MXPA05013402A - Method of altering the binding specificity of plasma proteins by oxidation-reduction reactions - Google Patents

Abstract

The binding specificity of at least one plasma protein suspended or dissolved in a liquid medium it altered by exposing the protein to an oxidizing agent or an electric current sufficient to alter its binding specificity. A masked protein such as an autoantibody can be recovered from blood or blood products or extracts by oxidizing the protein to change its binding specificity.

Description

METHOD FOR ALTERING THE SPECIFICITY OF JOINING PLASMA PROTEINS THROUGH OXIDATION REACTIONS- REDUCTION The present application claims the benefit of the filing date of United States Provisional Application No. 60 / 476,607, filed on June 9, 2003. The provisional application is incorporated by reference herein.

TECHNICAL FIELD The present invention relates to a method for altering a binding specificity of a plasma protein having a binding specificity that can be altered by oxidation-reduction reactions. The present invention also relates to a method for obtaining autoantibodies by uncoated autoantibodies naturally present in the blood, plasma or serum of normal subjects.

BACKGROUND OF THE INVENTION The term "autoimmune disease" refers to a group of diseases in which the immune system mistakenly attacks the cells, tissues and organs of the person's own body. Typically, autoimmune diseases include antibody binding of the body's own components, such as common proteins and lipids. Antibodies that bind to self-compounds (or, more typically, compounds that are so common found in each organism) are referred to as autoantibodies. As an example, the binding of phospholipid autoantibody and / or phospholipid-binding plasma proteins is related to diseases such as systemic lupus erythematosus (SLE), recurrent deep and arterial venous thrombosis, pulmonary embolisms, recurrent spontaneous abortion, thrombocytopenia, chorea , epilepsy, livedo, idiopathic pulmonary hypertension, rheumatological conditions and a host of collagenous diseases. Other diseases related to autoantibodies include multiple sclerosis, Crohn's disease, discoid lupus erythematosus, Hashimoto's thyroiditis, psoriasis, diabetes and rheumatoid arthritis. There are approximately 80 different autoimmune diseases, and as a group, these diseases affect millions of people. A conventional theory with respect to the etiology of autoimmune diseases has been that these diseases are caused by an overproduction of autoantibodies in the sick individual, possibly due to overexpression of a gene encoding said autoantibodies. According to this theory, the blood of an affected individual contains a high level of the particular autoantibody that causes the disease, whereas the blood of a normal individual does not contain any autoantibody or only a trivial amount. This theory is apparently supported by conventional tests, where abundant autoantibodies can be detected in the blood, or blood products, such as plasma or serum, from subjects having an autoimmune disease, whereby only an amount of zero or a Minimum autoantibodies can be detected in the blood or blood products of subjects who do not have an autoimmune disease. The present invention is based on the important discovery, reported herein, that the blood of normal individuals in fact contains a significant number of autoantibodies, in a wide variety of types and specificities. It is possible to detect and isolate these autoantibodies from the blood or a blood product of a normal individual if the blood or a blood product is treated by oxidation, for example, with an oxidizing agent or electric current, in accordance with a method described. at the moment. This discovery of autoantibodies in significant quantities in normal blood was not previously reported and, to the best knowledge of the inventor, the existence of said autoantibodies in significant quantities in normal blood was completely unknown before the present invention. Without being supported by any particular theory, it is clear that if autoantibodies can be obtained by manipulating normal blood taken from people who do not have any symptoms of autoimmune diseases, then it must be that the normal people's immune system routinely creates and circulates these autoantibodies, but in some form where they are covered or blocked, or otherwise prevented from having any harmful effects. The discovery of autoantibodies in significant quantities in normal individuals arises from the question of why antibodies are not detected in a standard test (typically based on the binding of an antibody to their corresponding antigen) and why antibodies do not cause symptoms of disease in normal individuals. Based on the above experiments described herein, an initial tentative explanation for how normal blood may contain autoantibodies without said antibodies being detected through ordinary screening procedures and without such antibodies that cause disease, was that the autoantibodies in individuals normal were somehow hijacked after they were produced. For example, sequestration may be in the form of macromolecules such as low or high density lipoproteins (LDL, HDL) or some other type of microparticles, vesicles, or micelles that may have the ability to maintain the autoantibodies formed in cord around and separated from other components of the bloodstream. According to this theory, the autoimmune disease can be triggered, not by the production of autoantibodies per se, but by the breakdown, rupture or lack of formation of the macromolecules, microparticles, vesicles or micelles that sequester the autoantibodies. This theory seems to be supported by the initial experiments wherein the autoantibodies were obtained from blood samples or serum samples after totally drastic manipulation of the samples including agitation and heating. In subsequent experiments, described herein, however, it was shown that simpler methods of the invention, such as exposure of blood or a blood product to an oxidation agent or DC electric current, may be sufficient to obtain autoantibodies to from normal blood, and that the procedure is reversible. In addition, it has been found that autoantibodies can be obtained by treating commercial Ivlg products, which may be free of any type of macromolecular scavenger entity. Based on these experiments, a more likely theory of how normal blood can contain autoantibodies without these antibodies being detected through ordinary screening procedures and without such antibodies causing disease, is that the antibodies circulate freely along with other antibodies but that the autoantibody antigen binding site becomes blocked or inactive somewhat in normal individuals. According to this theory, the autoimmune disease can be triggered by oxidation so as not to block the antigen-binding site of autoantibodies. In addition, this theory suggests a more general mechanism by which the binding specificity of certain plasma proteins can be altered.

An immediate practical use of the discovery that forms the basis of the present invention is that it allows an almost unlimited supply of autoantibodies to be obtained, whose autoantibodies can be used as standards in diagnostic equipment for laboratory diagnostics of autoimmune diseases and other diseases related to aPL. Previously, the collection of large quantities of antibodies for commercial use has been difficult since it was thought that autoantibodies should be obtained from individuals with an autoimmune disease or that they test positive for autoantibodies in standard tests. The amount of such blood that can be obtained from phlebotomy of individual patients or by pooling blood from a group of patients known to test positive for autoantibodies is limited. Other methods for obtaining autoantibodies, such as the selection of phage libraries, as described in the U.S.A. No. 5,885,793 can be difficult and prolonged. Testing blood samples for the presence or absence of coated antibodies may have important diagnostic value as it can be presaged or predicted that antibodies may appear subsequent to oxidative stress in particular individuals.

BRIEF DESCRIPTION OF THE INVENTION It is an object of this invention to provide a method for altering a binding specificity of a plasma protein having a binding specificity that can be altered by a change in its redox state. It is a further object of the present invention to provide a method for obtaining autoantibodies from blood, plasma or serum of normal individuals. It is a further object of the present invention to provide a method for treating a subject having an autoimmune disease by administering to the subject an antioxidant sufficient to inactivate autoantibodies in said subject. It is a further object of the present invention to provide a method for treating a subject having an autoimmune disease by inactivating autoantibodies from said subject extracorporeally. It is a further object of the present invention to provide a product comprising a biological fluid or an extract containing proteins from a biological fluid that has been exposed to an oxidation agent or sufficient DC electric current to alter the binding specificity of minus one protein contained in it. It is a further object of the present invention to provide a blood, plasma or serum sample from one or more persons who were negative for the presence of autoantibodies in routine clinical tests and who have been treated so that the blood, plasma or serum demonstrates subsequently the presence of autoantibodies.

These and other objects are achieved by a method for altering a binding specificity of at least one circulating protein in a biological fluid or in an extract containing proteins from a biological fluid, the circulating protein having a binding site with a specificity of junction that can be altered by a change in a redox state of the protein, by exposing the protein in the fluid or biological extract to an oxidation agent or to a direct electric current (DC) to effect the alteration of the binding specificity of the circulating protein. The objectives are furthermore achieved by a method comprising the steps of providing a composition comprising at least one plasma protein suspended or dissolved in a liquid medium, the plasma protein has a binding specificity that can be altered by a change in its redox state, and exposing the composition to an oxidation agent or to an electric DC potential sufficient to effect the alteration of the binding specificity of the plasma protein. In another embodiment, the invention relates to a method for obtaining autoantibodies or other circulating proteins coated from a biological fluid or an extract of a biological fluid by exposing the autoantibody or other circulating protein coated in the fluid or biological extract to a oxidation agent or to an electrical DC current sufficient to alter the binding specificity of the autoantibody or other circulating protein coated so that the autoantibody or other circulating protein covered is capable of binding to an antigen or ligand, becoming detectable and recoverable from the biological fluid or extract, and recovering the autoantibody or other circulating protein covered by the biological fluid. In another embodiment, the present invention relates to a method of treating an autoimmune disease by administering to a subject having an autoimmune disease an amount of an antioxidant sufficient to inactivate the autoantibodies in the subject. A treatment of a person who has an autoimmune disease may include extracorporeal blood treatment to reduce the uncovered proteins and replace them as coated proteins. In another embodiment, the present invention relates to a method for selecting a biological fluid or extract from a normal individual to determine which autoantibody is covered and thereby construct a potential antibody profile of autoantibodies that can cause autoimmune diseases in those individuals if they are exposed or not covered by oxidation or an electromotive force. As a particular non-limiting example, blood, plasma or serum, or a blood extract such as a mixture of immunoglobulin, may be exposed to an oxidation agent or DC electric current to effect the alteration of the binding specificity of at least an autoantibody contained in the blood, plasma, serum or extract, so that the autoantibody becomes detectable in and recoverable from the blood, plasma, serum or extract.

