Title of Invention	“IMMUNOGLOBULIN G (IgG) CONCENTRATE DEPLETED OF ANTI-A AND ANTI-B ANTIBODIES AND OF POLYREACTIVE IgGs”
Abstract	The present invention relates to an immunoglobulin G concentrate for therapeutic use. in which the respective contents of anti-A and anti-B antibodies are in accordance with a negative result in the in vitro indirect Coombs test. This IgG concentrate also has a polyreactive IgG content of between 0.01% and 0.1%, in particular between 0.07% and 0.1%, relative to the total content of IgG.

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The present invention relates to an immunoglobulin G concentrate (lg(j) depleted of ami-A (AcaA) and anti-B (AcaB) antibodies and having strongly reduced polyreactivity, and to a method to obtain said concentrates. The use of fractions of human plasma enriched in immunoglobulins (Ig) for the treatment of various infections or congenital deficiencies has been known since the development of Conn's ethanol precipitation method (Conn et al, 1946, J. Am. Chem. Soc. 68,459; Oncley et al, 1949, J. Am Chem. Soc. 71,541). There is an increasing need to produce highly purified Ig concentrates, for injection via intravenous route (IglV), obtained from human plasmas for example. The complex structure of immunoglobulins (four polypeptide chains joined by disulfide bridges), and the variety of antibodies present in the plasma mixture from several thousand donors, are currently factors which do not promote the biotechnological development of immunoglobulins. Although monoclonal antibodies are produced by genetic engineering, their extreme specificity amounts to a disadvantage for therapeutic applications in which polyspecificity appears to be a necessity. Additionally, numerous pathologies, of autoimmune origin lor example, i:re¬currently treated with IgG concentrates which has led to a shortage thereof in Hum K and the United States in recent years. Methods to obtain immunoglobulins and in particular IgG concentrates, in addition to selective precipitation ot the proteins by etha:iol. ma> also comprise various other treatments such as precipitation by polyethylene glycol, controlled proteolytic enzyme treatment... intended to remove aggregates of immunoglabulm polymers which may activate the complement system with associated risl.s of anaphylactic reactions. Also, the presence of dimers in IgGIVs has been correlated with arterial tension drops in vivo (Bleeker W.K. et al. Blood, 95, 2000, p.185o-1861). An alternative route to ethanol precipitation has been described by Steinbuch a al (Rev. Franc, et. Clin, et Biol. 1969, XIV, 1054) which has recourse :o precipitation by octanoic acid. This acid precipitates most plasma proteins and leaves the immunoglobulins in the supernatant. Purification of these immunoglobulins is obtained by passing through an anion exchanger, DEAE-ccllulose, under conditions which do not retain the IgGs. The fraction of non-retained IgG is then concentrated. Various methods have also been developed to increase the purity of the products using chromatographic techniques. Particular mention may be made of patent applications EP 0 703 922 and WO 99/64462, which describe the association of at least two successive chromatographic steps, one by anion exchange, the other by cation exchange. The specificity of these methods is provided by the propery of the anion exchangers whereby they do not retain immunoglobulins (i. under conventional chromatography conditions, but instead they fix most of the other proteins co-purified during the pre-purification steps. Similarly patent application WO 02/092632 can be cited, filed by the Applicant, which discloses the preparation of Ig concentrates using a single chromatography step on anion exchanger, conducted at alkaline pH, for their retention on the chromatographic medium. However, numerous scientific publications indicate that the injection of IgGs obtained by the above fractionating techniques can cause, sometimes severe, accidental haemolysis in patients undergoing treatment. As examples, the following publications can be cited: Buchta C et al, Biologicals, 33,2005,41-48, Wilson J.R. ei al, Muscle & Nerve, 29(9), 1997, 1142-1145, Copelan E.A. et al, Transfusion, 26, 1986, 410-412 and Misbah S.A. et al, Drug Safety, 9, 1993, 254-262. The study of effects on the blood of patients with hameolysis, performed using the direct Coombs test (direct Coombs test - DCT) in particular, has shown that the red blood cells are coated with immunoglobulins directed against antigens A, B or D present on their surface, thereby causing their haemolysis. This is why the IgGs available on the¬market are obtained from selected plasmas to avoid the presence of ann-l) immunoglobulins or other antibodies high in anti-A or anti-B litres. Buchta et al, cited above, considered different approaches to achieve a signification reduction in anti-A antibodies, originating iron; B and O booJ group donors, and in anti-B antibodies derived from A and 0-group donors, in plasma derivatives such as IgGs. with a view to minimizing the risks of hacmol) sis that :iv direcil) correlated with the levels of these antibodies, when treating patients with these plasma derivatives. It was notably envisaged to choose the donors, to remove the anti-A and anti-B antibodies, to produce derivatives of blood plasma originating from a specific group, group A and/or B, and to exclude from batches those plasmas having a high titre in anti-A and anti-B antibodies. Some approaches are considered to be non-realistic on account of the cost or complexity of' the steps to be taken. It is noted that anti-A and anti-B antibodies are partly removed during the ethanol fractionation mentioned above. Since the needs for IgGs are constantly increasing, there is a need for increasingly larger pools of donors which, statistically, will include greater numbers of O-group donors. As a result, blood derivatives such as IgGs will contain quantities of anti-A and anti-B antibodies that are too high for their removal by conventional fractionation. These increasing needs prevent contemplating the possible selection of group AB donors only, in order to ensure a low content of anti-A and anti-B antibodies. Each batch of purified IgG concentrates or preparations is controlled for anti-A and anti-B antibodies using test 2.6.20 of the European Pharmacopeia (1997) which is an in vitro application of the indirect Coombs test (indirect Coombs test - ICT). The ICT test consists of adding to a suspension of red blood cells, coated with anti-A or anti-B antibodies of IgG type contained in the IgG concentrates, a solution of antibodies (antiglobulins) directed against motifs of human IgG. These antibodies