determining the level of autoantibodies directed against oxidized LDL, characterized in that it uses fractions of LDL oxidized by myeloperoxidase ex-vivo. 33. Method for determining the sensitivity to oxidation by MPO of LDL present in the plasma, characterized in that it uses monoclonal antibodies obtained from a fraction of LDL oxidized by myeloperoxidase ex-vivo. 34. Method according to any one of the three preceding claims, characterized in that the fraction is fraction B. 35. A diagnostic method for the prevention of cardiovascular accidents involving at least one of the three methods of claims 31 to 33. 36. Diagnostic method for assessing the severity of a cardiovascular risk of a patient, characterized in that it is based on the following three parameters: - circulating level of LDL oxidized by myeloperoxidase - rate of autoantibodies directed against LDL oxidized by MPO - sensitivity to oxidation by MPO of LDL present in the patient's plasma. 37. Pure standard for fraction B oxidized by myeloperoxidase, characterized in that it is obtained from said fraction by passage through an immunoaffinity column comprising an anti-fraction B monoclonal antibody. 38. Diagnostic application kit comprising at least - a monoclonal antibody against fraction B of LDL oxidized by myeloperoxidase. 39. Kit according to claim 35 also comprising a standard or a range of standards consisting of compositions according to claims 1 to 9. 40. Test, in particular for determining the cardiovascular risk of a patient, characterized in that it comprises the following stages: ~ fixation of oxidized LDL on a solid substrate fixation of a monoclonal antibody · anti-fraction B to the said fixed oxidized LDL association of the oxidized LDL from the plasma to the said antibody and release of the monoclonal antibody - determination of the monoclonal antibody remaining fixed - determination of the LDLox concentration. 11. Treatment method consisting in treating the blood or blood fractions of a patient in order to remove the oxidized LDL fraction B thereof by contact with immobilized anti-oxidized LDL fraction B antibodies, for example on an immunoabsorption column , before being returned to the patient.