BRIEF DESCRICPION OF THE DRAWINGS Figure 1 is a graph showing a forward dispersion profile (size) and lateral dispersion (granulation) of the cell monocyte population as defined by the density gradient human white blood cells by flow cytometry. Figures 2A-2D are flow cytometry histograms showing monocyte activity of several sera. In histograms, antibody activity, if present, is measured by shifts in the mean channel values (log scale) along the horizontal axis. Figure 2A shows the monocyte reactivity of pooled normal human sera (NHS). Figure 2B shows the monocyte activity of a serum from a single normal subject. Figure 2C shows the monocyte activity of a blood sample of the subject shown in Figure 2B that was treated according to the method described in the starting section of the examples. Figure 2D shows the monocyte activity of positive control sera. Figure 3 a graph showing the amount of aPS, aCL, aPE and aPC (as measured by optical density, OD) detected in a series of Ivlg preparations that were incubated with hemin, as a function of the amount of serum of human (in μl) added to the preparations. Figure 4 is a graph showing the amount of aPS, aCL, aPE, and aPC (as measured by OD optical density) detected in a series of diluted human serum preparations that were incubated with hemin, as a function of the amount of hemin (in μl) added to the preparations. Figure 5 is a graph showing the amount of aPS, aCL, aPE, and aPC (as measured by multiples of the average, MoMs) detected in a series of Ivlg preparations that were incubated with hemin and vitamin C, as a function of the amount of vitamin C (in μl) added to the preparations. Figure 6 is a graph showing the amount of aPS, aCL, aPE, and aPC (as measured by multiples of the average MoMs), detected in a series of Ivlg preparations that were incubated with heme solubilized with NaOH, hematoporphyrin IX ( hplX) solubilized with DMSO, hplX solubilized with NaOH, NaOH only, DMSO only, and hemin solubilized with DMSO. Figure 7 is a graph showing the amount of aPS (as measured by OD optical density) detected in a series of Ivlg preparations that were incubated with increased amounts of hemin and increased amounts of hemin and hemopexin (hpx). Figure 8 shows the Western blots obtained for three cells used with Ivlg treated with hemin and Ivlg used as primary antibodies, together with a blot where a conjugate labeled with anti-human HRP was used as a control.

Figures 9A and 9B are graphs showing the amount of aPS, aCL, aPE, and aPC dependent on aPL and independent of aPL (as measured by multiples of the average, MoMs) detected in a series of Ivlg preparations where the electrodes connected to a 9 volt battery were immersed in a buffered solution of phosphate salts containing Ivlg for 2 minutes. Figure 10 is a graph showing the amount of aPS, aCL, aPE, and aPC (as measured by multiples of the average, MoMs) detected in a series of Ivlg preparations where the electrodes connected to a 6 volt battery are They were immersed in a buffered solution of phosphate salts containing Ivlg for 60 seconds. Figure 11 is a graph showing the amount of aPS, aCL, aPE, and aPC (as measured by multiples of the average, MoMs) detected in a series of Ivlg preparations where the electrodes connected to a 6 volt battery are submerged in a buffered solution of phosphate salts containing Ivlg, as a function of immersion time. Figures 12A, 12B and 12C are graphs showing the amount of aCL, aPE and aPS respectively (as measured by multiples of the average, MoMs), detected in control solutions before and after exposure for 240 seconds to electrodes connected to a 6-volt battery. Figure 13 is a graph showing the amount of aPS and aCL, respectively (as measured by multiples of the average, MoMs), detected in serum diluted with PBS from a patient aPS and aCL-positive. In the experiment, the graphite electrodes connected to a 6 volt battery were immersed in the diluted serum for a variable time. Figure 14 is a graph showing the amount of aPS, aCL, aPE, and aPC (as measured by multiples of the average, MoMs), respectively, detected in serum diluted with PBS from an aPE-positive patient. In the experiment, the graphite electrodes connected to a 6 volt battery were immersed in the diluted serum for a variable time. Fig. 15 is a graph showing the amount of aPS, aCL, and aPE (as measured by the optical density, OD), respectively, detected in serum diluted with PBS from an aPE-positive patient. In the experiment, the 10% adult bovine plasma (ABP) used in determining the binding of aPL dependent protein was treated by immersing graphite electrodes connected to a 6 volt battery in ABP for a variable time.

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for altering the binding specificity of at least one plasma protein or circulating protein in a fluid or biological extract of a biological fluid. As used herein, the terms "circulating protein" and "plasma protein" are used to refer to a protein naturally found in the circulation system of animals. Examples of circulating proteins include antibodies and other plasma proteins. It should be understood that the method of the invention is not intended to be universally applied to all plasma proteins or circulating proteins, but is preferably applied to any plasma protein or circulating protein having the property of having the binding specificity that can be altered by a change in the redox state of the protein. The discovery by the inventor that circulating proteins exist, such as autoantibodies, having this property forms a basis of the invention. Examples of proteins without antibody that have been found to have a binding specificity that can be altered by a change in the redox state include kininogen and prothrombin and / or beta2 glycoprotein. The term "coated circulating protein" was coined again for the present invention to designate and describe a circulating protein which, in normal individuals, is present in the blood, but is not detected by conventional binding tests based on receptor-ligand binding. that their binding site, in normal individuals or in a sample taken from the normal individual, is covered or blocked or otherwise prevented from binding to an antigen and that, when a sample containing the circulating protein covered is treated at changing its redox state, such as by exposure to an oxidizing agent or electric current in accordance with a method of the present invention, becomes capable of binding to an antigen and becomes detectable in a sample. An example of a circulating coated protein is an autoantibody. As discovered by the inventor herein, autoantibodies circulate in significant amounts in normal blood, but are not detectable in conventional tests based on antibody-antigen binding. As discussed herein, an autoantibody becomes detectable and recoverable when the autoantibody is subjected to sufficient oxidation-reduction conditions to alter its binding specificity. Antibodies that have been discovered by oxidation include anti-phospholipid, anti-nucleolar (scleroderma associated), anti-lamellar (very bright in nuclear pores), anti-mitochondrial (cytoplasmic), and anti-centriole antibodies. In addition, it has been found that samples of blood, serum or Ivlg that were initially negative for HCV (hepatitis C virus) tested positive for HCV after a treatment in accordance with the present invention, suggesting that normal individuals have anti-HCV antibodies. covered in your circulation. The term "altering the binding specificity" of a protein refers to a method with which a protein is changed or altered, such as by oxidation or reduction, so that it becomes capable of specific binding of an antigen or ligand that does not had previously been able to specifically bind or become incapable of specific binding of an antigen or ligand that had previously not been capable of specifically binding. The term "discover" refers to a method is where the binding specificity of a circulating coated protein is altered so that the protein becomes detectable by a binding test based on the altered binding specificity. The term "autoantibody" refers to any antibody of natural occurrence produced by the immune system of an animal and that binds to a self-antigen, that is, to a compound or antigen produced by the animal itself. The term "biological fluid" includes any body fluid that contains circulating proteins, including plasma, serum, and whole blood, saliva, urine, lactating fluids and other secretions. The term "protein-containing extract of a biological fluid" refers to any preparation that is collected or separated from a biological fluid, such as immunoglobulin fractions. Blood, plasma serum that can be used in the present invention can be obtained again from an individual, or can be obtained from such sources as pooled blood or plasma preparations obtained by blood banks or other blood collection facilities. For purposes of the present invention, blood, serum or plasma may also be from collections that are outdated or otherwise substandard by blood banks or blood collection facilities. Although this description focuses on human blood, plasma and serum, the identical procedures of this invention can be applied to animal blood and should result in obtaining antibodies from analogous animals for purposes related to veterinary medicine. Preferably, the blood or serum used in the method of the invention is diluted to reduce the effect of any antioxidant that may be contained in the blood, plasma or serum. In the method of the present invention, the binding specificity of at least one circulating protein or plasma protein in a biological fluid is altered by exposing the protein to an oxidant or an electric current. For example, the binding specificity of a coated circulating protein can be altered so that the protein is discovered, i.e., capable of binding to an antigen that was not able to bind before the method was carried out. A protein that has had its altered binding specificity can then be isolated and recovered by any separation method based on the specific binding.

If an oxidation agent is used to carry out the method of the invention, the oxidation agent can be any compound that is capable of altering the redox state of a biological molecule. More specifically, the oxidation agent is a molecule that has the ability to be reduced by acting as an electron acceptor for other molecules that act as electron donors. Examples of oxidizing agents include, but are not limited to hemin, chlorophyll, or other ring compounds containing a strong oxidation metal, and K4O4. Typically, when an oxidation agent is used, a mixture of biological fluid or extract and the oxidizing agent must be incubated for a period, typically around a day or overnight. The oxidizing agent should be used at a sufficient concentration to alter the binding specificity of a protein having an altered binding specificity, but not at a concentration that can destroy the protein. In the case of autoantibodies, it has been discovered that different types of autoantibodies can interact differently with different antioxidants. For example, to discover aPC autoantibodies, the results are deficient with hemin and very good with KMn04.