bind to anti-A or anti-B antibodies attached to the red blood cells and thereby cause their agglutination through the formation of bridges between the IgGs. The assay for detection of anti-A or anti-B antibodies is directly inspired by this conventional test in haematologjcal serology (Coombs test). According to the European Pharmacopeia, IgIVs must not show any agglutination of A or B red blood cells under the ICT test at a dilution of 1:64, conducted with an IgG solution whose initial concentration is reduced to 30 g/1. This is why IgG samples to be tested must be diluted to obtain a titre i.e. the value of the last dilution which no longer causes agglutination. Negative ICT results on IglV solutions whose dilutions are lower than the 1:64 dilution, following the European pharmacopeia, indicate a low content of anti-A and anti-B antibodies which is acceptable. However, even with IgG concentrates giving a negative result for the test prescribed by the European pharmacopeia, i.e. those with a dilution ratio of less than 1:64, the risks of haemolytic reactions cannot be excluded (Buchta el al. cited above). It is to be noted that the American and Japanese pharmacopoeias make iu As mentioned above, anti-A and anti-B antibodies are partly removed dunis-the preparation of IgG concentrates by elhanol fractionation, however a residua! content is observed which may exceed the upper limit of the Kuropc.m pharmacopeia. Additionally, concentrates prepared following the method developed by the Applicant and described in its patent application WO 02/092652 lia\ e a higher content thereof than those obtained by ethanol fractionation. Additional purification of the IgG concentrates thus obtained, with respect to anti-A anti-B antibodies, is therefore necessary since some batches of IgG concentrates may have contents thereof that are higher than the threshold set by the European Pharmacopeia. One technique to remove these antibodies from IgG concentrates consists of purification by affinity chromatography using immunoadsorbants as medium, of oligosaccharide type similar to antigens A and B of the blood groups, suid oligosaccharides particularly being trisaccharides grafted on a chromatographic matrix. As example, mention may be made of the publication by Ma/id M.A. et at. J. Appl. Biomater., 3(1), 1992,9-15, which uses a chromatographic medium containing silica particles on which are grafted haptenes of synthesized oligosaccharides, characteristic of blood groups A and B, and in particular A-trisaccharides. Also. I loin M.S. et al (ASAIO J, 46(6), 2000, 702-706) describe the use of tubular fibrous membranes grafted with specific anti-A and anti-B antigens for the removal of anti-A and anti-B antibodies from whole blood. It is also reported that said chromatographic mediums are very stable, which limits release of these residual haptenes in the concentrates of interest. Patent application WO 01/27623 describes a method to obtain a plasma dc-specified in antibodies of blood groups A and B i.e. a plasma suitable for any receiver. These specificities are essentially carried by immunoglobulins M (IgM). De-specification is obtained by passing through an experimental affinity medium for group A, then through another affinity medium, also experimental, for group B. In the event of simultaneous presence of anti-A and anti-B (group O), successive passing through the two medium supports is necessary. One of the additional characteristics of IgG concentrates available on the market is their polyreactivity. It is to be recalled that polyclonal antibodies such as lgG» aic normally combined with a single epitope (antigenic motif) in umque fashion. However, this strict specificity of antibodies may sometimes extend to other antigenic motifs and show affinity for secondary epitopes with weaker bind ng however than for the nominal motif. Polyclonal IgGs may react to a greater or lesser e\te.i; witt: .ttruciures such as actio, myosin. trinitrophenyl-modified albumin In this respect, intravenous immunoglobulins (IglV) are preparations of polyclonal human IgGs containing: - munune antibodies directed against external antigens and resulting from a:-immunization process; - natural antibodies recognizing intracellular proteins, surface mcmbn.n..-antigens, circulating self proteins and the variable region of other antibodies. The latter arc called anti-idiotype antibodies (Kazatehkine M.D.. et al, Immunol. Rev.. 139.1994,79-107). Natural antibodies do not result from deliberate immunization (Coutinho A. et al, Curr. Opin. Immunol., 7,1995,812-818. They are polyreactive in that they express variable affinities for the self antigens (Bememan A. et al, EurJ.Immunol., 22. 1992. 625-631 and Lacroix-Desmazes et al, J. Immunol. Methods, 216,1988,117-137). Chemical treatments (6M urea. 1.3M sodium thiocyanatc and acid treatment pH - 2.0) of lgIVs can increase the polyreactive activity of these polyclonal immunoglobulins (Douvet J.P. el al, J. Autoimmun., 16(2). 2001. 163-172). I lowevcr, no beneficial effect has ever been demonstrated in patients treated with said immunoglobulins. To summarize, the polyreactive activity of the antibodies present in an IglV preparation can be due to: - the presence of natural antibodies contained in each individual plasma, - the presence of anti-idiotype antibodies contained in each individual plasma. - the polyreactivity of the antibodies generated by the production method. Therefore, some authors consider that polyreactivity is an intrinsic property of IgGs which are therefore naturally present in the human body. Others demonstrate that a method to purify monoclonal or polyclonal IgGs can reveal polyreactivity that is undetectable before purification. Indeed methods to produce IgGs from plasma also generate a polyreactivity which translates a> oxidative "stress" and partial carbonylation of IgGs during production. This is confirmed by Bouvet J.P et al, Journal of Autoimmunity, 16, 2001. pp. 163-172, for whom polyclonal IgGs become highly polyreactive after treatment with urea in particular. It is also indicated in this document that part of the efficacy of IgGs in clinical use is attributed to their polyreactivity. IT tallows that the polyreactivity of these IgG concentrates can therefore h. explained by the combined presence of natural polyreactive IgGs and of polyrcactiw lgGs obtained by usual purification methods and accounting for 0.5 to 1% of :.