WO2002024752A2

WO2002024752A2 - Monoclonal antibodies against lipid structures different from the double layer of cell membranes for defining physiological states in different cell types and detecting anti lipid particle antibodies in human beings and/or animals suffering from certain antiphospholipid antibody-related diseases

Info

Publication number: WO2002024752A2
Application number: PCT/MX2001/000069
Authority: WO
Inventors: María Guadalupe ORTEGA PIERRES; Sara Rocío FONSECA LIÑAN
Original assignee: Centro De Investigacion Y De Estudios Avanzados Del Instituto Politecnico Nacional
Priority date: 2000-09-01
Filing date: 2001-08-31
Publication date: 2002-03-28
Also published as: AU2001288136A1; WO2002024752A3

Abstract

The invention relates to the obtention of murine hybridomas that produce monoclonal antibodies against non double layer lipid particles also known as lipid particles in liposomes. The monoclonal antibodies are isotype IgM. Said antibodies were obtained by fusing spleen cells of female BALB/c mice that were immunized with liposomes of unilamellar phosphatidylcholine: phosphatidate (2:1) containing lipid particles induced by Mn+2 and P3X63AGBU.1 myeloma cell line. In the present invention, the use of said antibodies has been tested in ELISA assays to detect non dual layer lipid associations both in animal and plant cells. Hence its potential use in the prevention and treatment of diseases associated with the production of antiphospholipid antibodies.

Description

"MONOCLONAL ANTIBODIES AGAINST STRUCTURES

LIPIDS DIFFERENT FROM THE BICYPE OF CELL MEMBRANES

TO DEFINE PHYSIOLOGICAL STATES IN DIFFERENT TYPES

CELL PHONES AND FOR THE DETECTION OF LIPID ANTIBODIES PRESENT IN INDIVIDUALS AND / OR

ANIMALS WITH SOME DISEASES RELATED TO ANTIPHOSPHOLIPID ANTIBODIES "

FIELD OF THE INVENTION The present invention is related to obtaining hybridoma producing monoclonal antibodies that recognize lipids, and more particularly, to obtaining monoclonal antibodies against lipid structures other than bilayer as well as their use to determine the presence of these structures in particular physiological states of some cells, and in a system for detecting antibodies against lipid particles in samples of individuals with diseases related to the presence of antiphospholipid antibodies.

BACKGROUND OF THE INVENTION There are several studies in the state of the art in which evidence of antibodies that recognize lipids can be found. For example, they have been detected in the serum of patients with antiphospholipid antibody syndrome, as described by Asherson et al. in his book "The Antiphospholipid Syndrome" in 1996 (CRC Press, Boca Raton). Similarly, they have been obtained in animals that were experimentally treated with lipids by active immunization, as described by Alving in 1992 (Biochim. Biophys. Acta 1113: 307-322), or, in animals that received passive immunization antiphospholipid antibodies, as Tincani and Shoenfeld described it in 1996 in the book mentioned above.

Anti-lipid antibodies have been classified into two groups according to the method used for their determination. These groups are anti-cardiolipin antibodies and anti-coagulant antibodies.

Anti-cardiolipin antibodies are detected by methods where the lipid antigen, cardiolipin, is immobilized in a solid phase, as described by Harris et al. in 1985 (Clin Rheum. Dis. 11: 591-609), such as the solid phase immunoenzymatic assays and the immunoassay radio, better known by their acronym in English respectively as ELISA (enzyme-linked immunosorbent assay) and RIA (radioimmuno assay), which have been widely used in the prior art.

As regards anti-coagulant antibodies, these are detected by methods in which an increase in the coagulation time of blood plasma samples is measured in vitro, as established by Bevers, et. to the. in 1991 (Thromb. Haemost. 66: 629-632), such as the methods of activated partial thromboplastin time (TTPA), coagulation time of Russeli, protein C and Protein S, among others. In these tests, the anticoagulant antibodies bind to the phosphatidylethanolamine lipid, which is an intermediate factor in the coagulation cascade, so that by decreasing the concentration of this lipid, by the immune reaction, the time in that coagulation occurs.

As regards anti-cardiolipin antibodies, they have the disadvantage that they cross-react with other anionic lipids such as phosphatidylserine and phosphatidylglycerol. Due to this lack of specificity, the aforementioned antibodies in general are known as antiphospholipid antibodies.

Antibodies against phosphatidylethanolamine have also been detected in the serum of patients with antiphospholipid syndrome, as well as antibodies against phosphatidylcholine in patients with hemolytic anemia, as described by Sugi and Mcintyre (Blood 86: 3083-3089) and Arvieux et al. (Thromb.

Haemost 74: 1120-1125), respectively, in 1995.

On the other hand, some studies have shown that the binding of antiphospholipid antibodies to the lipid antigen increases in the presence of a plasma protein. For example, in 1990, McNeil and colleagues found that the binding of antibodies to cardiolipin increases in the presence of β ₂ -glycoprotein or apolipoprotein H (Proc Nat Acad Sci . USA 87:... 4120-4124).

Additionally, some anti-cardiolipin antibodies bind to β ₂ -glycoprotein directly, as described roubey et al. in 1995 (J. Immunol. 154: 954-960).

This suggests that anti-cardiolipin antibodies can recognize an epitope on the β ₂ glycoprotein that is exposed in the β ₂ glycoprotein-cardiolipin complex, and which also bind directly with the β ₂ glycoprotein, but with very low specificity, of agreement with Pengo et al. (1995, Thromb. Haemost. 73: 29-34).

According to the various studies carried out, it can be concluded that the binding of antiphospholipid antibodies with other lipid antigens is also associated with proteins. Sugi and Mclntyre (op. Cit., 1995) found that proteins called cininogens are involved in the binding of antibodies to phosphatidylethanolamine, while proteins that bind phosphatidylserine, such as prothrombin, protein C, protein S and annexin V, they have been implicated in the binding of anti-coagulant antibodies, as established in 1994 by Nakamura and his collaborators (Biochem. Biophys. Res. Commun. 205: 1488-1493), as well as by Roubey (Blood 84: 2854-2867) . These studies indicate that the antigen of some antiphospholipid antibodies is actually a complex formed by specific phospholipids and proteins. However, other studies have identified anticardiolipin antibodies that do not require β ₂ -glycoprotein in its reaction with cardiolipin by ELISA. Such studies were conducted by McNeil and his collaborators in 1989 (Br. J. Haematol. 73: 506-513), as well as by Pengo and Biasiolo in 1993 (Thromb. Res. 72: 423-430).

On the other hand, anti-cardiolipin antibodies, purified by affinity chromatography, do not exhibit anti-coagulant activity (McNeil et al., 1989; Shi et al., 1993, Blood 81: 1255-1262), although other studies have shown that both types of antibodies are removed by absorption with cardiolipin (Pengo and Biasiolo, 1993; Pierangeli, 1993, Br. J. Haematol. 85: 124-132).

In general of the aforementioned studies, you can Note that the antiphospholipid antibodies that have been described in human patients and experimental animals have a very varied specificity towards lipid antigens, which could be attributed to the methods of detecting said antibodies. In such methods, the structure and molecular association, as well as the chemical properties that lipidio antigens have in nature, have not been considered. Consequently, in the lipid antigens that have been used, phospholipids are bound to artificial solid supports as in the cases of ELISA and RIA, or in a molecular association that is not fully characterized, as in the tests of increased coagulation time

There are very few studies in which the molecular structure of phospholipids has been considered when used as antigens. For example, the works of Rauch et al. of 1989 and in 1998 (Thromb. Haemost. 62: 892-896 and Thromb. Haemost. 80: 936-941, respectively) and Berard et al. (J. Lab. Clin. Med., 1993, 122: 601-605), found that the serum of some patients with generalized lupus erythematosus is inhibited in their anticoagulant activity by the associated phosphatidylethanolamine in the tubular phase Hn and that this inhibition does not It was presented when the phospholipid was associated in bilayer. However, the properties of the cell membrane cannot be related to those of associated phospholipids in tubular form, because the tubular lipid association Hn is practically incompatible with the vesicular structure of the cell membrane, as established by various authors. That is, in the lipid antigens used in these studies, phospholipids They are found in a molecular form that does not correspond to that found in cell membranes.

In the studies in experimental animals, treated by active or passive immunization, the methods of detection of antiphospholipid antibodies that have been described for humans are applied. Further; the different organs and tissues are analyzed by anatomical, histopathological, immunofluorescence, and even fetal loss studies and consequently the lesions produced in the fetuses and in the placenta of the animals, females, under study. Studies of this type have been carried out by Tincani and Shoenfeld (Op cit. 1996) as well as by Shoenfeld and Ziporen (Lupus 7: SI 58-SI 61, 1998).