If a DC electric current is used to carry out the method of the invention, the method can be carried out by any means for supplying an electric current such as by immersing positive and negative electrodes in a conductive solution containing the sample to be treated. . Typically, a solution containing a biological fluid can be exposed to an electrical potential of a sufficient magnitude and of sufficient duration to alter the binding specificity of a protein having an altered binding specificity. It has been found that positive results can be obtained by exposing a solution to an electrical potential of 6- 24 volts for a few seconds to a few minutes. As discussed in the examples, a prolonged exposure to an electric current may result in reversibility of the binding specificity alteration.

Attempts to produce positive results using an AC current have not been successful.

Without being bound to a specific theory, it is preferred, in the case of an autoantibody, that the autoantibody be exposed to the oxidizing agent or electrical current in an amount or for a time sufficient to oxidize an antigen-binding site in a Fab portion. of the autoantibody.

A particular protein of interest is one that has a binding specificity that can be altered by changing its redox status and the effectiveness of any set of conditions to alter the binding specificity of the particular protein of interest can be easily determined by ELISA or other assays. ligand -receptor. Said tests may be carried out before and after a protein is subjected to redox conditions to observe if the procedure has altered the binding specificity of the protein. For example, the best oxidation agent to recover a specific autoantibody can be easily determined by simple experimentation.

A further aspect of the present invention is the possibility of treating a subject having an autoimmune disease, either by administering to the subject an amount of an antioxidant sufficient to inactivate the autoantibodies in the subject or by taking a blood sample from the subject, exposing the blood sample to an antioxidant or electric current sufficient to inactivate the autoantibodies in said blood sample, and return the blood sample to the subject.

A further aspect of the present invention is a method for selecting a biological fluid from a normal individual or extract to determine which autoantibodies are covered and thereby construct a potential antibody profile of autoantibodies that could cause the autoimmune disease in that individual if exposed. or it is discovered by oxidation or an electromotive force. For example, in general terms, a sample of blood, plasma or serum from a subject can be tested to determine a quantity and / or type of detectable autoantibodies in the sample. Hereinafter, a sample of the subject's blood, plasma or serum can be treated by exposing the sample to an oxidizing agent or a DC electric current, and the subject's blood, plasma or treated serum sample can be tested to determine an amount and / or type of detectable autoantibodies in the treated sample. Hereinafter, the amount and / or type of autoantibodies detectable in the sample before the treatment step can be compared to the amount and / or type of autoantibodies detectable in the sample after the treatment step.

It has been found that samples of blood, plasma, serum or Ivlg untreated and the blood, plasma or serum or Ivlg samples treated in accordance with the method of the present invention can be lyophilized and transported or stored. When the samples are reconstituted, they retain their respective activity.

EXAMPLES Having described the invention, the following examples are given to illustrate specific applications of the invention, including the best known mode for carrying out the invention. The examples are presented in approximate chronological order and thus show a progress in understanding the components and procedures required to achieve the effects of the invention. These specific examples are not intended to limit the scope of the invention described in the application. With respect to each of the examples 1-17 described herein, unless otherwise indicated, the following procedure was typically used: a 10 ml sample of whole blood or 5 ml of serum or plasma from a subject normal aPL-negative and 4-5 ml of packed red blood cells were added to a container containing 3 ml of Biomerieux brand bacterial culture growth medium (containing at least the following ingredients: distilled water, digestion broth of casein-soybean, yeast extract, dextrose, sucrose, hemin, menadione (vitamin K3), pyridoxal HCl (vitamin B6), and sodium polyantolsulfonate (SPS) and charcoal.Thereafter, the mixture was incubated, shaken or stirred, at 37 ° C for a period of 18-22 hours.After incubation and centrifugation, a sample of the incubated blood or serum / RBC was tested for the presence of antiphospholipid antibodies (aPL) using an ELISA format. Ehensivo that provides 24 aPL test results separately. The test procedure is described in greater detail in the following publications, incorporated herein by reference; Wagenknecht DR, et al., The Evolution, Evaluation and Interpretation of Antiphospholipid Antibody Assays, Clinical Immunology Newsletter, Vol. 15, No. 2/3 (1995) p. 28-38 and Mcintyre JA, et al., Frequency and Specificities of Antiphospholipid Antibodies (aPL) in Volunteer Blood Donors, Immunobiology 207 (1): 59-63, 2003. Table 1 shows the 24 specific aPL specificities that were tested by using the comprehensive domestic aPL ELISA format. Four specificities were evaluated, 1) aPS = antiphosphatidylserine, 2) aCL = anticardiolipin, 3) aPE = antiphosphatidylethanolamine, and 4) aPC = antiphosphatidylcholine. For each of these aPL specificities, three immunoglobulin isotypes were searched for IgG, IgA and IgM. Each specificity and each isotype were evaluated in the presence (dependent) and absence (independent) of a pH regulating diluent supplement, 10% adult bovine plasma (PBL), containing phospholipid-binding plasma proteins) or albumin 1% bovine serum (BSA, which is free of phospholipid-binding plasma proteins), respectively. The final dilution of the subject's blood samples is between 1/50 and 1/100.

TABLE 1 Domestic aPL ELISA * PS CL PE PC igG IgG igG igG igA igA IgA igA igM igM igM igM * In the presence (dependent) and absence (independent) of phospholipid-binding plasma proteins Table 1 is a table indicating the particular antiphospholipid antibodies (aPL) that were tested using the enzyme-linked immunosorbent assay (ELISA) format used in many of the examples, described below. The results in the 24 specificities of aPL obtained for several experiments described herein are given in the accompanying drawings. Positive / negative findings are expressed in multiples of average (MoM) based on test plasma samples from 775 normal blood donors, as described in Mclntyre JA, Immunobiology, previously. The presence of +++ indicates strong antibody activity. The + and ++ markers indicate low and intermediate antibody activity, respectively. The drawings also provide the normal scale values for each aPL specificity and isotype combination. A positive result in the column indicated as PL-dependent "binding protein" means that the antiphospholipid antibody (aPL) is actually bound to a plasma protein that initially bound to a particular phospholipid indicated. Plasma proteins that can typically bind by PS and CL include the following: beta2-glycoprotein I, prothrombin, protein C, protein S, annexin V, and complement components factor H and C4 (see, for example, McIntyre, JA, Wagenknecht, DR and Faulk, WP Antiphospholipid antibodies: Discovery, definition, detection and disease, Prog. Lipid Res. 42 (3): 176-237, on page 182). The physiological nature of protein binding to plasma is not known precisely for all phospholipids, but such binding is thought to induce conformational changes in the plasma protein structure, thus exposing novel or cryptic epitopes that are then directed by autoantibodies of individuals. Plasma proteins that can typically be linked by phospholipid PE include the following: high and low molecular weight kininogenes, and factor XI and prekallikrein. The last two proteins can be detected by virtue of their fidelity in conjunction with high molecular weight kininogen. Plasma proteins that bind to PC have not yet been defined. In certain experiments, the aPL independent of plasma protein is observed (Table 3). One possible explanation for this activity is that it represents the presence of residual phospholipid-binding plasma proteins that are present in the original blood sample.

EXAMPLE 1 A blood sample from a normal subject was incubated and tested in accordance with the procedure described above. The aPL ELISA results are shown in Table 2. As shown in Table 2, the incubated blood sample shows a dramatic presence of autoantibody activity, as compared to normal, untreated blood shown in the normal scale column . In particular, the strong activity of autoantibodies is shown in the protein-dependent category for aPS (IgG), aCL (all isotypes), and aPE (IgG). The low or absent aPC IgG autoantibody activity was a characteristic discovery in early samples and in procedures where hemin was used as the oxidation agent. This result indicates that PC autoantibodies, especially the IgG isotype, are different and may not be activated in the same way as others. In subsequent experiments, it was found that important levels of aPC can be detected in samples that were treated with KMn04 (data not shown).

TABLE 2 Broth + blood unconditioned Antiphospholipid antibody (aPL) results PL binding protein Normal dependent scales Independent IgG 24 MoM +++ 3 MoM < 4MoM aPS igA 8M0M + 2 MoM < 3MoM igM 8 MoM 1 MoM < 5MoM igG 12MoM +++ 2 MoM < 4MoM aCL igA 12MoM +++ 2 MoM < 4MoM igM 15 M0M +++ 1 MoM < 6MoM igG 36 MoM +++ 7MoM + < 4MoM aPE 7MoM + 7 MoM < 3MoM igM 8M0M + 3 MoM < 5MoM igG 2 MoM 4 MoM < 4MoM aPC igA 1 MoM 3 MoM < 3MoM igM 6 MoM + 5 MoM + < 4 MoM Table 2 is a table summarizing the results of the aPL test for a blood sample from a normal aPL-negative subject, incubated according to the method described in the open section of the examples.

EXAMPLE 2 Blood samples taken from seven healthy subjects were incubated and tested in accordance with the procedure described above. In particular, the blood of the seven subjects was extracted in a period of 20 minutes and incubated for 20 hours under identical conditions. Table 3 is a table of compounds that shows the aPL seroconversion scale for these seven samples. These results show that there are variations in the levels of aPL detected as well as the isotypes present between different individuals. However, as shown by the invention, each individual has aPL antibodies that can be detected after incubation.