\\ polyvalent IgGs. The treatment of patients with IgG preparations may require the arministering of high doses of up to 1 to 2 g/kg. These dosages lead to short-term treatment e.g. in one day with quantities 7 to 10 times greater than the receiver's Physiological IgG quantities. As a result, this level of polyrceatiivc IgGs genereted by the- production method in IgG concentrates may cause adverse side effects such a.s fever, nausea or headache. Therefore a distinction must be made between the polyreactivity of antibodies due to natural and anti-idiotype antibodies which, as shown by the Applicant in its patent application EP 1 059 088 has advantages, from the polyrcuctivit} of tlu-antibodies generated by the production method. It has been shown by the Applio:n!i in patent application EP 1 059 088 that fractions of polyreactive IgGs naturally contained in the plasma, isolated from human polyvalent IgIVs, can advantageously be used to treat certain inflammatory diseases such as rheumatoid polyarthritis on account of the smaller dosage required for this fraction. Therefore, to avoid adverse reactions both regarding haemolysis of red blood cells and regarding side reactions which may occur with massive administering of IgGs during a course of treatment, there appears to be a need for IgG concentrates for therapeutic use, in particular for intravenous injection, significantly depleted of auti-A and anti-B antibodies and whose polyreactivity generated by the production method is preferably largely reduced compared with IgG concentrates current!) available on the market, whilst having at least identical efficacy with respect to immunotherapy. Therefore, the invention concerns a concentrate of immunoglobulins G for therapeutic use, characterized in that its respective contents of anti-A and anti-B antibodies give a negative result with indirect Coombs test in vitro. The invention also relates to a method to produce immunoglobulins G, with which it is possible not to generate these polyreactive antibodies which may be less well tolerated than natural and anti-idiotype antibodies. On this account, the IgIVs obtained with the method have a lower polyreactivity than the other tested IgIVs. Under the invention, the IgGs of these concentrates are advantageously polyclonal IgGs obtained from blood plasma or from a blood plasma fraction already enriched with IgGs. The IgG concentrates for therapeutic use have IgG concentrations that are frequently used at present, preferably between SO and 100 g/1. These concentrates are intended for clinical use, and may in particular be injected by intravenous route. For this purpose, they must be virally safe, and ma> optionally} contain excipicnts such as stabilizers compatible with this clinical use. The Applicant has found that it is possible to provide said IgG concern rules having anti-A and anti-B antibody contents that are much lower man those found in siuiidarJ IgG concentrates i.e. those obtained by ethanol fractionation and or using purification techniques associating chromatographies, as mentioned abo\c. and which have not undergone additional treatment to remove the antinbodies amd: consideration. Also their contents are wel' below the thresholds accepted by the-European Pharmacopeia, which very significantly limits the risks of haemolysis in some patients receiving treatment. When the IgG concentrates of the invention are subjected to tests intended to evaluate the quantities of anti-A and anti-B antibodies, it is obseivcd that the results of in vitro agglutination tests of A. B and'or AB red blood cells in the presence of anti-human IgG antibodies are systematically negative . notably at the initial concentration of 30 g/1 laid down by the method of the European Pharmacopeia. The conducting of the indirect Coombs test, defined previously, with IgG concentrates of the invention therefore leads to systematically negative results even with IgG samples as such i.e. non-diluted. It would therefore appear that content of these anti-A and anti-B antibodies in these concentrates is already non-detectable by the ICT test. If the level of anti-A and anti-B antibodies is very low in the lgG concentrates, the I AT test can no longer be applied, even more so under the conditions of the European Pharmacopeia since the agglutination reactions of the red blood cells no longer take place, even with the addition of anti-human IgG antibodies, since the density of anti-A and anti-B antibodies is too weak to allow bridges to be set up between the red blood cells by bonding of anti-A and anti-B antibodies fixed to the red blood cells and the anti-human IgG antibodies. It is possible however to verify the presence of these anti-A and/or anti-B antibodies in very low concentration by causing immunohaemolysis of the red blood cells which fixed these antibodies and by measuring depletion compared with a conventional concentrate which has not undergone treatment to remove anti-A and anti-B antibodies. Immmunohaemolysis is a specific immunological reaction which occurs when the antibody is attached to its target in the presence of complement factors. Activation of complement activity leads to the release of perforins which pierce the membrane of the red blood cell, allowing the haemoglobin to escape. All that is required subsequently is to use a sensitive method e.g. with radioactive tracers (see below) to measure the quantity of released haemoglobin, proportional to the quantity of anti-A and anti-B antibodies present. The Applicant has demonstrated in particularly advantageous manner that the IgG concentrate of the invention has a content of anti-A antibodies of no more than 23 ng/mg IgG, in particular between 19 and 23 ng/mg IgG. and a content of ant -B antibodies of no more than 20 ng/mg IgG, in particular between 12 and 20 ng/mn IgG. Advantageously, the Applicant has found that it is also possible to provide said IgG concentrates with a very low content of polyreactive IgGs, in particular that generated by the production method, thereby imparting a near non-polyreactive.