It is known that the molecular structure of the mammalian cell plasma membrane is of an association heteropolymer, consisting of phospholipids, glycolipids, cholesterol, proteins and glycoproteins, where lipids are generally found in the molecular association of bilayer. However, it is also known that lipids may have a different molecular association than bilayer, and that said association depends on the lipid geometry and the conditions at which they are found. Phospholipids and glycolipids are the only cell molecules that can self-assemble spontaneously in an aqueous medium and form specific molecular associations and constitute 60 to 70% of membrane lipids. If this characteristic is taken into account and the molecular form of lipids is found that the cylindrical lipids give a bilayer arrangement; Conical lipids are self-assembled in the form of tubes with the non-polar regions outward and the polar regions inward, this type of molecular association has been called hexagonal phase II (HH) (De Kruijff, 1987. Nature 329: 587-588; Rauch and Janoff, 1990. Proc. Nat. Acad. Sci, USA 87: 4112-4114; Baeza et al., 1995. Biochem Cell Biol. 73: 289-297). Inverted cone-shaped lipids give a micellar association, where the polar regions are located outside and the non-polar regions are located in the interior of the micelle. Conical and inverted cone lipids represent 30 to 40% of membrane lipids. In 1980 the Cullis group proposed the metamorphic mosaic model (Cullis et al., 1980: 1991), to try to explain the functions that different molecular associations can have in which lipids can be found in cell membranes. In this model, the membrane matrix is considered to be constituted by bilayer-associated cylindrical lipids, while conical lipids in general are also found in the bilayer molecular arrangement due to the presence of cylindrical lipids that stabilize this association, but It is proposed that conical lipids may form non-bilayer arrangements transiently when induced to be added to these associations by different molecules. In addition, in this model it is proposed that associations of non-bilayer or lipid particles could participate in cellular functions such as transport of ions and polar molecules through the cell membrane, at connection points between membrane vesicles, in membrane fusion processes. , such as endo and exocytosis, in the insertion of polar proteins in the membrane, in the formation of compartments and in the formation of pores through the bilayer that can allow the passage of polar molecules (Cullis et al., 1980; De Kruijff 1987; Cullis et al., 1991).

Similarly, it is known that in the presence of divalent cations, of drugs, such as chloropromazine and procainamide, of non-polar peptides, of proteins such as that of bacteriophage M13, of cholesterol, of lanthanum ions, as well as changes of temperature and pH, the conical lipids form different molecular associations to the bilayer, which are of a transitory nature, because when the concentration of the factors that induce their formation decreases, or the temperature decreases or the pH changes, the conical lipids return to the bilayer association, which is observed as a smooth surface in cryofracture analysis (Turnois, et al, 1990. Chem. Phys. Lipíds. 57: 327-340).

H 'and micellar lipid structures, as well as any other lipid structural arrangement that does not form a bilayer, are considered, for the purposes of the present invention, as lipid structures other than the bilayer, or, as lipid particles, regardless of the nature of the lipids that form them.

Lipids in general are poorly immunogenic molecules (Hughes, et al., 1986. J. Reumatol., 13 (3) 486-489; Alarcón-Segovi, 1991. Sem. Clin. Immunol., 1: 11-19), and of the two molecular associations that lipids can present in the membranes, it is considered that the bilayer will be the least immunogenic because it is the one that constitutes the membrane matrix (Kleinfeld, 1991. Membrane Fusion. Ed. Jan W. Dic H. Inc New York, pp 3-33).

However, it is known that these lipid structures, stabilized with divalent cations, induce the formation of antibodies that recognize the associated lipids in lipid particles and do not react with lipid in bilayer

(Aguilar, 1994. Master's Thesis).

In relation to the above, Baeza et al., In 1995 (Biochem. Cell Biol.

73: 289-297), reported the development of liposomes with structural arrangements different from the bilayer, as well as an antigenic activity thereof when obtaining polyclonal antibodies. By means of cytofluorometric analysis of the immune reaction it was possible to identify the presence of lipid structures in the liposomes described therein, using polyclonal antibodies against lipid particles obtained from sera of mice. Notwithstanding the foregoing, the immunization of the mice was performed by the introduction of artificially formed lipid particles so that an excess of lipid particles will cause the desired immune reaction (Nilsson et al; 1987. J. Immunol, Methods 99: 67-75 ). To date, it is believed that molecular associations other than bilayer or lipid particles would also be poorly immunogenic when they are present in nature, for example in human and animal cells, because they are transient and therefore would not be detected. by the immune system.

Additionally, from the analysis of all the above-mentioned studies, it can be observed that cardiolipin is the only lipid that has itself been able to react with antibodies present in patients presenting with antiphospholipid syndrome or related diseases, and that phospholipids normally present in the cell membrane mentioned above, they need to be associated with proteins to react with the antibodies of said patients, or they need to be associated in an arrangement molecular incompatible with the cell membrane (Hughes, 1991. Sem. Clin. Immunol. 1: 5-9).

In this regard, the presence in sera of patients with the anti-phospholipid syndrome of anti-cardiolipin antibodies, an itochondrial lipid, anti-nuclear antibodies and anti-DNA antibodies, is indicative of the existence of previous events that cause damage to the membranes. , with the breakdown of the cells and the exposure of the intracellular components to the immune system, causing the corresponding reaction and, therefore, the syndrome (Asherson et al., 1996. J. Rheumatol. 15 (2) 1742-1745; Hughes, 1991. Sem. Clin. Immunol. 1: 5-9). However, to date there have been no studies to determine what are the events that cause the breakdown of the cell membrane. In this context they propose to give hypotheses that could explain the formation of antiphospholipid antibodies.

The first is that factors not yet described cause the destruction of the cell membrane, which promotes the formation of lipid particles from the membrane lipids that come into contact with the immune system together with the intracellular components, with the consequent formation of anti-lipid, anticardiolipin and antinuclear antibodies. The second hypothesis is to assume that lipid particles are formed in the cell membrane before their destruction, so anti-lipid particles antibodies would be formed that would destroy the membrane, exposing the cellular components to the immune system and subsequently giving rise to the formation of anticardiolipin and antinuclear antibodies. To date, neither of the two hypotheses has been demonstrated, so having a system that allows detecting non-bilayer arrangements of conical phospholipids in the cell membrane is essential. For this reason, a method for obtaining hybridomas that produce specific monoclonal antibodies against lipid structures other than the bilayer of cell membranes was proposed. These monoclonal antibodies constitute a very specific and irreplaceable biological reagent for the unequivocal detection of non-bilayer arrays in non-cellular (liposome) and cellular systems, since the. Polyclonal antibodies that can be obtained against these structures may give nonspecific reactions. On the other hand, in the production of monoclonal antibodies, liposomes can be used in which it is possible to reproduce the molecular arrangements that lipids can have on cell membranes (Baeza et al., Op. Cit., 1994; Ibañez, et al., , op. cit., 1996). Also in the liposomes its lipid composition can be controlled and induce changes of the lipid molecular associations and since the monoclonal antibodies that are obtained do not contain proteins only recognize the non-bilayer associations.

On the other hand, the use of monoclonal antibodies is proposed as an unambiguous indicative and positive control necessarily included in a system for detecting antibodies against lipid particles in biological samples of individuals who have diseases related to the presence of antiphospholipid antibodies. Likewise, the detection of anti-lipid particles antibodies can also be performed according to. the scheme described for humans, plants and in experimental animals in which the formation of anti-lipid particles antibodies.

OBJECTIVES OF THE INVENTION

Taking into account that the specific detection of no-bilayer arrangements of conical phospholipids in cell membranes is very necessary to determine the metabolic processes in which they could participate, as well as the factors that induce and stabilize them in such a way that they are recognized by the immune system as auto antigens and induce autoantibodies in individuals with diseases that are related to the presence of antiphospholipid antibodies, it is an object of the present invention to obtain hybridomas producing monoclonal antibodies that specifically recognize the lipid molecular associations of non- bilayer formed by different conical lipids.

A further object of the present invention is to provide a method for obtaining specific monoclonal antibodies against lipid structures other than bilayer cell membranes.

Another object of the present invention is to provide these antibodies for the detection of non-bilayer lipid molecular associations formed by different conical lipids in the membranes of animal and plant cells that exhibit particular physiological states.

As another object of the present invention, the specific biological elements necessary (monoclonal antibodies, liposomes containing defined lipids) are provided for the detection of lipid antiparticle antibodies in early stages of diseases in humans and / or animals that are related to the presence of antiphospholipid antibodies, as well as in experimental models, such as mice, in which antiphospholipid antibodies are developed by any method.