TABLE 3 Table 3 is a table of compounds summarizing the results of the aPL test of blood samples from seven normal aPL-negative individuals. incubated in accordance with the method described in the open section of the examples.

EXAMPLE 3 In a first experiment, a serum sample from a normal subject was incubated and tested in accordance with the basic procedure described above. In the incubation mixture, horse red blood cells (RBC) were used instead of human RBC. The aPL ELISA results are shown in Table 4. As shown in Table 4, the important aPL activity was obtained, particularly with respect to aPS (IgGylgM) and aCL (IgAylgM).

TABLE 4 Horse RBC, human serum, broth Antiphospholipid antibody results (aPL) PL-binding protein Normal dependent scales IgG independent 14 MoM +++ 1 MoM < 4MoM aPS IgA 5MoM + 1 MoM < 3MoM igM 19MoM +++ 1 MoM < 5MoM igG 2 MoM +++ 1 MoM < 4MoM aCL igA 13 MoM +++ 1 MoM < 4MoM igM 27 MoM +++ 1 MoM < 6MoM igG 1 MoM 2 MoM < 4MoM aPE igA 1 MoM 1 MoM < 3MoM igM 1 MoM 4 MoM < 5MoM igG 1 MoM 1 MoM < 4MoM aPC igA 1 MoM 1 MoM < 3MoM igM 1 MoM 1 MoM < 4MoM Table 4 is a table summarizing the results of the aPL summary test for a serum sample from a normal aPL-negative subject, incubated according to the method described in the open section of the examples, with the characteristic that red horse cells (RBC) were replaced by human RBC in the procedure. In a second experiment, a horse serum, instead of human serum, was incubated with human RBC and tested in accordance with the basic procedure described above. The aPL ELISA results are shown in table 5. As shown in table 5, aPL activity was not obtained. (The ELISA test used in this experiment used antibody probes labeled with human antibody-specific alkaline phosphatase to detect aPL, to know if the horse aPL contained in the sample was unknown).

TABLE 5 Human RBC, horse serum, broth Antiphospholipid antibody results (aPL) PL-binding protein Normal dependent scales Independent IgG 1 MoM 1 MoM < 4 MoM aPS IgA 1 MoM 1 MoM < 3 MoM IgM 1 MoM 1 MoM < 5 MoM IgG 1 MoM 1 MoM < 4 oM aCL IgA 1 MoM 1 MoM < 4 MoM IgM 1 MoM 1 MoM < 6 MoM IgG 1 MoM 1 MoM < 4 MoM aPE ÍgA 1 MoM 1 MoM < 3 MoM igM 1 MoM 1 MoM < 5 MoM igG 1 MoM 1 MoM < 4 MoM aPC igA 1 MoM 1 MoM < 3 MoM igM 1 MoM 1 MoM < 4 MoM Table 5 is a table summarizing the results of the aPL test of an incubation of a serum sample that is carried out according to a method described in the open section of the examples, except that the horse serum is replaced by human serum. The results shown summarized in Tables 4 and 5 unequivocally demonstrate that all aPLs that were obtained during the seroconversion procedure of the present invention originate from human serum and are not released from human RBC, since the first experiment uses RBC of horse, which is free of human antibodies, instead of human RBC, and still shows positive results, whereby the second experiment uses horse serum in the presence of human RBC and shows negative results.

EXAMPLE 4 A blood sample from a normal subject was incubated and tested in accordance with the basic procedure described above, except that the incubation was carried out at room temperature (22 ° C), rather than at an elevated temperature. Table 6 shows that the sample did not undergo seroconversion when incubated at room temperature. These results suggest that the seroconversion procedure may be sensitive to temperature.

TABLE 6 Table 6 is a table summarizing the results of the aPL test for a blood sample from a normal aPL-negative subject, incubated according to the method described in the open section of the examples, except that the incubation was carried out at room temperature (22 ° C).

EXAMPLE 5 A sample of blood from a normal subject was incubated and tested in accordance with the basic procedure described above, with the feature that Degalan 0.7mm (plastic) beads were used as the particulate solid in the incubation mixture instead of carbon. Since coal was used in initial experiments showing seroconversion, this experiment was carried out to determine if carbon plays an important role in seroconversion. Table 7 shows that the sample presented seroconversion even when plastic beads were used instead of carbon. These results suggest that the role of carbon is mechanical, rather than chemical, in nature, and that any particulate solid, such as plastic, resin or glass beads, can be used. Without wishing to be limited to any particular theory, the theory can be made that the particulate component acts as an abrasive on the RBC membrane, probably causing release of the NO ion from RBC, either by interacting with the RBC protein AE1 / band 3 or with the transition molecules of SNO-hemoglobin or both. The possibility of mechanical abrasion is supported by the observation in Example 6, where the results of the negative test are shown for an incubation mixture that does not shake or shake. Particulate solids can also serve as a mechanical function to aid in the release of autoantibodies.

TABLE 7 Table 7 is a table summarizing the results of the aPL test for a blood sample from a normal aPL-negative subject, which was incubated in accordance with the method described in the open section of the examples, with the characteristic of that pearls of 0.7 mm were used Degalan (plastic) as a particulate solid in the incubation mixture.

EXAMPLE 6 A blood sample from a normal subject was incubated and tested in accordance with the basic procedure described above except that the incubation mixture remained stationary, instead of shaking or shaking. Table 8 shows that the sample did not suffer seroconversion when it remained stationary. These results suggest that movement can facilitate interaction between solid particles and RBC. Stationary incubation conditions did not facilitate the release of aPL, although a small amount of movement such as that produced by the transport of the samples to the incubator can produce small amounts of an aPL release.

TABLE 8 Blood in a stationary vessel at 37 ° C Antiphospholipid antibody results > (aPL) PL binding protein Normal dependent scales Independent IgG 1 MoM 1 MoM < 4MoM aPS IgA 1 MoM 1MoM < 3MoM IgM 2 MoM 1 MoM < 5MoM IgG 1 MoM 1 MoM < 4MoM aCL oR 1 MoM 1 MoM < 4MoM IgM 7MoM + 1 MoM < 6MoM igG 1 MoM 1 MoM < 4MoM aPE igA 1 MoM 1 MoM < 3MoM igM 4 MoM 3 MoM < 5 oM igG 1 MoM 3 MoM < 4M? M aPC igA 1 MoM 1 MoM < 3M? M igM 3 MoM 7MoM + < 4 oM Table 8 is a table summarizing the results of the aPL test for a blood sample from a normal aPL-negative subject, which was incubated according to the method described in the open section of the examples, except that the Incubation mixture remained stationary, instead of being shaken or shaken.

EXAMPLE 7 A blood sample from a normal subject was incubated and tested in accordance with the basic procedure described above, with the added feature that after incubation and removal of RBC and carbon by centrifugation, the incubation mixture was heated at 56 ° C for 30 minutes. Table 9 shows that the amount of aPL detected was significantly increased by this procedure.

TABLE 9 Effect of heating at 56 ° C Antiphospholipid antibody results (aPL) PL-binding protein Normal dependent scales Independent IgG 47 MoM +++ 69 MoM +++ < 4MoM aPS IgA 12 MoM +++ 18 MoM +++ < 3M? M igM 3 MoM 1 MoM < 5 μM IgG 13 MoM +++ 16 MoM +++ < 4MoM aCL igA 16 MoM +++ 22 MoM +++ < 4MoM igM 12 MoM +++ 16 M? M +++ = 6MoM igG 48 MoM +++ 44 MoM +++ < 4MoM aPE 8 MoM + 8 MoM + < 3MoM IgM 2 MoM 1 MoM < 5MoM igG 5 MoM + 6M0M + < 4 MoM aPC igA 1 MoM 2 MoM < 3M? M igM 1 MoM 3 MoM < 4MoM Table 9 is a table summarizing the results of the aPL test for a blood sample from a normal aPL-negative subject, which was incubated according to a method described in the open section of the examples, with the added feature that the incubation mixture was heated at 56 ° C for 30 minutes.

EXAMPLE 8 A blood sample from a normal subject was incubated and tested in accordance with the basic procedure described above, with the characteristic that a bacterial culture growth medium from a different provider (Becton Dickinson, Sparks, MD) was used in place of growth medium of bacterial culture of Biomerieux. Table 10 shows that the sample presented seroconversion in the Becton Dickinson medium, indicating that the method of the present invention does not depend on a growth medium of bacterial culture from a particular source.

TABLE 10 Becton Dickinson culture vessel * Antiphospholipid antibody (aPL) results PL-binding protein Normal dependent scales Independent IgG 11 MoM +++ 3 MoM < 4MoM aPS IgA fails Fault < 3MoM IgM 4 MoM 1 MoM < 5MoM IgG 6M0M + 4 MoM < 4MoM aCL 5MoM + 5MoM + < 4 MoM IgM 9MoM + 1 MoM < 6MoM IgG 32 MoM +++ 1 MoM < 4MoM aPE IgA 2MoM + 1 MoM < 3MoM igM 10MoM +++ 1 MoM < 5 MoM igG 1 MoM 1 MoM + < 4MoM aPC igA 1 MoM 1 MoM < 3MoM igM 1 MoM 1 MoM < 4MoM Replace Biomerieux Table 10 is a table summarizing the results of the aPL test for a blood sample from a normal aPL-negative subject, incubated according to the method described in the open section of the examples, with the characteristic that a growth medium of bacterial culture from a different supplier (Becton Dickinson, Sparks, Md) was used in place of the growth medium of bacterial culture of Biomerieux.