-charactcr to these concentrates which arc just as efficient for the treatment of immunotherapies as the prior art IgG concentrates. This notable absence in these IgGs of a polyreactivity character due to the production method substantially reduces the risks of side effects which may arise subsequent to treatments requiring h i-n dosages. It is also advantageously possible to correct adverse effects arising from the presence of polyreactive IgGs in the IgG concentrate due to the production method, this presence generated in particular by oxidative "stress" and carbonylution during purification methods. Preferably, the residual content of polyreactive IgGs is comprised between 0.01% and 0.1%, in particular between 0.07 and 0.1%. Under the invention. b> content of polyreactive IgGs is meant a molar or weight percentage. This content is determined using methods described by the Applicant in patent application 1 0S9 088. The IgG concentrates of the invention are dierefore defined by a notable absence of anti-A and anti-B antibodies in the active ingredients, which arc directed against the epitopes present on the red blood cells. The IgG concentrates may be in liquid or lyophilised form, in the presence of suitable stabilizers, and may be stored for later use. These stabilizers arc advantageously those developed by the Applicant in its patent application WO 2004/091656 A2, namely a mixture of a sugar alcohol, preferably mannitol, sorbitol or their isomers, of glycine and of a non-ionic detergent such as Tween®80, Tweenflfi 20, Triton® X100 or Plutonic® F68, all three compounds being pharmaceutical ly acceptable. The concentrations of the formulation were determined by the Applicant to stabilize liquid and/or lyophilised forms.. Preferably, the final mannitol concentrations in the concentrates lie between 30 g/1 and SO g/1, that of the detergent between 20 and 50 ppm. and that of glycine between 7 g/1 and 10 g/1. The concentrations of these compounds repiesent the m,: concentrations in the IgG concentrates. Said IgG concentrates, for therapeutic use, may in particular be injected \ i: intravenous route as indicated previously. For this purpose, the IgG concentrates : the invention must be virally safe using a conventional solvent-detergent treatment for example known in the prior art. e.g. using a mixture of TweenSf 80TnBP )) Triton® X 100/TnBP, and/or filtering steps for optional removal of viruses and/.or other macromolcculcs which may not have been removed by the solvent-deterge.n viricidc treatment e.g. the prion - the agent responsible for transmissible spongifor n encephalopathy. The invention also concerns a method to obtain an IgG concentrate such us mentioned above, comprising the following steps: a) preparing an IgG concentrate by ethanol fractionation and/or chromatographic separation, associating a viral inactivation step, b) immunoaffinity chromatography by percolating said IgC concentrate through a mixture of medium supports whose matrixes are grafted with oligosaccharide groups which have antigenic similarity with blood groups A and IB. and c) filtering to remove viruses and/or particles of a size greater than 20 nm It was found in remarkable manner by the Applicant that not onl> can this method advantageously be implemented on an industrial sale, but also that In combining the steps leading to the preparation of IgG concentrates with a specific step to remove anti-A and anti-B antibodies, it is possible to obtain an IgCi concentrate of the invention, for therapeutic use, which also preferably comprises a content of polyreactive IgGs that is less than 0.1% relative to the total IgG content. Additionally, in said concentrate the content of undesired anti-A and anti-B antibodies is well below the lower limit of the test described in the European Pharmacopeia, even giving a negative result for the ICT test on said non-diluted samples. Preferably, step a) of the method may itself be a method to obtain IgG concentrates such as those previously mentioned. It concerns ethanol fractionation developed by Conn et al or chromatographic separation such as described i'or example in EP 0 703 922 and WO 99/64462. Particular preference is given to the methods developed by the Applicant in patent applications WO 94/29334 and WO 02/092632 Al, and more particularly to that described in WO 02/092632 AI. In this case, step a) of the method of the invention comprises prc-purificuM.n in precipitation of lipid contaminants from blood plasma or from an IgG-ennered fraction of blood plasma, single chromatography on an anion exchange lesin conducted at alkaline pH. selective elution of the IgGs in one step using a suitable buffer at a ph comprised between 4 and 7. Step a) of the method comprises viral inactivalion treatment, preferably using a solvent-determent as described by Horowitz in patent US 4 764 369. This is judiciously curried out before a, or when applicable, before the subsequent chromatographic sk-p performed in particular to remove the chemical residues of this treatment. The collected IgG fraction is already sufficiently concentrated, and can then undergo additional concentration steps by ultrafiltration and sterilizing filtration. This concentrate is then subjected to imniunoaffiniiy chromatography ■»; a mixture of two medium supports grafted with antigen groups having similarity wh\ blood groups A and B, preferably on a column loaded with said medium mixture. Preferably, the chromatographic medium consists of a natural crosslinked polymer matrix, of agarose type, on which spacers or coupling arms are grafted, which in turn are grafted with oligosaccharides these advantageously being trisaccharides corresponding to the epitopes of blood groups A and B. In particular. vcry good results arc obtained using said medium whose tnsacchuridcs. corresponding to the epitope of blood group A. have the structure N-acetylgalactosamine (GalNAc) - Galactose (Gal) - Fucosc (Fuc). and those corresponding to the epitope of blood group B have the structure Galactose-Galactose-Fucose. Said medium is highly advantageously a gel or rosin commercially available under the trade name GLYCOSORB ABO® from Glycorex Transplantation AS (Sweden). By way of example, if this medium is used, the trisaccharide corresponding to the epitope of blood group A has the following structure: (Formula Removed) N-acetylgalactosamine (GalNAc) Galactose (Gal) Fucose (Fuc) For example, if this medium is used, the trisaccharide corresponding to the epitope of blood group B has the following structure: (Formula Removed) Advantageously, the medium mixture grafted with antigen groups similar to blood group A and blood group B has a respective proportion comprised between 25/75 and 75/25 (v/v). It is effectively possible to adjust the proportion of the two medium supports in the column to the donor population according to the distribution of its blood groups. For usual use, the column is preferably loaded with a 50/50 (v.