The present invention provides as an additional object an in vitro diagnostic kit or instrument for the detection of antibodies against early lipid particles in early stages of diseases presenting such antibodies in animals, plants and humans.

The present invention also has the object of providing pharmaceutical compositions containing therapeutically effective amounts of the monoclonal antibodies for the treatment of diseases related to lipid anti-particle antibodies, by blocking antibodies against lipid particles or by stabilizing cell membranes. As well as its therapeutic uses and applications in the treatment of said diseases.

DETAILED DESCRIPTION OF THE INVENTION As described above, the presence of lipid particles in cell membranes may be transient or may be more stable due to pathological or deficient conditions that occur in an individual. If this occurs, it is possible that non-bilayer associations can be immunogenic and thus induce the formation of antibodies with the consequent damage to the membranes of cells and tissues, in which these associations are presented. In this context it is necessary to detect the presence of non-bilayer molecular arrangements formed by phospholipids Conical in cell membranes in particular physiological states, determine the metabolic processes in which they could participate and the factors that can induce and stabilize them.

It is also important to be able to detect antibodies that are produced in experimental animals and in humans who are suffering from diseases related to the production of antiphospholipid antibodies.

In the latter context, by using an experimental model in BALB / c mice, which were immunized with lipid particles, the production of anti-lipid particles antibodies could be detected at an early stage after the immunization of the animals began. It is important to note that the detection of these antibodies was only possible through the use of specific monoclonal antibodies against lipid particles (Aguilar et al. J. Biol. Chem. 274; 25193-25196) and a liposomal ELISA system ( Baeza et al., 1995. Biochem. Cell Biol., 73: 289-297).

These antibodies were detected before those produced towards cardiolipin, than the lupus anticoagulant and than antinuclear antibodies. These data suggest that anti-bilayer anti-array antibodies when presented at early stages after immunization constitute the first phase in the development of clinical manifestations in immunized mice. These animals developed alopecia and butterfly wing lesions similar to those described for some autoimmune diseases in humans as well as deposits of immune complexes and pathological alterations of different organs. It should be noted that for the purposes of the present invention, disease related to antiphospholipid antibodies is understood as any disease that has antiphospholipid antibodies at any stage of development. Some of these diseases are mentioned below, but not limited to: primary or secondary antiphospholipid antibody syndrome (SAAF); autoimmune diseases such as vasculitis, rheumatoid arthritis and systemic lupus erythematosus (SLE); diseases that cause an increase in cell division, such as neoplasms of the type of carcinoma in the liver or ovary, lymphomas, leukemia or myeloproliferative disorders; viral infections such as infectious mononucleosis and acquired immunodeficiency syndrome; diseases caused by bacteria, such as syphilis; and, diseases caused by protozoa such as malaria.

Additionally, the presence of antiphospholipid antibodies has been related to myocardial infarction and senescence (Galli et al., 1990), so the detection of anti-lipid particle antibodies also allows the establishment of cellular physiological states, where cellular physiological states they will fall within the concept of diseases related to antiphospholipid antibodies for the purposes of the present invention. The methods used for the purposes and purposes of the present invention are described below, which are considered within the scope of protection of the claimed invention.

Therefore, one aspect of the present invention is the obtaining of hybridomas that produce specific monoclonal antibodies that recognize non-bilayer lipid molecular associations, formed by different conic lipids. For this, syngeneic mice of the strain are used

BALB / c females of 2 months old. From the immunized mice, it is possible to obtain the monoclonal antibodies by any known means, preferably by fusion of murine origin P3X63 Ag8U.1 cells with immune mouse spleen cells using polyethylene glycol for cell fusion with the consequent obtaining of a hybridoma. In a specific embodiment, the hybridoma is obtained according to the following stages: A) A first stage of immunization of mice intrasplenic with an effective dose of lipids such as phosphatidylcholine liposomes: phosphatidate (2: 1), where said liposomes contain lipid particles induced with Mn ²⁺ .

B) A second stage of immunization of mice intraperitoneally with the same liposomes and with the same dose as those used for the first stage of immunization.

C) A third stage of immunization of mice intravenously with the dose of liposomes used in the first stage of immunization. D) A stage of fusion of spleen cells of immunized mice as described above with a mouse myeloma called P3X63 Ag8U.1 derived from the P3X63 Agδ line obtained by Kohler and Milstein from the MOPC21 myeloma of female BALB / c mice. This line does not synthesize strings. gamma but produces kappa chains. This fusion is carried out at least four days after the intravenous immunization step to obtain at least one hybridoma producing a monoclonal antibody against lipid particles. E) A step of selecting hybridomas that show acceptable immunoreaction by detecting antibodies against lipid particles generated by them by means of cytofluorometry and / or the ELISA method. In a further embodiment, the first stage of immunization comprises administering the liposomes at least 2 times with intervals of 1 week, and the second stage of immunization comprises introducing the liposomes at least 4 times with intervals, of 2 weeks, by the method described by Nilsson et al. in 1987 (J. Immunol. Methods, 99, p. 67-75). In another additional embodiment, the mice used for immunization are selected from syngeneic strains, preferably using mice of the female BALB / c strain 2 months old.

In a preferred embodiment of the present invention, spleen cells are obtained as described by Ortega-Pierres et al. (1984. Parasitology, 88: 359-369), by disintegrating the mouse spleen in a cell culture medium specific for the cultivation of Dulbeco Modified Eagle Medium hybridomas (DMEM Hibrimax), preferably incomplete, followed by various purification steps and a Erythrocyte lysis, preferably by ammonium chloride. The hybridoma obtained by the aforementioned method was deposited in the National Collection of Microorganism Crops (CNCM) of the Pasteur Institute in Paris, France in accordance with the provisions of the Budapest Treaty for the deposit of biological material in the field of patents, remaining said hybridoma registered, and consequently, the monoclonal antibody that is produced therein, under the CNCM H308 I-2537 deposit.

As regards the method for performing the fusion, the one described by Ortega-Pierres et al. (op. cit., 1984), which consists of using spleen and myeloma cells with a viability greater than 85%, which are centrifuged and mixed in a 1: 1 cell ratio, to be subsequently subjected to the action of the polyethylene glycol 4000 fusing agent and after being washed they are grown in cell culture plates previously seeded with macrophages. In relation to the selection method, ELISA is preferably used to identify the monoclonal antibody producing clones. After selection, the positive clones are expanded in vitro and in vivo (inoculation of animals) to obtain monoclonal antibodies.

In a particular embodiment of the invention there is included a method for the characterization of monoclonal antibodies that recognize non-bilayer lipid molecular associations in terms of isotype and specificity.

In the typing of monoclonal antibodies a commercial immunoenzymatic system (Boehringer Mannheim) is used that contains the reagents necessary to carry out the development of the technique.

Regulator for gluing the antigen, washing regulator, detergent, mouse anti-immunoglobulin antibodies IgA, IgG, IgGI, lgG2a, lgG2b, IgM and light gamma and kappa anti-chains all conjugated to peroxidase, said method is characterized by the following steps:

A) In a first stage, in supernatants of hybridoma cultures, they are diluted 1: 10 in a wash regulator and 50 μl of these dilutions are added to each immunoplate well. and B) In a second stage, an incubation of the plates is carried out at 30 'at 37 ° C.

C) A step of eliminating the solution preferably by suction and washing of the immunoplate.

D) In the third stage 200 μl of the wash regulator are added and the plates are incubated 15 'at 37 ° C. E) A stage of washing the plates with regulator of washing and suction thereof.

F) A fourth stage in which 50 µl of the peroxidase-conjugated antibodies are added to each well in 1:10 dilution, incubating at 37 ° C for 30 '. G) A stage of washing the plates with regulator of washing and suction thereof.

H) A fifth stage in which an effective amount of the peroxidase substrate solution is added by incubating at 37 ° C for 30 'by means of an effective amount of sulfuric acid. I) A stage of analysis of the plates in an ELISA reading device preferably at 492 nm. The use of the methodology results in the typing of monoclonal antibodies as of the IgM class. In a preferred embodiment, the specificity of the monoclonal and polyclonal antibodies that recognize non-bilayer lipid molecular associations by liposomal ELISA is determined in this modality, a monoclonal antibody of the IgM isotype with specificity totally different from that of the monoclonal antibodies against lipid particles.

The ELISA assay is carried out using liposomes that have L-α phosphatidylcholine and L-α phosphatidic acid according to the method described for liposomal ELISA, in this modality the hybridoma supernatant containing monoclonal antibodies against lipid associations without dilute or a 1: 2 dilution, sera collected from animals immunized with lipid particles are diluted 1: 50.