EXAMPLE 9 A blood sample from a normal subject was incubated and tested in accordance with the basic procedure described above, with the characteristic that the incubation occurred under anaerobic conditions (under nitrogen) instead of under aerobic conditions (in the presence of oxygen and C02) . Table 11 shows that the sample presented seroconversion even under anaerobic conditions and that the method of the present invention does not depend on an aerobic environment.

TABLE 11 Broth + blood from the anaerobic culture vessel Antiphospholipid antibody (aPL) results PL binding protein Normal dependent scales Independent IgG 16 MoM +++ 10MoM +++ < 4MoM aPS IgA 3 MoM 2 MoM < 3 MoM igM 9MoM + 5 MoM < 5MoM igG 8M0M + 3 MoM < 4MoM aCL oG 4MoM + 1 MoM < 4MoM igM 17MoM +++ 3 MoM < 6MoM igG 33 MoM +++ 10MoM +++ = 4 MoM aPE igA 4MoM + 1 MoM < 3MoM igM 9M? M + 3 MoM < 5 oM igG 1 MoM 2 MoM < 4M? M aPC igA 1 MoM 1 MoM < 3MoM igM 3 MoM 5MoM + < 4MoM Table 11 is a table summarizing the results of the aPL test for a blood sample from a normal aPL-negative subject, incubated according to the method described in the open section of the examples, with the characteristic of that the incubation occurred under anaerobic conditions.

EXAMPLE 10 A blood sample from a normal subject was incubated and tested in accordance with the basic procedure described above, with the characteristic that K562 cells (a human hematopoietic tumor cell line) were used in place of red blood cells. In addition, only 11.3 million K562 cells were present in the culture medium, compared to 3-4 mis of packaged RBC typically used in the method of the invention. Table 12 shows that the sample presented seroconversion. Other experiments have shown that samples that are incubated with other types of isolated cells, lymphocytes, monocytes and neutrophils typically did not show seroconversion of aPL. In particular, the white blood cells of the lymphoid and myeloid series do not support the release of aPL nor a cell line of porcine B lymphocytes designated as L14 (data not shown). These results suggest that hemoglobin may be a key component in the incubation mixture, since K562 and RBC cells contain hemoglobin, and lymphocytes, monocytes and neutrophils do not.

TABLE 12 Total cell count 11.3x10 Table 12 is a table summarizing the results of the aPL test for a blood sample from a normal aPL-negative subject, incubated according to the method described in the open section of the examples, with the characteristic that K562 cells (a human tumor cell line) were used instead of RBC.

EXAMPLE 11 A blood sample from a normal subject was incubated and tested in accordance with the basic procedure described above, except that the bacterial culture growth medium was replaced by cell culture medium used to grow human cells: RPMI. Table 13 shows that seroconversion did not occur. This experiment shows the importance of some ingredient in the bacterial culture medium for the purpose of this invention. Although RPMI is a culture medium designated for human cells, it does not support the release of aPL when it is replaced by a fresh broth. The lists and comparisons of the ingredients in the two broths of the microbiological container with RPMI show that RPMI lacks hemin and menadione (a provitamin K made by a man) called vitamin K3. It is known that hemin is an iron porphyrin (Fe +++) chelator derived from RBC, and menadione is a fat-soluble vitamin. This indicates that redox reactions may play a role in the release of autoantibody.

TABLE 13 Table 13 is a table summarizing the results of the aPL test for a blood sample from a normal aPL-negative subject, except that the bacterial culture growth medium was replaced with a cell culture medium used for make human cells grow EXAMPLE 12 A sample of placental cord blood was incubated and tested in accordance with the basic procedure described above. The placental cord blood was removed after the birth of the baby, but before the placenta detached from the wall of the uterus. Neither the mother's blood nor the baby's cord blood showed the presence of aPL in conventional laboratory tests. When processed in accordance with the invention described herein, the strong aPL antibody was shown to be present in the cord blood samples, as shown in table 14. The antibodies were only IgG, an observation that is compatible with antibodies of maternal origin. Since the mother transports IgG to the fetus before birth, this experiment seems to indicate that covered maternal autoantibodies transported to the fetus via Fc? specialized in the trophoblast (Fc? Rn) remain covered by the fetus in the fetal blood. Since the mother's blood and cord blood were shown to be aPL-negative prior to seroconversion by the method of the invention, and since IgM or IgA immunoglobulins were not detected, these findings support the concentration of that IgG aPL observed in cord blood subsequent to seroconversion are of maternal origin. It is also of interest that the trophoblast expressing Fc? Rn does not express the HLA antigens.

TABLE 14 Table 14 is a table summarizing the results of the aPL test for a cord blood sample from a normal aPL-negative mother and a baby.

EXAMPLE 13 A plasma sample from a normal subject was incubated and tested in accordance with the basic procedure described above; with the characteristic that sodium nitroprusside (SNP, 200 micromolar) was used in place of RBC in the incubation mixture. Table 15 shows that the sample presented seroconversion.

Since SNP is a potent nitric oxide (NO) donor, these results provide supporting evidence that the NO radical is not involved in the release of autoantibody and also supports a theory that RBC and solid particulates fulfill a role of provide a donation of NO from RBC. Other reactions mediated by free radicals in addition to sodium nitroprusside can also cause autoantibody release.

TABLE 15 Replace RBC with sodium nitroprusside * Antiphospholipid antibody (aPL) results PL-binding protein Normal dependent scales Independent igG 18 oM +++ 5MoM + < 4 MoM aPS igA 8M0M + 4MoM + < 3MoM igM 1 MoM 1 MoM < 5 oM igG 7MoM + 1 MoM < 4 MoM aCL igA 7 MoM 1 MoM < 4 MoM igM 1 MoM 1 MoM < 6 MoM IgG 23 MoM +++ 9MoM + < 4MoM aPE igA 3 MoM 4 MoM + < 3 oM igM 1 MoM 1 MoM < 5MoM igG 1 MoM 2 MoM < 4MoM aPC igA 2 MoM 1 MoM < 3MoM igM 1 MoM 1 MoM < 4 MoM * 200μM Table 15 is a table summarizing the results of the aPL test for a blood sample from a normal aPL-negative subject, incubated according to the method described in the open section of the examples, with the characteristic that Sodium nitroprusside (SNP) was used instead of RBC in the incubation mixture.

EXAMPLE 14 A blood sample from a normal subject was incubated in accordance with the basic procedure described above and tested for lupus anticoagulant activity. The anticoagulant or lupus inhibitor is another type of aPL and is typically only detected by functional laboratory tests. The results in Table 16 show a strong lupus anticoagulant (LA) in the seroconverted blood taken from an individual lupus inhibitor negative and processed by the method of this invention. Although initially corrected by adding normal plasma to the seroconverted broth in the dRWT test, incubation for 1-2 hours resulted in the reappearance of the inhibitor. This time structure is proposed as the time taken for LA or uncovered antibodies to bind the relevant phospholipid-binding plasma proteins introduced by the mixing study. It also eliminates the possibility of coagulation of factor deficiencies since a 1: 1 mixture provides sufficient levels of coagulation factors to correct coagulation times in a factor deficient sample. The diluted prothrombin time (dPT) was not corrected in the presence of normal plasma and increased prolongation of coagulation times was observed after incubation with normal plasma, which is indicative of a strong lupus inhibitor.

TABLE 16 Lupus anticoagulant activity Whole blood broth Immediate mixture Mix Incubating 1: 1 1: 1 1-2 hours dRWT 46.7 sec * 104.4 sec dPT 42.5 sec + 48.7 sec * normal = 28 - - 49 sec normal = 29.6 - 41.8 Table 16 is a table summarizing the results of a lupus anticoagulant activity test for an incubated blood sample according to the method described in the open section of the examples. The blood sample was obtained from a subject whose blood is negative lupus anticoagulant before seroconversion by the method of the present invention.

EXAMPLE 15 Blood samples from five normal subjects were incubated in accordance with the basic procedure described above and were tested by fluorescence microscopy for the presence of other types of autoantibodies. Serum and plasma samples from these five individuals were negative before processing in accordance with the teachings of the invention. Table 17 indicates additional autoantibody specificities identified when using the Hep-2 cell line. Anti-nucleolar (associated scleroderma) was identified, anti-lamellar (very bright in nuclear pores), anti-mitochondrial (cytoplasmic) and anti-centriole. The results show that autoantibodies released by the method of the present invention can also be detected by a different detection methodology, fluorescence microscopy, as opposed to ELISA-based tests. The results confirm that many types of autoantibodies in addition to aPL are covered in the blood of individuals whose serum and plasma were negative for these antibodies in routine laboratory tests. From these results, it can be expected that many of the autoantibody specificities are expected to be found when analyzing blood processed by this invention.