\) mixture of each specific medium mentioned above. Analysis columns 15 to 25 cm in length can be used and 0.5 to 1 cm in diameter. For application on a pilot scale. columns with a length of 40 to 60 cm and width of 40 to 60 mm can be used. In this case it is possible to load the column with 600 ml of immunoaffinity medium. Said medium is stored in 1M NaOH between two cycles of use. Before use. it is washed with water. The immunoaffinity chromatography column is then loaded iwth IgG concentrate, preferably to a proportion of 0.2 to 4 litres, in particular 1 to 2 litres, per millilitre of medium. The specificity of said medium does not require prior packing of the IgG fraction i.e. any IgG fraction or concentrate obtained by plasma fractionating techniques known in the prior art is suitable. Percolation of the concentrate does not entail any eluting mechanism. Therefore, irrespective of the manner in which the IgG concentrate is obtained, it is percolated through the column, optionally using a pump. This percolation allows retention of the anti-A and anti-B antibodies and of the polyreactive IgGs. Advantageously, the column is then washed with water to collect the IgGs still present in the dead volume of the column. After percolating the IgG concentrate, an IgG fraction is obtained depleted of anti-A and anti-B antibodies and of polyreactive IgGs derived from the production method. The anti-A and anii-B antibodies are retained on their antigenic motif of ll e chromatographic medium which modifies their conformation. Therefore the polyreactive IgGs generated during the production method arc also retained on the sites exposed by this conformational change. The affinity of these polyreactive IgGs rerained secondarily is much lower than thai of the anti-A and anti-B antibodies. Their elution is possible by fractionation, after passing the IgGs, through tiie use of an elution buffer containing for example an alkaline-carth metal salt for having a concentration of between 0.1 and 1.5 M at a pi I of 3-8.6. After step b), the method may comprise concentration steps by ultrafiltration and sterilizing filtration. The chromatographic column and the medium arc then washed with an aci-j solution such as glycine-MCl. pH 2.8. for desorption of the anti-A and anti-B antibodies retained on the medium. This medium is then rinsed with water and treated with a 1M NaOH solution. The IgG concentrate highly depleted of anti-A and anti-B antibodies and of polyreactive IgGs is then subjected to filtration to remove any viruses which may have resisted the solvent-detergent treatment and/or to remove other particles of size greater than 20 ran such as prions, IgG polymers generated during production steps. micelle lipopolysaccharidcs, aggregated nucleic acids and proteins. Said treatment is advantageously a nanoflltration implemented with filters of decreasing porosity from 100 to IS nm, in particular three filters arranged in scries and with decreasing retention thresholds of 100, SO and 20 nm. After step c), the method may comprise an additional step to add stabilizers firstly to ensure the stability of the IgG concentrates during their storage, and secondly to allow lyophilisation to prevent denaturing of the IgGs in the various phases associated therewith. Preferably, a single stabilizing formulation is added, that is phannaceuticaily acceptable, meeting the objective of stabilizing the two envisaged storage forms of the IgGs i.e. liquid form or lyophilised form, and of maintaining and even improving the therapeutic efficacy of these IgGs as described in patent application WO 2004/091656 A2. According to other embodiments, selective collection of other immunoglobulins is also possible, as described in patent WO 02/092632 Al. The IgG concentrates are optionally subjected to a subsequent concentration step by ultrafiltration, followed by sterilizing filtration, and can be stored in bottles preferably at temperatures in the region of 4°C. As largely explained above, the IgG concentrates of the invention have anti-A ant-B antibody contents that are well below the thresholds accepted by the European Pharmacopeia. Therefore, assay method 2.6.20 (1997) described therein ma> prove to be insufficiently sensitive to detect the antibodies under consideration present at very low levels in the IgG concentrates of th-j invention. It is there Lire essential to develop assay methods for these antibodies requiring a lower detection threshold than that of the 1CT test in the European Pharmacopeia applied to the detection of anti-A and anti-B antibodies. Said assay method for anti-A and/or anti-B antibodies in the IgG concentrates of the invention comprises the steps consisting of: a) preparing and calibrating a suspension of red cells of bload group- A B and/or O Rhesus+, b) preparing solutions of monoclonal anli-D antibodies over a range of concentrations from 0 to 200 ng/ml in a biologically acceptable buffer. c) contacting said red blood cells with samples of IgG solution:; or with the solutions of monoclonal anti-D antibodies, and incubating: the mixtures of red blood cells thus obtained for a pre-determined time, d) adding to each red blood cell mixture a fragment of anti-human IgG antibody F(ab')2 labelled with a fluorochrome, and incubating said red blood cells. e) subjecting each mixture of red blood cells obtained at step d) to flow cytometry, 0 determining the content of anti-A and/or anti-I) antibodies in the IgG concentrates. One embodiment of said method to determine the content of anti-A and/or anti-I) antibodies may comprise the preparation of a 1% v/v suspension of red cells of blood group A. B and/or O in a PBS buffer, of pH between 7.0 and 7.4. containing 0.8 to 1.5 wt.% of bovine serum albumin BSA. The red blood cells of the suspension arc counted in a usual flow cytometry device, whose functioning is known to those skilled in the art, then the suspension is calibrated to 37 to 43.106 red corpuscles/ml of suspension. Solutions of monoclonal anti-D antibodies are prepared, whose concentrations range from 0 to 200 ng/ml buffer, preferably a PBS buffer of pH between 7.0 and 7.4, optionally containing 0.8 to 1.S wL% bovine serum albumin BSA. Each solution thus prepared is assayed by absorptiometry to determine its molar extinction coefficient (»:). The IgG concentrates of the invention are then adjusted to a concentration in the range of values of from 1 to 5 mg/ml, preferably 1 mg/ml, using a PBS buffer of pH between 7.0 and 7.4, containing 0.8 to 1.5 wt.% bovine serum albumin BSA. A volume of SO to 100 ul of the suspension of red cells of each blood group is placed in each well of a microplate, e.g. a 96-well microplate, followed by SO to 100 ul of IgG solution in this suspension of red blood cells, or SO to 100 ul of anti-D antibody solutions in this suspension of red blood cells. The whole is left to incubate for a time comprised between lh30 and 2h30. in particular for 2h at temperatures usually lying between 30 and 40°C. preferably 37°C. The different mixtures of red blood cells thus obtained arc then preferably washed with the PBS buffer containing the preceding BSA. and are centrifged: then, to each mixture of red blood cells contained in a mierowcll plate. 