The results show that the monoclonal antibodies have a greater reactivity with the non-bilayer structures induced by Mn ²⁺ , while the polyclonal antibody binds with less avidity and recognizes the smooth liposome structures. The non-specific monoclonal antibody does not show any reaction with smooth liposomes or having a non-bilayer structure. Because in the routine tests to measure antiphospholipid antibodies, plates covered with lipids dissolved in ethanol are used, the binding of the monoclonal antibody to phospholipids was determined purified as L-α phosphatidylcholine, L-α phosphatidic acid and cardiolipin. The results show that the monoclonal antibody does not interact with plates coated with these lipids while the polyclonal antibody does. These results strongly suggest that monoclonal antibodies interact with non-flat lipid surfaces.

In a preferred embodiment of the purification of monoclonal antibodies, the boric acid precipitation method of the monoclonal antibodies present in the ascites of animals inoculated with the hybridomas is used, characterized by the following steps: A) A first stage consists in adding boric acid (2%) to the ascites fluid that contains the monoclonal antibodies. In this, for each 20 ml of boric acid, 2 ml of the ascites liquid are added and the solution is stirred for 2 hrs in an ice bath to allow the precipitation of immunoglobulins. B) In a second stage the suspension is centrifuged at 45,000 X g for 20 min. at 4 ° C.

C) In a third stage the precipitate of monoclonal antibodies is resuspended in a minimum volume of phosphate saline. D) In a fourth stage the solution containing the antibody is extensively dialyzed with phosphate saline to remove excess boric acid.

E) In a fifth stage the amount of protein in the solution containing the antibodies is determined, preferably by the method of Lowry (Lowry et al, 1951. J. Biol. Chem. 193-265-275).

F) In a sixth stage the protein is analyzed by electrophoresis in polyacrylamide gels (SDS-PAGE) by the method described by Laemmli (Laemmli, 1970. Nature, 227: 680-685). Figure LA and B.

Another aspect of the present invention is a method for determining whether a subject that does not have anticardiolipin, lupus anticoagulant, anti-DNA or antinuclear antibodies, has a disease related to the presence of antiphospholipid antibodies, wherein said method comprises detecting directly or indirect the presence or absence of antigens containing lipid particles in a sample of the subject; and observe whether or not the lipid particles are detected, where the presence of the lipid particles indicates that the subject is developing a disease related to the presence of antiphospholipid antibodies. In a preferred embodiment, the detection of lipid particles is carried out indirectly by the use of an antigen containing lipid particles that is reacted with the subject's serum in order to determine if said serum contains anti-lipid particles antibodies, such determination, preferably performed by using at least one technique selected from cytofluorometry, immunofluorescence and ELISA.

In a specific embodiment, the antigen containing lipid particles is selected from neoplastic cells and liposomes, where liposomes are formed from at least one lipid capable of changing geometry by means of temperature changes, presence of divalent ions and /or presence of drugs, said lipid being preferably selected from phosphatidate; cardiolipin; phosphatidylglycerol, phosphatidylinositol; phosphatidylcholine; phosphatidylserine; sphingomyelin; and, diglycosyldiacylglycerides. In a preferred embodiment, the lipid is found in abundance in the cell membrane. In a specific embodiment, the lipids used to form the liposomes are selected according to their availability in the cell membrane, preferably using a cylindrical lipid in combination with a conical lipid in a molar ratio between 1: 1 and 4: 1. In a further embodiment, a combination of phosphatidylcholine with egg yolk phosphatidate in a 2: 1 molar ratio is used.

In another additional embodiment, in addition to the subject's serum, it is reacted with the antigen at least with a monoclonal antibody against lipid antiparticles to confirm the presence or not of the lipid antiparticle antibodies in the subject's serum. In another preferred embodiment, the detection is carried out directly by reacting the subject's cells with at least one monoclonal antibody against lipid particles, preferably by using the indirect immunofluorescence technique.

In a further embodiment, in addition to the subject's cells, at least one antigen containing lipid particles is reacted with the antibody at the same time, preferably selected from neoplastic cells and liposomes of at least one lipid capable of changing geometry by means of temperature changes, presence of divalent ions and / or presence of drugs, said lipid being preferably selected between phosphatidate; cardiolipin; phosphatidylglycerol, phosphatidylinositol; phosphatidylcholine; phosphatidylserine; sphingomyelin; and, diglucosyldiacylglycerides.

To obtain the liposomes that are used in various embodiments of the present invention, the reversible phase evaporation method described by Baeza et al. Is preferably used. (op. cit.) and subsequently treated with a lipid particle forming agent, preferably selected from divalent cations, drugs and combinations thereof, wherein the treatment to form lipid particles is preferably performed by incubating the liposomes with a effective amount of the lipid particle forming agent at a temperature of 25 to 40 ° C, said effective amount preferably being a lipid molar ratio: lipid particle forming agent from 1:50 to 1: 1000.

Regarding the methods of detecting lipid antiparticle antibodies in patient sera by using antigens containing lipid particles, or detecting lipid particles in patient cells by using anti-lipid particle antibodies, below. Preferred techniques for such detections will be described.

On the one hand, the ELISA-liposomal method. It comprises the following steps: A) A first step of addition, and incubation, in which an effective amount of an antigen suspension is added to each well of an ELISA immunoplate with high antigen binding property and said immunoplate Incubate at room temperature for 0.5 to 1.5 h. B) A second stage of addition and incubation, in which an effective amount of a blocking solution is added to each of the 10 wells of a high antigen-binding ELISA immunoplate and said immunoplate is incubated.

5 at room temperature for 0.5 to 1.5 h.

C) A step of removing the blocking solution, preferably by suction.

D) A third step of addition and incubation, in which an effective amount of the antibody carrier is added to each

10 one of the wells of the ELISA immunoplate in a carrier dilution. Blocking solution from 1: 5 to 1:75, said immunoplate incubating for 0.5 to 1.5 h at a temperature between 35 and 40 ° C.

E) A first step of washing the immunoplate with the blocking solution, preferably repeating 4 times.

F) A fourth stage of addition and incubation, in which an effective amount of an antibody selected preferably from goat anti-IgG, IgA and IgM or IgM anti-goat antibodies is added to each well of the ELISA immunoplate

20 mouse, conjugated to peroxidase, in a final dilution between 1: 25 and 1: 2000, said immunoplate incubating in the dark for 0.5 to 1.5 hours at a temperature between 35 and 40 ° C.

G) A fourth stage of addition and incubation, in which an effective amount of the peroxidase substrate is added to each of the ELISA immunoplate wells and said plate is incubated for 0.1 to 0.5 hours at a temperature between 35 and 40 ° C, the peroxidase reaction being stopped by means of an effective amount of sulfuric acid. H) A step of analyzing the plates in an ELISA reading set, preferably at 492 nm. In a specific embodiment, the antigen suspension is obtained by suspending the antigen in a pH regulating solution, at pH 7, in a ratio of 0.001 to 0.05 moles of antigen per liter of pH regulating solution.

Likewise, the blocking solution comprises a pH regulator, pH 7, and 4% by weight of a solution with a high protein content, preferably gelatin, with or without an effective amount of a lipid particle forming agent, ' preferably with the effective amount and the lipid particle forming agent used to form the antigen.

In a preferred embodiment, the effective amount of the antigen suspension in step A) is from 50 to 100 µl.

On the other hand, the liposomal cytofluorometry method (Baeza et al., Op. Cit.), As the name implies, is applicable in those cases where the antigen is a liposome, regardless of the origin of the antibody carrier, and comprises The following stages:

A) A first step of addition and incubation, in which the antibody carrier is added to an antigen suspension, said carrier being at a dilution from 1: 5 to 1: 75, the mixture obtained being incubated for 0.5 to 1.5 h at a temperature between 35 and 40 ° C.

B) A first step of washing the antigen with a pH 5 regulating solution, pH 7, with or without an effective amount of a lipid particle forming agent, preferably with the effective amount and the forming agent used to obtain the antigen.

C) An antigen recovery step, preferably by centrifugation.

D) A second step of addition and incubation, in which an effective amount of an antibody preferably selected from goat anti-IgG, IgA and IgM or anti-mouse IgM anti-mouse IgM antibodies is added to the centrifuged antigen

A fluorescent substance or substrate, preferably fluorescein isothiocyanate (FITC), to have a final dilution between 1: 25 and 1: 3500, the obtained mixture being incubated for 0.5 to 1.5 hours in the dark at a temperature between 35 and 40 ° C .

E) A second stage of antigen washing with a pH regulator 20, pH 7, with or without an effective amount of a lipid particle forming agent, preferably with the same amount and the same forming agent used to obtain the antigen.