TABLE 17 Autoantibodies identified by immunofluorescence Fluorescence patterns observed in Hep-2 cell lines Anti-nucleolar (associated scleroderma) Anti-lamellar (very bright in nuclear pores) Anti-mitochondrial (cytoplasmic) Anti-centriole (meaning unknown) Table 17 is a table indicating other types of autoantibodies that have been identified in blood samples that are incubated in accordance with the method described in the open section of the examples. The indicated antibodies were identified by immunofluorescence microscopy.

EXAMPLE 16 A blood sample from a normal subject was incubated in accordance with the basic procedure described above and tested for reactivity with monocytes using flow cytometry and anti-human IgG fluorescent conjugated antibodies. The comparative test was performed with normal human serum pool untreated (NHS) with serum from the same normal subject used with the invention and with positive control human serum. (The treated blood did not show autoreactivity with lymphocytes and neutrophils, these data are not shown). Figure 1 shows the forward dispersion (size) and lateral dispersion (granulation) profile of the monocyte population of a normal cell subject as defined by flow cytometry. This cell monocyte population was confirmed by showing reactivity with CD 14 monoclonal antibodies. Figure 2A shows anti-monocyte activity with NHS. The average reactivity shown is 743.50 on a linear scale. Figure 2B shows the auto-anti-monocyte activity of the normal subject's serum; this subject has no antibody activity to autologous monocytes. The average reactivity shown is 737.00. Figure 2C shows the auto-anti-monocyte activity of a blood sample from the subject shown in Figure 2B after being treated in accordance with the method of the invention. The average value shown is 864.00, indicating a strong auto-anti-monocyte activity. Despite the fact that the plasma processed in accordance with the teachings of the invention was used at a 1/8 dilution, it showed more activity with monocytes than the undiluted positive control sera did. Thus, this example demonstrates that blood or serum samples processed according to the method of this invention release autoantibodies that specifically signal monocytes. The same results were documented for four additional samples from other individuals when processed in accordance with the teachings of the invention.

EXAMPLE 17 Comparative tests for the presence of antinuclear antibodies (ANA) using a RELISA® selection test were carried out in untreated cord blood serum; cord blood incubated in accordance with the method of the present invention, without agitation; cord blood treated in accordance with the method of the present invention with agitation; untreated serum from a healthy ANA-negative donor (identified as ACS) and serum from the same healthy ANA-negative donor that was incubated in accordance with the method of the present invention. As shown in Table 18, a significant amount of ANA was identified in cord blood and serum samples that were treated by the method of the present invention. From the results of tables 16 and 17, you can It is expected that many of the autoantibody specificities expected to be found by analyzing blood processed by the invention.

TABLE 18 ANA antibodies identified by ImmunoConcepts Laboratories * Using the RELISA® selection test Sample Units Serum cord 0 Stationary cord 27 Cord agitation 75 ACS serum 0 Shake + warm ACS 90 * Sacramento, California 1 < 10 units = neg 10-15 units = limit Table 18 is a table that summarizes the results of a Antinuclear antibody (ANA) test of several samples using a RELISA® selection test.

EXAMPLE 18 To understand the role of red blood cells in the phenomenon of autoantibody release, the experiments were designed to replace red blood cells with simpler ingredients that can mimic the action of red blood cells. In the present experiment, the red and carbon globules were replaced with sodium nitroprusside (SNP) and ferric chloride. This substitution was made because sodium nitroprusside is a powerful nitric oxide producer, and it is known that RBC are carriers of NO. "Ferric chloride (FeC supply solution, 25 μM) was added to a substitute for iron in hemoglobin Culture bottles containing the growth medium of bacterial culture and 5 ml of human or serum plasma and varied concentrations of sodium nitroprusside (SNP, 200 μm) and exogenous ferric chloride (final concentration 4.1 μm) were used instead of red blood cells and charcoal, they were incubated at 37 ° C and then heated at 56 ° C for 30 minutes.The samples showed aPL seroconversion, but only IgG (data not shown) .The results suggest that NO "It can be involved in the non-coating of the antibody, and suggests that the mechanical action of a solid phase material in the culture bottle breaks red blood cells and releases NO." Alternatively, the release or modify The NO molecule can allow the hemoglobin molecule to participate in redox reactions. EXAMPLE 19 In an effort to determine whether the effect of not covering autoantibodies was due to the breakdown of macromolecular structures containing autoantibody within the serum or blood or whether it was due to direct changes in the specificity of antibody binding by themselves, they were carried out a series of experiments in which commercial intravenous immunoglobulin (Ivlg) was replaced by human or serum plasma. Ivlg is a fraction of precipitated plasma alcohol pooled from multiple donors, typically 1, 000 - 10,000 donors. Typically, Ivlg contains mainly IgG, and mostly lacks IgA, IgM and other plasma proteins. When Ivlg is tested untreated for the presence of autoantibodies by the ELISA test, the results of the test are negative. Due to its preparation, Ivlg is also free of lipoprotein micelles, vesicles or other macromolecular structures. Therefore, if Ivlg was positive for the presence of autoantibodies after an incubation treatment, it could be that the autoantibodies were obtained by an alteration of IgG antibodies already present in the preparation of Ivlg and not by a decomposition of structures or vesicles, hiding the autoantibodies. In the examples that follow, the commercial preparation of Ivlg used was lyophilized Ivlg (Immune Globulin Intravenous (Human) Gammar-PI.V., Aventis Behring, Kankakee, Illinois). A commercial preparation of 5 grams of lyophilized Ivlg was reconstituted in sterile phosphate buffered saline (PBS, 100 mg / ml). 1.7 ml of reconstituted Ivlg solution was added to a culture bottle containing the growth medium of bacterial culture (without red blood cells or carbon) and incubated at 37 ° C for 20 hours. The incubated mixture showed seroconversion and the presence of aPL IgG (data not shown). (As expected, only IgG, not IgA or IgM was detected). In similar experiments, the autoantibodies were detected in a mixture that was incubated at room temperature in a shaking vessel, but the results were not as good as at 37 degrees (the results are not shown). Heating the mixture of the bacterial growth medium Ivlg above 37 ° C did not result in additional increases in autoantibodies. As a control, complete Ivlg was analyzed from the bottle for aPL and other autoantibodies, and the results were negative.

EXAMPLE 20 In Example 19, it is shown that autoantibodies can be obtained by incubating a preparation of commercial Ivlg in a bacterial growth medium. The next step was to try to determine which ingredients in the growth medium of bacterial culture play a role in producing detectable autoantibodies. First, Mg in 2% Tryptic Soy Broth (TSB), (containing peptones in a 17 to 3 ratio of pancreatic digestion of casein to soybean papaya digestion, respectively) (the remainder being water incubated at 37 ° C for 20 hours with shaking The incubated mixture was tested for the presence of aPL, and the result was negative, then Ivlg was incubated in a test tube in soy broth, sodium nitropuside (SNP) and hemin (a protoporphyrin containing iron (ferric) at 37 ° C for 20 hours with shaking.The amounts used were 60 microliters of Ivlg, 5 microliters of SNP and 5 microliths of hemin in a total of 1 ml of soy broth The incubated mixture was positive for the presence of aPL, particularly Aps (15 MoM) and aPE (41 MoM). (The data is not shown).

EXAMPLE 21 A series of experiments were performed to determine if incubation with hemin alone was sufficient to cause the appearance of autoantibodies in Ivlg or in plasma or serum. Lyophilized reconstituted ivlg (at a concentration of 100 mg / ml) was added to and incubated in a phosphate buffered solution (PBS) with hemin for 20 hours at 37 ° C. The amounts used were 300 μl of Ivlg solution and 5 μl of hemin solution (75 mg) in a total volume of 1 ml. As shown in Table 19, the incubated mixture showed significant amounts of aPS and aPE IgG, and, to a lesser extent, aCL IgG. When serum or plasma were incubated with hemin under similar conditions, no autoantibodies were detected.

TABLE 19 Antiphospholipid antibody (aPL) results * PL binding protein Normal independent dependent IgG 31 MoM +++ 4 MoM < 4 MoM aPS IgA 1 MoM 1 MoM < 3 MoM IgM 1 MoM 1 MoM < 5 MoM igG 7MoM + 1 MoM < 4MoM aCL oR 1 MoM 1 MoM < 4MoM igM 1 MoM 1 MoM < 6MoM igG 32 MoM +++ 3 MoM < 4 MoM aPE igA 1 MoM 1 MoM < 3MoM igM 1 MoM 1 MoM < 5MoM igG 5 MoM + 1 MoM < 4 MoM aPC igA 1 MoM 1 MoM < 3MoM igM 1 MoM 1 MoM < 4 MoM Table 19 is a table summarizing the aPL test results for a sample of Ivlg that was incubated with hemin in a Tris regulator.