50 to 100 µl I-\ab'2) goat ami-human IgG antibody are added labelled with a fluorochrome e.g.. phycoerythrin. present in the previously defined PBS and BSA buffer. The whole is left to incubate for around 20 to 3D minutes in the dark. The different mixtures of red blood cells thus obtained arc then washed and subjected to flow cytometry using any suitable apparatus available on the market containing a device to detect fluorescence of the analyzed compounds. The mean fluorescence intensity (MFI) is given in relation to the eoncentration of monoclonal anti-D antibodies, and the linear regression equation is obtained usiny Excel software. Then, for each sample, the concentration in anti-D antibody equivalent is obtained using the linear regression equation. Since triple batches of the samples were assayed, the mean concentration is determined and the coefficient of variation is calculated using Excel software. The content of anti-A and anti-B antibodies in the IgG concentrates of the invention can be deduced therefrom, which is advantageously the content given above Preferably, one assay method for the anti-A and anti-B antibodies in the above IgG concentrates is conducted by flow cytometry adapted to the context of the invention, whose principle is based on the use of human red cells of blood group A or B, according to the desired specific determination of anti-A and anti-B antibod\ content, using detection of a fluorescence signal proportional to the content of these antibodies. Said assay method comprises the steps consisting of: a) preparing and calibrating a suspension of red cells of blood group A or B, b) contacting said red blood cells with diluted samples of IgG solutions, and incubating the mixture obtained for a pre-determined time, c) incubating said red blood cells in the presence of an anti-IgG antibody labelled with a fluorochrome, and d) subjecting the suspension of red blood cells obtained at step c) to flow cytometry. A 1% (v/v) suspension of red clls is prepared of blood group A or B in a PBS buffer, of pH between 7.0 and 7.4, containing 0.8 to l.S wt.% of bovine scrum albumin BSA. The red blood cells of the suspension are counted in a usual flow cytometry device, whose functioning is known to those skilled in the art. and the suspension is calibrated at 37 to 43.106 red blood cells/ml of suspension. A volume of SO to 100 ul of suspension is placed in each well of a 96-\\ell microplate. followed by SO to 100 ul of different IgG solutions diluted by increments of two by two from a solution of 30 g/1 until an IgG solution of 0.234 g/1 is obtained. Th-* whole is lctt to incubate for between lh30 and 2h30. in particular 2h. at a temperature usually ranging from 3010 40°C, preferably 37°C. The red blood cells arc then washed with the PBS buffer coitiaini0nc the preceding USA. and are centrifuged, then to each wel! 50 to 10') ul of l(ab'); goat anti-human IgG antibody is added, labelled with a fluorochrome such as phycoerythrin. The whole (step c)) is incubated for around 20-30 min. away from light. The suspension obtained is then washed and subjected to flow cytometry using.-any suitable apparatus available on the market comprising a fluorescence deteetion device for the analyzed compounds. For example, the contents of anti-A and anti-B antibodies of three IgG concentrates called Bl, B2 and B3, respectively prepared by ethanol fractionation following Cohn's method (cited above) (Bl), according to patent application WO 02/092632 (B2) and according to patent application WO 02/092632 followed b immunoaffinity chromatography (B3) for depletion in anti-A and anti-B antibodies, and are indicated in Table 1 below. The results arc given relative to the control litre of anti-A and anti-B antibodies in sample Bl whose content of these antibodies was arbitrarily set at 1 as reference. Table 1 (Table Removed) The results in this table show firstly that the contents of anti-A and anti-B antibodies of the IgG concentrates (Bl) prepared following Cohn's method, contain around four times less thereof man IgG concentrates (B2) prepared following the method described in WO 02/092632. In addition, the subsequent treatment of these IgG concentrates on specific immunoaffinity columns reduces the titre of anti-A antibodies by a factor close to 5, and of anti-B antibodies by a factor close to 7 (B3). Another method to determine the content of anti-A and anti-B antibodies which can advantageously be applied, consists of in vitro lysis with the complement, known to those skilled in the art, but which has been specifically designed for the needs of the invention. Said assay method comprises the steps consisting of: a) radio-labelling a suspension of papain-treated red blood cells chosen from among the blood groups A, B, AB and O, previously counted, using a suitable radioactive marker, b) contacting the radiolabeled red blood cells with samples of a predetermined volume of IgG concentrates, c) adding an identical volume to the volume in step b) of norma! serum o d) incubating the mixture obtained at step c) lor a pre-dctermined time, and e) measuring the radioactivity of the incubated solution obtained. A 1% (v/v) suspension of papain-treated red blood cells is prepared of blood group A, B, AB or O, which is then counted in a Malassj/. cell to obtain 100 red blood cells. 100 µCi of slCr (1 volume per 1 volume of red blood cells) are added. The whole is incubated for between 1 and 2 hours, and the radiolabeled red blood cells are then washed between 4 and 6 times. The radiolabelled red blood cells are then contacted with samples of IgG concentrates at a concentration of preferably between 1 and 3 mg/ml, in particular 1.2 mg/ml per 4-6.106 radiolabelled red blood cells, in a volume of 100 ul for example. An identical volume to the preceding volume, e.g. 100 µl of normal serum from blood group AB is then added to the preceding mixture to provide the different complement factors. The reaction mixture obtained is then incubated, preferably for a time comprised between 3 and Sh. in particular 4h. at a temperature usually comprised between 30 and 40°C, preferably 37°C. The reaction mixture is then preferably centrifuged, and die radioactivity of the incubated solution is measured using suitable, commercially available devices. The measured radioactivity of the solution is proportional to the extent of haemolysis of the treated red blood cells, and therefore to the content of anti-A and anti-B antibodies. By way of example, the extent of hameolysis obtained for the red cells of blood groups A, B and AB, considering an IgG concentrate of the invention (B3) and an IgG concentrate of the prior art (CI) having the lowest haemolysis levels amongst all concentrates available on the market, are indicated in following Table 2. Table 2 (Table Removed) The following examples illustrate embodiments of the present invention, without limiting its scope however. EXAMPLE 1 A 40 g/1 sample of IgG concentrate (132) is obtained following the method described in WO 02/092632. A chromatography column SO cm in length and 44 mm in diameter is load.