F) A suspension and analysis stage, in which the antigen is suspended in a developer solution, preferably selected from FACS Flow (Beckton Dickinson Co.) and

Haema Une 2 (Serotono-Baker Diagnostics, 1 NC), in a ratio from 0. 1 to 10 moles of the antigen in 1000 ml of the said solution. solution being preferably filtered previously with a 0.22 μm diameter filter: pore, analyzing the mixture obtained in a flow cytometer, preferably with 488 nm argon laser beam.

In a preferred embodiment, the antigen suspension is obtained by suspending the antigen in a pH regulating solution, at pH 7, at a rate of 0.5 to 5 moles per liter of pH regulating solution.

As regards the methods of detecting non-lipid particles in cells in particular physiological states, the indirect immunofluorescence method for cells, applicable when the antigen is a cell, comprises the following steps:

A) A first stage of addition and incubation, in which an effective amount of the antigen, preferably IxIO ³ cells, is placed in an object cover inside a cell culture box and incubated in an atmosphere containing an effective amount of C0 ₂ at a temperature between 35 and 40 ° C until reaching 90% cell confluence.

B) A first washing step consisting of washing the antigen with a suitable culture medium, preferably repeating 2 times, and with a phosphate regulating solution, pH 7.4, in sterile conditions.

C) A second stage of addition and incubation, in which an effective amount of an antibody carrier is added to the antigen, preferably 50 to 200 μl undiluted, or, with a maximum dilution of 1: 2, incubating in a atmosphere containing an effective amount of C0 ₂ for 0.5 to 1.5 h at a temperature of 35 to 40 ° C.

D) A second washing stage consisting of. wash the antigen with a phosphate regulator solution, pH 7.4, at least 3 times.

E) A third stage of addition and incubation, in which

(add an effective amount of an antibody to the antigen

. preferably selected from goat anti- antibodies

IgG, IgA and IgM or anti Fe mouse IgM, conjugated to FITC, the antigen being incubated in an atmosphere containing an effective amount of C0 ₂ for 0.5 to 1.5 h at a temperature of 35 to 40 ° C.

F) A third washing step consisting of washing the antigen with a phosphate regulator solution, pH 7.4, preferably repeating 3 times.

G) An analysis stage in which the coverslip is mounted on a slide to be observed in a microscope with epifluorescence and Nomarski optics.

In a specific modality, the effective amount of C0 ₂ is 5% in volume with respect to air, while the effective amount of phosphate regulatory solution is 10 ml.

It should be noted that in the description of said techniques the term "antibody carrier" refers to any fluid capable of containing antibodies against lipid particles, such as a serum, a solution or a suspension, while the term "antigen" is refers to those structures that may contain lipid particles such as liposomes or cells.

Additionally, it is important to note that in a specific embodiment of the present invention, before starting any detection method, an inactivation of the system is carried out by temperature, preferably subjecting it to temperatures between 50 and 60 ° C for 15 to 60 minutes.

Another aspect of the present invention is an in vitro diagnostic kit or instrument for diseases related to antiphospholipid antibodies useful for performing the method of the present invention that includes at least one reagent indicating the presence of lipid particles and / or antibodies. lipid anti-particles in a sample of a subject that does not have anticardiolipin, lupus anticoagulant, anti-DNA or antinuclear antibodies; means for allowing the reaction of the sample with the reagent; and, means to make said reaction evident.

In a preferred embodiment, the reagent is selected from liposomes with lipid particles on their surface, neoplastic cells and monoclonal antibodies against lipid particles. The preferred way of applying the present invention is by supplying the monoclonal antibodies presented herein or the pharmaceutical composition containing them, wherein a therapeutically effective amount is provided with a convenient unit dose regimen to an individual suffering from diseases related to antiphospholipid antibodies, said regimen can be adjusted in accordance with the degree of affliction.

The preparation of a pharmaceutical composition that includes the monoclonal antibodies of the invention can be carried out by using standard techniques well known to those skilled in the art in combination with any of the pharmaceutically acceptable carriers described in the state of the invention. technique, including, but not limited to starch, glucose, lactose, sucrose, gelatin, malt, rice, wheat flour, chalk, silica gel, magnesium stearate, sodium stearate, glyceryl monostearate talc, sodium chloride, glycerol, propylene glycol, water, ethanol and the like. These compositions may take the pharmaceutical form of solutions, suspensions, tablets, pills, capsules, powders and prolonged release formulation and the like.

The above description and the following examples are intended to illustrate particular ways of carrying out the invention and should not be considered as limiting the scope of protection thereof.

EXAMPLES

Example 1. Obtaining hybridomas by fusion of P3x63Ag8U.1 cells with Spleen cells of a mouse BALB / c producing antibodies against lipid particles.

The spleen of a female BALB / c mouse in whose serum sample anti-lipid particles antibodies were detected in a 1:40 dilution, was extracted under sterile conditions and placed in a petri dish, adding 6 ml of incomplete DMEM medium . With blunt-tipped tweezers, it disintegrated until a cell suspension was obtained, which was transferred to a 15 ml falcon tube and left to rest so that the thick debris settled. The cell suspension was then transferred to another falcon tube and centrifuged at 17 x g for 7 min. It was then decanted and the cells were resuspended by gentle agitation and diluted by dropwise aggregation of 10 ml of incomplete DMEM medium. The cell preparation was centrifuged as indicated, decanted and 4 ml of 0.16 M NH4CI was added to lyse the erythrocytes. In this step the tube was incubated at 37 ° C and rotated gently for 4 min. Subsequently, 6 ml of incomplete DMEM medium was added and centrifuged at 17 xg for 7 min, it was decanted and the cell package was resuspended in 10 ml of incomplete DMEM medium that was kept at room temperature until use (Kohier and Milstein, 1975) .

On the other hand, the P3x63Ag8U.I myeloma cells were collected from the culture boxes in falcon tubes and aliquots were taken that were treated with the blue trypan dye and counted, as were the spleen cells of the mouse BALB / c immune, in a Neubauer chamber. The viability of both cell types was greater than 85%. The cell populations of P3x63Ag8U.I and immune mouse spleen were centrifuged at 17 xg for 5 min. Later mixed in a 1: 1 cell ratio, with 36 x 106 cells of each type and washed with 10 ml of incomplete DMEM medium. Once this was done, it was decanted, the culture medium was removed and the cell packet disintegrated gently. Subsequently, 1 ml of the polyethethylene glycol 4000 solution was added dropwise over 1 min, stirred manually for 1.5 min and 1 ml of incomplete DMEM medium was added in 30 sec, with slow tube rotation. Next, 3 ml of incomplete DMEM was added for 30 sec with tube rotation, then 16 ml of the same medium was added for 1.5 min and with gentle agitation, finally the volume was taken to 40 ml and incubated without stirring for 5 min. at room temperature. It was subsequently centrifuged at Í7 xg for 5 min. It was decanted and washed once with 40 ml of incomplete DMEM medium. The cell packet was resuspended in 30 ml of the DMEM-HAT selection medium, 100 µl aliquots of this cell suspension were taken and three cell culture plates were placed in the 96 wells of each cell 24 hours before the fusion. seeded with macrophages. The culture plates were incubated at 37 ° C, in an atmosphere with 5% C0 ₂ . At 5 and 8 days after the fusion, the hybrids were fed with 50 μl of DMEM-HAT medium, at 11 days the medium was changed to 100 μl of DMEM-HT. When hybrids developed, supernatants were taken and analyzed by the ELISA-liposomal method. Of all the hybrids that gave positive results, cell samples were taken and frozen at -70 ° C in liquid nitrogen. Subsequently, 8 hybrids were chosen that gave the highest intensity immunoreaction and transferred in duplicate to plates of 24-well cell culture, to obtain a greater amount of supernatants and to determine the presence of anti-lipid particles antibodies by ELISA-liposomal.

Hybridomas that gave higher titers of lipid antibody were cloned back into 96-well cell culture plates. The hybrids that were developed were analyzed to detect those producers of anti-lipid antibodies by ELISA-liposomal, and those that gave the highest titers of these antibodies were grown in 250 ml bottles for the mass production of supernatants containing said antibodies.

Likewise, the hybridomas were inoculated into BALB / c mice to obtain ascites fluid containing a high concentration of monoclonal antibodies. Monoclonal antibodies were characterized as for the isotype and purified by precipitation with boric acid for use in liposomal ELISA, cytofluorometry and indirect immunofluorescence assays.

Example 2. Determination of the specificity of monoclonal and polyclonal antibodies that recognize non-bilayer lipid molecular associations by liposomal ELISA. In this test the specificity of monoclonal antibodies against lipid particles was analyzed. For this, the ELISA assay was used using liposomes with defined lipids, monoclonal and polyclonal antibodies against lipid particles and a monoclonal antibody of the IgM isotype with specificity totally different from that of the monoclonal antibodies against lipid particles (Ortega-Pierres, et al., op cit. 1984).