EXAMPLE 22 The fact that positive results for the presence of autoantibodies could be obtained when Ivlg was incubated with hemin, while negative results were obtained when serum or plasma were incubated with hemin, suggesting that the serum or plasma could contain substances that inhibit or interfere with the procedure of obtaining autoantibodies. In a series of experiments, Ivlg was incubated in a Tris pH regulator with hemin, for 20 hours at 37 ° C, similar to the procedure of Example 21 with the added feature that an increased amount of human serum (the inventor) was added. to lots before incubation. Each lot was separately analyzed for the presence of aPS, aCL, aPE and aPC autoantibodies, and the results are shown in Figure 3. The results shown in Figure 3 demonstrate that increased amounts of serum have an inhibitory effect on the release of antiphospholipid antibodies. Similar results were shown with serum replacement plasma (data not shown). One possible explanation for these results is that hemin, which contains an iron molecule in the ferric state and which is known as an active oxidation agent, can act to oxidize a binding site of certain immunoglobulin molecules so that the site Altered binding is able to bind auto-antigens. This procedure can be inhibited by substances, perhaps antioxidants, in the blood.

EXAMPLE 23 Human serum (the inventor) was diluted 1/10 in Tris pH buffer. In a series of experiments, the diluted serum, in batches of 1 ml, was incubated with an increased amount of hemin, specifically, 0 μl, 10 μl, 25 μl and 50 μl. (Previously, it was found that hemin by itself was not sufficient to cause the release of autoantibodies from blood or serum, although it was sufficient to cause such release of Ivlg. Therefore, the purpose of diluting the serum was to dilute the effect of any interfering substance found in the blood, such as antioxidants). The batches were analyzed for the presence of aPS, aCL, aPE and aPC autoantibodies, and the results are shown in Figure 4. The results shown in Figure 4 show that although no significant amounts of autoantibodies were detected in the diluted serum when they added 0 or 10 μl of hemin, important quantities were detected with 25 μl of hemin. For an unknown reason, the amounts of autoantibodies detected were lower with 50 μl of hemin.

EXAMPLE 24 The next series of experiments were designed to determine if an antioxidant such as vitamin C, which is present in the blood, can inhibit the release of autoantibodies. In a series of experiments, Ivlg was incubated in a pH regulator of Tris with hemin, with the added feature that an increased amount of ascorbic acid (vitamin C) was added to the pH buffer containing hemin and allowed to mix for 30 minutes. minutes before adding Ivlg and before incubation. As shown in Figure 5, there was approximately 78% inhibition of aPE-induced aPE release with 1 mg of vitamin C, an amount representing a physiological concentration of vitamin C. There is a biphasic curve with aPS release that gives the possibility that vitamin C at low concentrations can act as an oxidizing agent, but becomes an antioxidant (reducing) agent at higher concentrations.

EXAMPLE 25 The following series of experiments was designed to determine whether the vehicle in which hemin dissolves has an impact on the results obtained and whether the iron atom in hemin is necessary. In a series of experiments, Ivlg was incubated in a pH regulator of Tris with hemin, or with other additives. In particular, in one example, hemin was solubilized with NaOH. In another example, it was solubilized with DMSO. In other examples, hematoporphyrin IX (hplX), which is the same molecule as hemin, but without iron (Fe +++), was used instead of hemin and solubilized with NaOH or DMSO. In other examples, NaOH and DMSO were tested as controls (without hemin or HplX). As shown in Figure 6, the use of hemin solubilized with NaOH produced positive results for the presence of autoantibodies, while hemin + DMSO, hplX + NaOH, hplX + DMSO, NaOH alone and DMSO alone did not produce positive results.

EXAMPLE 26 To further establish that hemin caused the oxidation of antibodies, equimolar amounts of hemopexy (Hpx) were added to the hemi-mix IvIGgPBS. Hpx is an antioxidant molecule with an extraordinarily high binding affinity for heme iron. Freeze-dried Hpx purchased from SciPac (Kent, England) was reconstituted in PBS at 10 mg / ml. Figure 7 shows the redox data APS that result from adding increased concentrations of hemin to opposite IvIgG with the addition of equimolar concentrations of Hpx. Since there is a 1: 1 binding interaction between hemin and Hpx, Hpx was able to negate the redox capacity of ferric iron present in hemin.

EXAMPLE 27 To illustrate the wide range and activity of autoantibodies that can be obtained by the treatment of Ivlg oxidation, a series of Western blots were fixed using used cells from 3 different cell lines using Ivlg treated with hemin or Ivlg untreated as antibodies Primary and using conjugate labeled with antihuman HRP as a control (HRP = horseradish peroxidase) The blots are shown in Figure 8. The "B" lysate is a B cell line called Raja from a patient with lymphoma. The "T" lysate is a cell line derived from T-lymphocytes called Jurkat again from a patient with leukemia. The U87MG lysate is a cell line of blasts of gleoblastoma (brain cancer). The reduced used ones were run in the gene at a concentration of 50 mg / ml. To obtain the preparation of Ivlg treated with hemin, 75 μg of hemin was combined with 1 ml of PBS containing 6 mg of IvIgG. The incubation lasted 20 hours at 37 degrees. In Figure 8, the blot in which Ivlg treated with hemin was used as the primary antibodies was labeled "IgG test; the blot where the untreated IvgG was used as the primary antibodies was marked" Control "and the blot to which HRP-labeled anti-human conjugate without primary antibodies was labeled "Secondary." The treated hemin and untreated IgG preparations were diluted 1/1000 respectively, The conjugate labeled with anti-human HRP was used as a dilution of 1. / 5000. These data clearly demonstrate that Ivlg treated with hemin has abundant activity towards human cellular components compared to untreated IvIgG and conjugated control, which does not do so.

EXAMPLE 28 The following experiment was carried out to determine whether oxidation agents other than hemin, and in particular, oxidation agents that do not contain iron, can be effective in not covering autoantibodies. A mixture of 25 μg of potassium permanganate (KMn0) at a concentration of 100 μM, and 2 mg of Ivlg in a total volume of 1 ml of phosphate buffered saline was incubated overnight at 37 ° C. In the incubated mixture, aPC and aPS can be detected. ACL was usually detected, but not aPE (data not shown). It was subsequently determined that one reason why aPE was not detected is because KMn0 alters the phospholipid PE antigen used in the ELISA test.

EXAMPLE 29 After it was shown that autoantibodies may not be covered by oxidation reactions, the next question was whether electrochemical methods, such as an electromotive force from a battery, could achieve the same effect. Ivlg was dissolved in a phosphate-buffered saline solution, and, in separate experiments, galvanized steel, copper, or stainless steel electrodes were connected to the positive and negative terminals of a 9-volt battery and immersed in solution for 1-2. minutes During this period, bubbling was noted in the solution and the PBS solution changed color (blue when copper wires were used, brown when stainless steel and green wires were used when galvanized steel wires were used). As shown in Figures 9A and 9B, the treated solution that tested positive for the presence of aPS, aCL, aPE and aPC autoantibodies in an aPL-dependent test, and positive for the presence of aPS, aPE and aPC autoantibodies in a test independent of aPL.

EXAMPLE 30 To avoid the interaction of metals with the solution and thus determine the effect only of an electric current, graphite electrodes were used instead of the metal electrodes. Graphite is inert, but it is capable of passing electrons in conductive solutions without participating in the reactions. Ivlg was dissolved in a phosphate-buffered saline and graphite electrodes connected to the positive and negative terminals of a 6-volt battery were immersed in the solution for 60 seconds. As shown in Figure 10, the treated solution tested positive for the presence of aPS, aPE and aPC autoantibodies.

EXAMPLE 31 In experiments involving the application of electric current to solutions of Ivlg in phosphate buffered saline, a significant increase in pH was noted, possibly due to the formation of NaOH. To maintain the reactions at physiological pH levels, a cell culture medium, RMPI, was substituted for the buffered saline phosphate solution. The next series of experiments was carried out to determine the effects of exposure time for electric current upon discovery of the autoantibodies. Ivlg was dissolved in RMPI, a cell culture medium and graphite electrodes connected to the positive and negative terminals of a 6 volt battery were immersed in the solution for a variable amount of time. As shown in Figure 11, the maximum release of dependent aPL was obtained after 60 seconds after exposure to the current. Interestingly, between 2 minutes and 4 minutes, the amount of aPL declined or disappeared.

EXAMPLE 32 Since the previous experiment showed that aPL antibodies can be obtained from Ivlg after exposure to an electric current, but that Ivlg antibodies disappeared after further exposure to the current, the next question was whether not covering the autoantibodies could be reversible by an electric current. That is, can a positive control serum be treated so that autoantibodies are no longer detectable? In separate experiments, aCL positive control serum at a dilution of 1: 400, aPE positive control serum at a dilution of 1: 75 and aPS at a dilution of 1: 400 were exposed to an electric current when immersing graphite electrodes connected to the positive and negative terminals of a 6-volt battery for up to approximately 240 seconds. As shown in Figures 12A-12C, each control serum becomes negative for its respective specificity.

EXAMPLE 33 Based on the results in Example 32, the next question that was questioned was whether autoantibodies in a patent that have an autoimmune disease can be re-covered if the patient's serum is exposed to an electrical current. Serum of a patient having high levels of aPS and aCL was diluted 1/400 with phosphate buffered saline (the dilution in PBS was in an amount that can achieve an OD value of 1,000 in 10-15 minutes) and the electrodes of graphite connected to the positive and negative terminals of a 6-volt battery were immersed in the solution for a variable time. As shown in figure 13, the amount of aCL and aPS detectable in serum samples of the autoimmune patient declined significantly after 30 seconds and was no longer detectable after 2 minutes. These experiments were repeated for other patient antibodies and the same result was obtained (data not shown).