-with a 50/50 (v/v) mixture of GLYCOSORB ABO medium grafted with trisaccharides corresponding to epitopes of blood group A and blood group I), and is then subjected to a prior washing step with 1200 in! water. The B2 IgG concentrate is injected to the proportion of 0.2 l/ml of medium using a pump. Once this volume has percolated through the column, the column is washed with a minimum volume of water for an injectable preparation (IP) to collect the IgGs present in the dead volume of the column. A B3 IgG concentrate is collected at around 40 g/i depicted of anti-A and anti-B antibodies and of polyreactive IgGs, which is then subjected to ultrafiltration to bring me concentrate to 60 g/1 and to nanofiltration to remove viruses on three filters arranged in series and having decreasing retention thresholds of 100. 50 and 20 nm. The stabilizing excipients consisting of a mixture of glycine (7 g/l). munnitol (30 g/l) and 20 ppm Twecn 80® are dissolved in the IgG concentrate at 60 g/l and the IgG concentration is adjusted to SO g/l using PI water, then the concentrate is subjected to sterile filtration and divided into bottles. EXAMPLE 2 : Quantification of anti-A/Bs in IelVs 1) Principle of the assay 1-1) Preparation of human red blood cells The suspensions of human red cells of blood group A Rhesus+, B Rhesus- or O Rhesus+ are normalized to a concentration of 40 x 106 red blood cells/ml in PBS buffer + 1% BSA at pH - 7.4. 1-2) Preparation of the monoclonal anti-D range A preparation of monoclonal anti-D (called R297) is assayed for Optical Density (OD) at 280 nm against its PBS buffer of pH 7.4. The molar extinction coefficient (e) of the protein is calculated relative to its composition in different amino acids, and the concentration (C) in monoclonal anti-D is obtained by applying the formula: C = OD/E1 in which 1 = width of the vessel to conduct OD measurement. A range of 0 to 200 ng/ml of monoclonal anti-D antibodies is produced at 12 points (200; ISO; 100; 75; SO; 25; 12.S; 6.2S; 3.13; 1.S6; 0.78 and 0 ng/ml). 1-3) Preparation of immunoglobulin solutions Different intravenous immunoglobulins available on the market were IVMJJ. The chief characteristics of these immunoglobulins are detailed in the table beluu: (Table Removed) * obtained following the method described in WO 02/092632 follows! In the immunuafiiniy step described in Example 1. ** obtained following the method described in WO 02/092632 without the immunoaffinity step described in Example 1. The different immunoglobulin preparations arc adjusted to a concentration of 1 mg/ml using a PBS + 1% BSA buffer with a pH of 7.4. 1-4) Sensitization of red blood cells In a round-bottomed microplate the following are deposited in the wells: - SO ul of the suspension of A Rhesus+, B Rhesus+- or O Rhesus • red blood cells, with 40 x 106 red blood cells/ml, - 50 ul of the anti-D range or 50 ul of the (IglV) samples to be assayed. The samples to be assayed are deposited in triple batches. The plates are then incubated 2 hours at 37°C under stirring. 1-5) Washings The plates are centrifuged 1 minute at 770 g. The supernatant is discarded by inverting, then 200 ul of PBS + 1% BSA is added to each well. The operation is repeated 3 times. 1-6) AddUion of conjugate and washings A goat anti-human IgG F(ab'2) (Fc specific) labelled with phycoerythrin (PH) (Beckmann Coulter, Ref: PN IM0550) is diluted to 1/20* in PBS + 1% BSA buffer. pH 7.4, men 50 ul of the solution are deposited in each well. The plate is then incubated 20 to 30 minutes at room temperature in the dark. Three successive washings are carried out as described under paragraph 1-5). 1-7) Flow cytometry reading The suspensions of red blood cells are read off the flow cytometer (Beckmann Coulter FC500) using a suitable programme. Reading is conducted on 50 000 events, and the apparatus automatically calculates the mean fluorescence intenssity) (Ml-1) o' each dot or sample. 1-8) Interpretation of results The MFI is obtained in relation to the concentration of monoclonal anti-l) antibody, and the linear regression equation is uUained u.,mg Excel soltware. laod.. for each sample, the concentration in equivalent anti-D antibodies is obtained using-the linear regression equation. Since the samples were assayed in triple, the mean concerntration is determined and the coefficient of variation (CV) is calculated us :i-Excel software. 2) Results 2-1) Concentration in anti-A antibodies (Table Removed) ns = not significant 2-2) Concentration in anti-B antibodies (Table Removed) ns = not significant 3J Conclusions The affinity step truly contributes towards the removal of anti-A and anti-B antibodies. Amongst the different immunoglobulins tested that are available on the market, product IgNG2 is the product which contains the least number of anti-A and anti-B antibodies. EXAMPLE 3 A 1% (v/v) suspension of red cells of blood group A is prepared in PBS buffer. p!I 7.4 containing 1 wt.% bovine serum albumin (BSA). 50 ul of the suspend.m o! rod blood cells is taken and added to a How cytomcter tube (Beckmann-Coultcr l.p.cs XL)) togetlicr with 50 ul of an internal labelling solution measuring the flow. "1 he-suspension is calibrated at 40.106 red blood cells/ml. Light batches of IgG solutions are prepared by successive dilution by faeio .a of the IgG concentrate (v/v) (B3) obtained in example 1. the most concentrated batel Hiving 30 g/l. the most diluted having 0.234 g/1. A volunu* of 50 uJ of the suspected is then placed in each well of a 96-weI! microplate. followed by 50 ul of the dicarn: diluted IgG solutions. The whole is left to incubate for 2 h at a temperature of 37°C under stirring Each well is then washed with 200 ul of PBS buffer containing the preceding BSA, and the microplatc is centrifuged for 1 minute at 2000 rpin. After removing the supernatant. 50 ul of a solution of goat anti-human IgG F(ab'2) antibodies diluted to 1/20 with PBS-BSA are added, labelled with phycoerythrin fluorochrome (Beckmann Coulter). The whole is left to incubate for 30 min in the dark. The suspension obtained is then washed as previously. The residue of each well is dissolved in 100 ul of PBS-BSA. The volume contained in each well of the microplate is transferred to a tube in which 500 ul of Isoflow sheath fluid is added (Coulter) and then subjected to flow cytometry on Coulter-Beckmann Epics XL apparatus comprising software for data acquisition and analysis of results. Fluorometry is measured for each sample. The same procedure is carried out for red cells of blood group B. This operating mode is followed for three different batches of IgG (B3) and is also applied to three different batches of IgG prepared by ethanol fractionation according to Cohn's method (cited above) (Bl). The results obtained are given in Table 3 below: Table 3 (Table Removed) EXAMPLE 4 A 1% (v/v) suspension of papain-treated red cells of blood groups A, B, AB or O is prepared and counted in a Malassez cell to obtain 106 red blood cells. 