The ELISA assay is carried out using liposomes that have L-phosphatidylcholine and L-α phosphatidic acid according to the method described for liposomal ELISA, in this modality the hybridoma supernatant containing monoclonal antibodies against undiluted lipid associations was used or a 1: 2 dilution, sera collected from animals immunized with lipid particles are diluted 1: 50.

The results show that the monoclonal antibodies have a greater reactivity with the non-bilayer structures induced by Mn ²⁺ , while the polyclonal antibody binds with less avidity and recognizes the smooth liposome structures. The non-specific monoclonal antibody does not show any reaction with smooth liposomes or having a non-bilayer structure. Because in the routine tests to measure antiphospholipid antibodies, plates covered with lipids dissolved in ethanol are used, the binding of the monoclonal antibody to purified phospholipids such as L-phosphatidylcholine, L-α phosphatidic acid and cardiolipin was determined. The results show that the monoclonal antibody does not interact with plates coated with these lipids while the polyclonal antibody does. These results strongly suggest that monoclonal antibodies interact with non-flat lipid surfaces. Figure 2. Example 3. Detection of antibodies against lipids in bilayer or lipid particle associations in sera of individuals with primary or secondary antiphospholipid syndrome and control sera. Liposomes of phosphatidylcholine: phosphatidate (2: 1) in Tris-NaCI (10mM-1mM) alone or incubated with CaCI ₂ or MnCI ₂ 5mM for 30 min at 37 ° C were used as antigens.

Sera from patients with primary or secondary antiphospholipid syndrome were analyzed to determine the presence of antibodies directed against lipid particles. As negative controls, sera from blood bank donors were used.

Ultracentrifuge tubes (Beckman ultra-clear No.

344060) 14 x 95 mm. in which liposomes were placed at a concentration of 0.1 μmol of the conical lipid in 100 μl of TS regulator at pH 7.0; sera from patients or donors were subsequently added, which were previously decomplemented at 56 ° C for 30min, at a final dilution of 1: 80, these preparations were incubated for 1 h at 37 ° C. After incubation, the liposomes were washed with TS regulator alone or with 5 mM CaCl ₂ or MN Cl ₂ , and centrifuged 50 min at 202,000 x g. The supernatant was then removed, and the liposome tablet was resuspended in 100 μl of

TS pH 7.0. Subsequently the second anti-IgG antibody, IgA and

Human IgM conjugated to fluorescein isothiocyanate at a dilution 180, and incubated 1 h at 37 ° C in the dark; After incubation the preparations were washed as described above. Finally, the liposome tablet was resuspended in 1,000 µl of TS pH 7.0.

The antigens that were included were: of the second antibody conjugated to FITC and that of liposomes; the treatment described for the reaction of the antibodies of the patients' sera was applied to both except that the serum, or the second antibody conjugated to FITC for the liposome control was not added. When working with Mn ²⁺ or Ca ²⁺ , the following controls were added, the conjugate control with the divalent cation and the liposome control treated with the divalent cation. As a positive control, the reaction of the H-308 monoclonal antibody with liposomes carrying lipid particles was used.

The liposomal preparations obtained were placed in tubes for 12 x 75 mm cytometry (Elkay Products, Inc.) and read on the FACScalibur cytofluorometer equipped with a 488 nm argon laser beam (Beckton, Dickinson Co.). phosphates for cytometry, and the following parameters were used in the cytofluorometer: FSC-H E00, SSC-H at 401 V. FL1-H at 748 V, a threshold of FSC-H at 52. Finally, the data analysis was done obtained with the Cellsquest program (Beckton Dickinson). The results of the immunoreaction analysis of the sera of patients with generalized lupus erythomatous (LGE), with antiphospholipid antibody syndrome (SAAF) or with SAAF secondary to LGE using calcium-treated liposomes showed a fluorescence 20 to 40 times greater than the reaction of the control sera with those liposomal antigens. Serums from blood bank donors did not react with lipid particles since the fluorescence results obtained were similar to those of autofluorescence of control liposomes incubated in Tris NaCI and treated with calcium. Example 4. Detection of lipid particles in eukaryotic cells. The detection of particles in C5337 neoplastic cells from pancreatic cancer was carried out by the indirect immunofluorescence method.

. A sterile coverslip was placed in a P60 cell culture box for each test that was performed; Approximately 1X10 ⁶ cells of the pancreatic cancer cell line were added which were allowed to adhere and grow.

When approximately 90% cell confluence was obtained on the coverslips, the cells were washed twice with incomplete DMEM medium and once with two ml of sterile pH 7.4 phosphate regulator. Subsequently, the cells were fixed with 1 ml of 0.2% glutaraldehyde solution - 2% formaldehyde for 5 min. and washed 3 times with two ml of phosphate regulator. Then 200 mi were added. of the undiluted H308 hybridoma supernatants and incubated 1 hr at 37 ° C. Subsequently, the cultures were washed 3 times with 2 ml of phosphate regulator and 200 µl of a 1: 100 dilution of the FITO conjugated mouse IgM anti-Fc antibody were added. These preparations were incubated 1 h at 37 ° C then washed 3 times with phosphate regulator. Finally, the coverslips were mounted on slides with a phosphate regulator, sealed with transparent varnish and observed under an epifluorescence microscope and Nomarski optics (Nikon optiphot-2) with 20X and 40X objectives. The results showed that the neoplastic cells are basically marked in two ways: (1) cells in which areas with a strong fluorescence intensity located at small points were observed. In some cases it was found that the points of fluorescence corresponded to the possible junction zones between the cells. Likewise, cells that were marked on the entire surface were observed. In these cases the cell presented a round morphology which corresponds to cells not adhered to the culture plate indicating that these cells were dead or in apoptosis, or not adhered to other cells due to the process of cell division. The intensity of the fluorescence was higher above the nucleus but it was located practically throughout the cell surface (Figure 3), the controls that were included in this analysis were cells that were only incubated with the second antibody showing a very faint fluorescence that may correspond to nonspecific antibody binding.

In accordance with the above, it can be seen that the use of antibodies obtained from lipid structures other than the bilayer to determine cellular physiological states and for the diagnosis and / or treatment of diseases have been devised to allow early detection of diseases related to antiphospholipid antibodies, and it will be apparent to any person skilled in the art that the modalities presented herein are only illustrative but not limiting of the present invention, since numerous changes of consideration in their details are possible without departing from the scope of the invention. Even when a specific embodiment of the invention has been illustrated and described, it should be emphasized that numerous modifications to it are possible, such as the use of different strains of mice, lipids to obtain them. liposomes, immunization methods and methods of obtaining hybridomas, various reagents for the instrument of diagnosis and / or various diseases related to antiphospholipid antibodies. Therefore, the present invention should not be considered as restricted except as required by the prior art and by the spirit of the appended claims.

BRIEF DESCRIPTION OF THE FIGURES Other particularities and advantages of the invention will be apparent from the following detailed description, of the preferred objectives and modalities, of the appended claims and of the accompanying figures, wherein:

Figure 1 shows an electrophoretic analysis in polyacrylamide gels in the presence of sodium dodecyl sulfate. (Monoclonal antibodies are precipitated with boric acid, precipitated antibodies are analyzed on 10% polyacrylamide gels in the presence of SDS, the molecular weight of the proteins used as standards are specified in the same figure;

Figure 2 A and B represents the detection of antibodies against lipid particles by the liposomal ELISA method. A) Unilamellar liposomes containing PC / PA 2: 1 are incubated with TBS regulator (10mM TRIS HCI and 1mM NaCI pH7) only or the same regulator added with 5mM of MnCI ₂ or 3mM CaCI ₂ . These liposomes were used as an antigen in liposomal ELISA (see description). Supernatants containing the monoclonal antibodies were used undiluted or diluted 1: 2. Liposomes were tested using monoclonal antibodies, polyclonal antibodies of a BALB / c mouse immunized with lipid particles or with an IgM isotype monoclonal antibody specific for T. spirals antigens (Ortega-Pierres and cois). The absorbance of the reaction in the wells is determined at 492 nm. B) The prepared lipids of L-α phosphatidic acid and L-α phosphatidylcholine and cardiolipin were used as antigens in an ELISA test to test the reactivity of monoclonal and polyclonal antibodies against lipid particles, the absorbance of immunoplate wells determined at 492 nm.

Figure 3 depicts the immunoreactivity of the H308 monoclonal antibody with C5337 human pancreatic cancer cells. The cells are incubated with the monoclonal antibody and with the second antibody conjugated to FITC and observed by fluorescence microscopy (A) and by interference microscopy of Nomarski (B). The numbers indicate: (1) fluorescence points above the cell nucleus, (2) possible areas of union between cells and (3) round morphology cells. The photographs were taken with a 20X objective.