EXAMPLE 34 In an early experiment, a blood sample from a patient who had high and very specific titre IgA aPE was exposed to hemin in a routine microbiology culture bottle. It was observed that after exposure to hemin his IgA aPE disappeared, and the emergence of IgG aPS, aCL and more particularly, IgA aPE was detected in aPL ELISA. At that time, an explanation for that phenomenon was not readily apparent. With the discovery of a faster procedure not to cover, using electric current, it becomes possible to confirm the earlier results with another patient who has aPE superior. In this experiment, the serum of a patient having higher aPE was diluted in PBS by 1/75 and the graphite electrodes connected to the positive and negative terminals of a 6 volt battery were immersed in the solution for a variable time. As shown in Figure 14, aPE becomes undetectable (covered) within 30 seconds of a 6-volt DC current application, with a concomitant non-cover and detection of PS and aCL IgG. The new non-covered aPLs spiked around 30 seconds only to be covered again after 2-4 minutes of exposure. An important technical aspect addressed by the previous experiment was that the patient's aPE was further treated from the plasma protein diluent used in the test, and in the current case, 10% adult bovine plasma (PBL). In other experiments not shown, the diluted patient serum was exposed to 6 volt EMF conditions before adding the plasma proteins used in the ELISA diluent. The important aspect of these experiments was to show that the effects of EMF are applied to patient antibodies and not to EMF changes in the plasma proteins used in the diluent. These experimental data support the observations that redox reactions determine the appearance and disappearance of different antibody specificities. Also what is learned from these experiments is that the effects of redox appear to be limited to the antibody binding site, the Fab portion of the antibody molecule. This is because the conjugates labeled with heterologous antihuman antibody used in ELISA are not affected as the conjugates continue to recognize different antibody heavy chain targets (Fe portions) of the antibody molecules. Thus, since the human antibody is not consumed or destroyed by redox, the most reasonable explanation is that the antibody binding site in the Fab portion of the antibody molecule contains accessible electrons that can participate in the oxidation process /reduction.

EXAMPLE 35 The following experiments were carried out to observe whether plasma proteins other than autoantibodies can have altered binding specificity by oxidation-reduction. In these experiments, a solution of 10% adult bovine plasma (PBL), the same solution containing phospholipid-binding proteins that was used to determine the binding of protein dependent aPL, was exposed to an electrical current of a 6 volt battery for a variable time. The treated PBL samples were then used in ELISA tests with patient serum aPS-, aCL and aPE-positive to observe if the treatment of PBL could affect the ELISA result. As shown in Figure 15, at time zero (untreated PBL), the positive patient serum gives the aPL response in PBL that is observed routinely. Since 10% ABP is exposed to oxidation-reduction (EMF) over time, the amount of aPL detected decreases and after 2 minutes, the aPE positive serum is no longer positive. These results indicate that the plasma proteins that are responsible for the reactivity of aPL in the patient are altered by exposure to electric current. For example, since kininogen is the plasma protein responsible for providing a positive ELISA signal for aPE-dependent reactions (the kininogen binds to PE, subsequently the antibody binds to the kininogen, however the aPE does not bind PE or kininogen independently ), this shows that the kininogen in the ABP sample is altered by redox exposure. aCL is also negative after 240 seconds of exposure and since this patient serum requires protombrin and / or beta2 glycoprotein (or both may be involved) to produce a positive signal in aPL ELISA, these two proteins must also be altered by the reactions of Redox The same two plasma proteins are involved in the aPS example. Obviously, many modifications and variations of the present invention are possible in view of the above teachings. Therefore, it should be understood that, within the scope of the appended claims, the invention may be practiced otherwise than as specifically described.

Claims (5)

1. NOVELTY OF THE INVENTION CLAIMS 1. - A method comprising the steps of: providing a composition comprising at least one plasma protein suspended or dissolved in a liquid medium, the plasma protein has a binding specificity that can be reversibly altered by a change in its redox state , and exposing the composition to an oxidation agent or sufficient electrical potential to reversibly effect the alteration of the binding specificity of said plasma protein.
2. 2. The method according to claim 1, further characterized in that said liquid medium is whole blood, serum or plasma diluted or undiluted.
3. 3. The method according to claim 1, further characterized in that the composition comprises intravenous immunoglobulin (Ivlg) suspended or dissolved in a liquid medium.
4. 4. The method according to claim 1, further characterized in that the plasma protein is an antibody of isotype IgG, IgA or IgM.
5. 5. The method according to claim 1, further characterized in that the plasma protein is an autoantibody of isotype IgG, IgA or IgM. 6. - The method according to claim 1, further characterized in that the plasma protein is a plasma protein different from an antibody. 7. The method according to claim 1, further characterized in that the oxidation agent is hemin. 8. The method according to claim 1, further characterized in that the oxidation agent is KMn0. 9. The method according to claim 1, further characterized in that the oxidation agent is chlorophyll. 10. The method according to claim 1, further characterized in that the oxidation agent is a molecule that has the ability to reduce by acting as an electron acceptor for other molecules that act as electron donors. 1 - A method comprising the steps of: providing a composition comprising a fluid or biological extract of a biological fluid, wherein the biological fluid or extract contains at least one circulating coated protein that has a binding site with a binding specificity which can be altered by a change in its redox state, exposing the composition to an oxidation agent or to an electrical potential sufficient to effect the alteration of the binding specificity of said coated circulating protein, thus not covering the circulating protein and detecting the protein circulating without coating in the composition or recovering the circulating protein without coating the composition. 12. - The method according to claim 1, further characterized in that the biological fluid is whole blood, serum, plasma or placental cord blood diluted or undiluted. 13. A method for obtaining and isolating an autoantibody from a biological fluid containing antibody or from an extract containing antibody from a biological fluid, said autoantibodies containing fluid or biological extract which, prior to the method being carried out, are not capable of binding to an autoantigen and therefore not detected by a test based on a receptor-ligand binding, the method comprises the steps of: exposing the biological fluid or extract to an oxidation agent or sufficient DC electric current to altering a binding specificity of an autoantibody so that said autoantibody becomes capable of binding to an antigen, thus becoming detectable and recoverable from the fluid or biological extract by a method of separation of receptor-ligand binding, and recovering the autoantibody of the fluid biological. 14. The method according to claim 13, further characterized in that the biological fluid is whole blood, serum or plasma diluted or undiluted. 15. The method according to claim 13, further characterized in that the extract containing antibody from a biological fluid is intravenous immunoglobulin (Ivlg). 16. The method according to claim 13, further characterized in that the oxidation agent is hemin or chlorophyll. 17. - The method according to claim 13, further characterized in that the oxidation agent is KMn04. 18. The use of an antioxidant for the preparation of a medicament for treating an autoimmune disease caused by oxidation of autoantibodies that do not cover an antigen binding site of the autoantibodies. 19. A method comprising the steps of testing a sample of blood, plasma or serum from a subject to determine a quantity and / or type of autoantibodies detectable in the sample, treating a sample of blood, plasma or serum of the subject when exposing the sample to an oxidation agent or DC electric current, test the blood sample, plasma or treated serum of the subject to determine a quantity and / or type of autoantibodies detectable in the treated sample, and compare the amount and / or type of detectable autoantibodies in the sample without treating with the amount and / or type of autoantibodies detectable in the treated sample. 20. A product comprising a biological fluid or an extract containing protein from a biological fluid that has been exposed to an oxidation agent or to an electric DC current sufficient to alter a binding specificity of at least one protein contained in the same, wherein the binding specificity is altered from the protein that does not have the binding capacity with respect to an antigen or ligand specific to the protein that has binding capacity with respect to the antigen or specific ligand. 21. - The product according to claim 20, further characterized in that said biological fluid is whole blood, serum or plasma. 22. The product according to claim 20, further characterized in that the extract containing protein from a biological fluid is intravenous immunoglobulin (Ivlg). 23. The product according to claim 20, further characterized in that at least one protein is an antibody. 24. The product according to claim 20, further characterized in that at least one protein is an autoantibody. 25. The product according to claim 20, further characterized in that the oxidation agent is hemin or chlorophyll. 26. The product according to claim 20, further characterized in that the oxidation agent is KMn04. 27.- A method comprising the steps of providing a composition comprising at least one plasma protein suspended or dissolved in a liquid medium, the plasma protein has a binding specificity that can be altered by a change in its redox state , and exposing the composition to an oxidation agent or sufficient electrical potential to effect the alteration of the binding specificity of said plasma protein, wherein the binding specificity is altered from the plasma protein that does not have the binding capacity with respect to a specific antigen or ligand to the plasma protein that has binding capacity with respect to the antigen or specific ligand. 28. A method comprising the steps of providing a composition comprising at least one plasma protein suspended or dissolved in a liquid medium, the plasma protein has a binding specificity that can be altered by a change in its redox state , and exposing the composition to an oxidation agent or sufficient electrical potential to effect the alteration of the binding specificity of said plasma protein, and to detect the plasma protein with an altered binding specificity.