Claims

1.- A method for obtaining hybridomas that produce specific monoclonal antibodies against lipid structures other than the bilayer of cell membranes or that recognize non-bilayer lipid molecular associations formed by different conical lipids, where said hybridoma is obtained by the following stages:

A) A first stage of immunization of mice intrasplenically with an effective dose of lipids such as phosphatidylcholine:phosphatidate (2:1) liposomes, where said liposomes contain lipid particles induced with Mn ²⁺ .

B) A second stage of immunization of mice intraperitoneally with the same liposomes and with the same dose as those used for the first stage of immunization. C) A third stage of immunization of mice intravenously with the dose of liposomes used in the first stage of immunization.

D) A fusion stage of spleen cells from mice immunized as described above with a mouse myeloma called P3X63 Ag8U.1 derived from the P3X63 line

Ag8 obtained by Kohler and Milstein from MOPC21 myeloma of female BALB/c mice. This line does not synthesize gamma chains but produces kappa chains. This fusion taking place at least four days after the immunization stage intravenously to obtain at least one hybridoma producing an anti-lipid particle monoclonal antibody. E) A step of selecting hybridomas that present an acceptable immunoreaction by detecting anti-lipid particle antibodies generated by them by means of cytofluorometry and/or the ELISA method.

2.- The method according to claim 1, wherein the first immunization step of section A) comprises administering the liposomes at least 2 times with intervals of 1 week, and the second immunization step of section B) comprises introducing liposomes at least 4 times with 2-week intervals.

3.- The method according to claim 1, wherein the mice used for immunization are selected from syngeneic strains, preferably using 2-month-old female BALB/c strain mice.

4.- The method according to claim 1, wherein the spleen cells are obtained by disintegrating the mouse spleen in a specific cell culture medium for the cultivation of hybridomas, preferably incomplete, followed by various purification steps and a lysis of the erythrocytes, preferably using ammonium chloride.

5.- A specific monoclonal antibody against lipid structures other than the bilayer of cell membranes, obtained from the method according to claim 1.

6.- The monoclonal antibody according to claim 5, wherein said monoclonal antibody specifically recognizes non-bilayer lipid molecular associations formed by different conical lipids in the membranes of plant and human animal cells.

1.- The monoclonal antibody according to claim 5, wherein _said monoclonal antibody allows the detection of anti-lipid particle antibodies in biological samples from individuals who present diseases related to the presence of antiphospholipid antibodies.

8.- The monoclonal antibody according to claim 7, wherein the detection of anti-lipid particle antibodies can be performed in humans, plants and animals.

9.- A hybridoma obtained from the method according to claim 1, which produces ^specific monoclonal antibodies against lipid structures other than the bilayer of cell membranes or that specifically recognize non-bilayer lipid molecular associations formed by different lipids. conical.

10.- The hybridoma according to claim 9, wherein said hybridoma is registered under the CNCM H308 I-2537 deposit.

11.- A method for the detection of anti-lipid particle antibodies in early stages of diseases in humans and/or animals, which are related to the presence of antiphospholipid antibodies.

12.- A method for the characterization of monoclonal antibodies that recognize non-bilayer lipid molecular associations, in terms of isotype, specificity and typing, characterized by the following steps: A) In a first stage, supernatants from hybridoma cultures are diluted 1:10 in wash buffer and 50 μl of these dilutions are added to each well of the immunoplate.

B) In a second stage, the plates are incubated for 30' at 37°C.

C) A step of removing the solution preferably by suction and washing the immunoplate.

D) In the third stage, 200 μl of the washing regulator are added and the plates are incubated for 15' at 37°C. E) A stage of washing the plates with a laying regulator and its suction.

F) A fourth step in which 50 μl of the peroxidase-conjugated antibodies are added to each well in a 1:10 dilution, incubating at 37°C for 30'. G) A plate washing stage with a washing and suction regulator.

H) A fifth stage in which an effective amount of the peroxidase substrate solution is added, incubating at 37°C for 30' using an effective amount of sulfuric acid. I) A stage of analysis of the plates in a reading device

ELISA preferably at 492 nm.

13.- The method according to claim 12, wherein the monoclonal antibodies are of the IgM class as a result of typing.

14.- The method according to claim 12, wherein the specificity of the monoclonal and polyclonal antibodies that recognize non-bilayer lipid molecular associations is carried out by the liposomal ELISA method.

15.- The method according to claim 14, wherein the liposomal ELISA method includes a monoclonal antibody of the ^* IgM isotype with a totally different specificity than that of monoclonal antibodies against lipid particles.

16.- A method for the purification of monoclonal antibodies that recognize non-bilayer lipid molecular associations, characterized by the following steps:

A) A first step consists of adding boric acid (2%) to the ascitic fluid containing the monoclonal antibodies. In this, for every 20 ml of boric acid, 2 ml of ascites fluid are added and the solution is stirred for 2 hours in an ice bath to allow the precipitation of immunoglobulins.

B) In a second stage, the suspension is centrifuged at 45,000 X g for 20 min. at 4°C.

C) In a third stage, the monoclonal antibody precipitate is resuspended in a minimum volume of phosphate saline solution.

D) In a fourth stage, the solution containing the antibody is exhaustively dialyzed with phosphate saline solution to eliminate excess boric acid. E) In a fifth stage, the amount of protein in the solution containing the antibodies is determined.

F) In a sixth step, the protein is analyzed by polyacrylamide gel electrophoresis (SDS-PAGE).

17.- A liposomal ELISA method, which includes the following steps:

A) A first addition and incubation step, in which an effective amount of an antigen suspension is added to each of the wells of an ELISA immunoplate with high antigen binding property and said immunoplate is incubated at room temperature. for 0.5 to 1.5 h.

B) A second addition and incubation step, in which an effective amount of a blocking solution is added to each of the 10 wells of an ELISA immunoplate with high antigen binding property and said immunoplate is incubated at room temperature for 0.5 to 1.5 h.

C) A step of removing the blocking solution, preferably by suction.

D) A third addition and incubation step, in which an effective amount of the antibody carrier is added to each of the wells of the ELISA immunoplate in a carrier:blocking solution dilution from 1:5 to 1:75, said immunoplate being incubated for 0.5 to 1.5 h at a temperature between 35 and 40°C. E) A first step of washing the immunoplate with the blocking solution, preferably repeated 4 times.

F) A fourth addition and incubation step, in which an effective amount of an antibody preferably selected from goat anti-IgG, IgA and IgM or anti-IgM Fe antibodies is added to each of the wells of the ELISA immunoplate. mouse, conjugated to peroxidase, in a final dilution between 1:25 and 1:2000, said immunoplate being incubated in the dark for 0.5 to 1.5 hours at a temperature between 35 and 40°C. G) A fourth addition and incubation step, in which an effective amount of the peroxidase substrate is added to each of the wells of the ELISA immunoplate and said plate is incubated for 0.1 to 0.5 hours at a temperature between 35 and 40°C, stopping the peroxidase reaction by means of an effective amount of sulfuric acid.

H) A step of analyzing the plates in an ELISA reading equipment, preferably at 492 nm.

18.- The method according to claim 17, wherein the suspension of the antigen in part A) is obtained by suspending the antigen in a pH buffer solution, at pH 7, in a ratio of from 0.001 to 0.05 moles of antigen per liter. of pH buffer solution.

19.- The method according to claim 17, wherein the blocking solution comprises a pH regulator, pH 7, and 4% weight by volume of a solution with high protein content, preferably gelatin, with or without an effective amount of a lipid particle-forming agent, preferably with the effective amount and lipid particle-forming agent used to form the antigen.

20.- The method according to claim 17, wherein the effective amount of the antigen suspension in step A) is from 50 to 100 μl.

21.- A liposomal cytofluorometry method, which includes the following steps:

A) A first addition and incubation step, in which the antibody carrier is added to a suspension of the antigen, said carrier being at a dilution from 1:5 to 1:75, the mixture obtained being incubated for 0.5 to 1.5 h at a temperature between 35 and 40°C.

B) A first step of washing the antigen with a buffer solution, at pH 7, with or without an effective amount of a lipid particle-forming agent, preferably with the effective amount and the forming agent used to obtain the antigen.

C) An antigen recovery step, preferably by centrifugation.

D) A second addition and incubation step, in which an effective amount of an antibody preferably selected from goat anti-IgG, IgA and IgM or anti-mouse IgM Fe antibodies conjugated to the centrifuged antigen is added. a fluorescent substance or substrate, preferably fluorescein isothiocyanate (FITC), to have a final dilution between 1:25 and 1:3500, the mixture obtained being incubated for 0.5 to 1.5 hours in the dark at a temperature between 35 and 40°. c. E) A second step of washing the antigen with a pH regulator, pH 7, with or without an effective amount of a lipid particle-forming agent, preferably with the same amount and the same forming agent used to obtain the antigen. F) A suspension and analysis step, in which the antigen is suspended in a developer solution, preferably selected between FACS Flow (Beckton Díckinson Co.) and

Haema Line 2 (Serotono-Baker Diagnostics, 1 NC), in a ratio of 0.1 to 10 moles of the antigen in 1000 ml of the solution, said solution being preferably previously filtered with a filter of 0.22 μm pore diameter, The mixture obtained is analyzed in a flow cytometer, preferably with a 488 nm argon laser beam.

22.- The method according to claim 21, wherein the suspension of the antigen is obtained by suspending the antigen in a buffer solution, at pH 7, in a ratio of 0.5 to 5 moles per liter of buffer solution. pH.

23.- An indirect immunofluorescence method for the detection of non-lipid particles in cells, applicable when the antigen is a cell, which includes the following stages:

A) A first addition and incubation step, in which an effective amount of the antigen, preferably IxIO ³ cells, is placed on a coverslip inside a cell culture box and incubated in an atmosphere containing an effective amount of

C0 ₂ at a temperature between 35 and 40°C until reaching 90% cell confluence.

B) A first washing step that consists of washing the antigen with a suitable culture medium, preferably repeated twice, and with a phosphate buffer solution, pH 7.4, under sterile conditions.

C) A second addition and incubation step, in which an effective amount of an antibody carrier is added to the antigen, preferably 50 to 200 μl undiluted, or with a maximum dilution of 1:2, incubating in a atmosphere containing an effective amount of C0 ₂ for 0.5 to 1.5 h at a temperature of 35 to 40°C.

D) A second washing step that consists of washing the antigen with a phosphate buffer solution, pH 7.4, at least 3 times.

E) A third addition and incubation step, in which an effective amount of an antibody preferably selected from goat anti-IgG, IgA and IgM or anti-mouse IgM Fe antibodies, conjugated to FITC, is added to the antigen. the antigen being incubated in an atmosphere containing an effective amount of C0 ₂ for 0.5 to 1.5 h at a temperature of 35 to 40°C.

F) A third washing step that consists of washing the antigen with a phosphate buffer solution, pH 7.4, preferably repeated 3 times.

G) An analysis step in which the coverslip is mounted on a slide to be observed under a microscope with epifluorescence and Nomarski optics.

24.- The method according to claim 23, wherein the effective amount of CO ₂ is 5% by volume with respect to air, while the effective amount of phosphate buffer solution is 10 ml.

25.- A method to determine a disease related to the presence of antiphospholipid antibodies in a subject who does not present anticardiolipin lupus anticoagulant, anti-DNA or antinuclear antibodies, where said method comprises directly or indirectly detecting the presence or absence of antigens containing lipid particles in a sample from the subject; and observing whether or not the lipid particles are detected, where the presence of the lipid particles indicates that the subject is developing a disease related to the presence of antiphospholipid antibodies.

26.- The method according to claim 25, wherein the detection of the lipid particles is carried out indirectly through the use of an antigen that contains lipid particles that is reacted with the subject's serum in order to determine whether anti-lipid particle antibodies exist in said serum.

27.- The method according to claim 25, wherein the determination is preferably carried out by using at least one technique selected from cytofluorometry, immunofluorescence and ELISA.

28.- The method according to claim 25, wherein the antigen containing lipid particles is selected from neoplastic cells and liposomes.

29.- The method according to claim 28, wherein the liposomes are formed from at least one lipid susceptible to change in geometry through changes in temperature, presence of divalent ions and/or presence of drugs.

30.- The method according to claim 29, wherein the lipid is preferably selected from phosphatidate; cardiolipin; phosphatidylglycerol; phosphatidylinositol; phosphatidylcholine; phosphaditylserine; sphingomyelin; and diglucosyldiacylglycerides.

31.- The method according to claim 30, wherein the lipid is found in abundance in the cell membrane.

32.- The method according to claim 30, wherein the lipids used to form the liposomes are selected according to their availability in the cell membrane, preferably using a cylindrical lipid in combination with a conical lipid in a molar ratio between 1: 1 and

4:1.

33.- The method according to claim 32, wherein uses a combination of phosphatidylcholine with egg yolk phosphatidate in a 2:1 molar ratio.

34.- The method according to claim 26, wherein the subject's serum is also reacted with the antigen with at least one anti-lipid particle monoclonal antibody to confirm the presence or not of the anti-lipid particle antibodies. in the subject's serum.

35.- The method according to claim 25, wherein the detection is carried out directly by reacting cells of the subject with at least one monoclonal anti-lipid particle antibody, preferably by using the indirect immunofluorescence technique.

36.- The method according to claim 35, wherein in addition to the cells of the subject, at least one antigen containing lipid particles, preferably selected from neoplastic cells and liposomes of at least one, is reacted with the antibody at the same time. least a lipid susceptible to change in geometry through changes in temperature, presence of divalent ions and/or presence of drugs.

37.- The method according to claim 36, wherein the lipid is preferably selected from phosphatidate; cardiolipin; phosphatidylglycerol; phosphatidylinositol; phosphatidylcholine; phosphatidylserine; sphingomyelin; and diglucosyldiacylglycerides.

38.- The method according to claim 37, wherein the lipids used to form the liposomes are selected according to their availability in the cell membrane, preferably using a lipid cylindrical in combination with a conical lipid in a molar ratio from 1:1 to 4:1.

39.- The method according to claim 38, wherein a combination of phosphatidylcholine with egg yolk phosphatidate is used in a 2:1 molar ratio.

40.- An in vitro diagnostic kit or instrument that comprises monoclonal antibodies for the detection of anti-lipid particle antibodies in early stages of diseases related to antiphospholipid antibodies that occur in animals, plants and humans, which includes at least one reagent indicating the presence of lipid particles and/or anti-lipid particle antibodies in a sample from a subject who does not present anticardiolipin, lupus anticoagulant, anti-DNA or antinuclear antibodies; means for allowing reaction of the sample with the reagent; and, means to make said reaction evident, where the reagent is selected from liposomes with lipid particles on their surface, neoplastic cells and monoclonal anti-lipid particle antibodies.

41.- A pharmaceutical composition comprising therapeutically effective amounts of said monoclonal antibodies for the treatment of diseases related to anti-lipid particle antibodies by blocking anti-lipid particle antibodies, in combination with a pharmaceutically acceptable carrier.

42.- A pharmaceutical composition comprising therapeutically effective amounts of said monoclonal antibodies for the treatment of diseases related to anti-particle antibodies lipids by stabilizing cell membranes, in combination with a pharmaceutically acceptable carrier.

43.- The pharmaceutical composition according to any of claims 41 or 42, wherein the diseases related to anti-lipid particle antibodies are selected from the group consisting of primary or secondary antiphospholipid antibody syndrome (SAAF); autoimmune diseases such as vasculitis, rheumatoid arthritis, and generalized lupus erythematosus (SLE); diseases that cause an increase in cell division, such as neoplasms such as carcinoma in the liver or ovary, lymphomas, leukemias or myeloproliferative disorders; viral infections such as infectious mononucleosis and acquired immunodeficiency syndrome; diseases caused by bacteria, such as syphilis; and, diseases caused by protozoans such as malaria.

44.- The pharmaceutical composition according to any of claims 41 or 42, wherein the diseases related to anti-lipid particle antibodies are selected in addition to myocardial infarction and senescence.

45.- The use of specific monoclonal antibodies against lipid structures other than the bilayer of cell membranes or that specifically recognize non-bilayer lipid molecular associations formed by different conical lipids, for the preparation of a medication for the treatment of related diseases with anti-lipid particle antibodies.

46. Use according to claim 45, wherein the diseases related to anti-lipid particle antibodies are selected from the group consisting of primary or secondary antiphospholipid antibody syndrome (SAAF); autoimmune diseases such as vasculitis, rheumatoid arthritis, and generalized lupus erythematosus (SLE); diseases that cause an increase in cell division, such as neoplasms such as carcinoma in the liver or ovary, lymphomas, leukemias or myeloproliferative disorders; viral infections such as infectious mononucleosis and acquired immunodeficiency syndrome; diseases caused by bacteria, such as syphilis; and, diseases caused by protozoans such as malaria.

47.- The use according to claim 46, wherein the diseases related to anti-lipid particle antibodies are selected in addition to myocardial infarction and senescence.