100 uCi or 51Cr are added (1 volume per 1 volume of red blood cells). The whole is left to incubate for 1 hour, and the radiolabeled red blood cells are then washed 5 times. The radiolabeled red blood cells are then contacted with samples of lgG concentrates (.B2) obtained in example 1, at a concentration of 1.2 my'ml per 5.1')' radiolabeled red blood cells, in a volume of 100 ul. An identical volume to the above i-f 100 ul norma! scrum from blood group AB is then added to the previous mixture to provide the different complement factor.-, The reaction mixture obtained is then incubated 4h at a temperature of 37J(" The reaction mixture is then cewrifuged for 1 minute at 2000 rp;u and the radioactiviiy of the incubated supernatant solution is measured, usuiy suitable devices available on the market. The measured radioactivity of the solution is proportional to me extent of haemolysis of die treated red blood cells, and consequently to the content of anti-A and anti-B antibodies. Identical procedure is followed with red cells of blood groups B, AB and (J. a 1 being Rhesus+, and with a sample of serum from group 0+. This operating mode is followed with three different batches of IgG (B2). In addition, the procedure is applied to three batches of commercial samples ol" IgG concentrates, denoted C2 to C4, and a sample of serum from group O-r- denoted C5 included as negative control. The measured radioactivity of the solution is proportional to the extent of haemolysis of die treated red blood cells, and consequently to the quantit} of anti-A and anti-B antibodies bound to the red blood cells. The haemolysis results are given in Table 4 below: Table 4 (Table Removed) The results obtained show that the IgG concentrate B3, which was subjected to affinity chromatography according to the invention, contains the lowest quantity of anti-A and anti-B antibodies, since the haemolysis percentages of the red blood cells originating from the different blood groups are the lowest. No haemolysis is observed with the red blood cells of phenotype O + included as negative control. EXAMPLE 5 Measurement of the polyreactivily of IgG concentrates B2 (before immunoaffinity chromatography) and after this chromatography (IgG concentrate B3) described in example 1. The measurement of the polyreactivity of these IgG concentrates is conducted following patent EP 1059 OSS Using two antigens which reaet with polyureaction IgGs. These arc myosin and albumin modified b> dinitrophenyl groups (l)M' Albumin). Table 5 gives the enrichment factors of die polyrceactive IgGs in samples 1)2. B3 and C4 of example 3 whose IgG content was arbitrarily set at 1 as reference These measurements were conducted on three different batches of the lg Table 5 (Table Removed) The results indicate that the B3 IgG concentrate of the invention contains 5 to K times fewer polyreactive lgGS than the prior art concentrate C4. EXAMPLE 6 Example comparing the efficacy of the B3 IgG concentrates depleted ol anti-A and anti-B antibodies and of polyreactive IgGs, with the Bl IgG conceniraics The test included mice deficient in FcyRI and FcyRIII receptors treated with a view to evaluating the immunomodulating activity of the IgG concentrates of the invention. These animals were used as model for thrombocytopenic purpura. As control an IgG concentrate (Bl) was used, obtained by ethanol fractionation according to Conn. The experimental protocol was the protocol described by Teeling J.L. ei al (Blood, 15/08/2001, vol. 98, number 4, pp. 1095-1099). The platelets, destroyed by injection of anti-platelet monoclonal IgGs from 9.10a/ml to 2.108/ml, rose to 7.10a/ml in those animals treated with IgG concentrates Bl and B3 at a therapeutic dose of 1 g/kg. The immunomodulating activity of the B3 IgG concentrate according to the invention was not modified by the immunoaffinity chromatography. 4 WE CLAIM 1. Concentrate of immunoglobulins G (IgG) for therapeutic use, characterized in that it has respective contents of anti-A and anti-B antibodies conforming to a negative result for the Coombs in vitro indirect test, in particular a content of anti-A antibodies of no more than 23 ng/mg IgG, and a content of anti-B antibodies of no more than 20 ng/mg IgG. 2. Concentrate of immunoglobulins G according to claim 1 also having a residual content of polyreactive IgGs of between 0.01 % and 0.1 %, in particular between 0.07 and 0.1 %, relative to the total IgG content. 3. Concentrate according to any of claims 1 or 2, containing stabilizers intended to allow the storage of said concentrate, wherein the stabilizers are a mixture of a sugar alcohol, preferably mannitol or sorbitol, of glycine and of a non-ionic detergent. 4. Concentrate according to any of claims 1 to 3 that can be injected via intravenous route. 5. Method to obtain an IgG concentrate comprising the steps of: a) preparing an IgG concentrate by ethanol fractionation and/or chromatographic separation, associating a viral inactivation step, b) immunoaffinity chromatography by percolation of said IgG concentrate on a mixture of media whose matrices are grafted with oligosaccharide groups having antigenic similarity with blood groups A and B, and c) filtration to remove viruses and/or particles of size greater than 20 nm. 6. Method according to claim 5, wherein step a) of the method of the invention comprises prepurification by precipitation of lipid contaminants from a blood plasma or from an IgG-enriched fraction of blood plasma. 7. Method according to any of claim 5 or 6, wherein step a) of the method of the invention comprises pre-purification by precipitation of lipid contaminants from a blood plasma or from an IgG-enriched fraction of blood plasma, single chromatography on an anion exchange resin conducted at alkaline pH and selective elution of the IgGs in one step using a suitable buffer of pH between 4 and 7. 8. Method according to any of claims 5 to 7, wherein the pre-purification is carried out by means of agents precipitants chosen among a group consisting of octanoic acid, calcium phosphate and bentonite. 24 9. Method according to any of claims 5 to 8, wherein the immunoaffinity chromatography of step b) is carried out by means of a mixture of two medium supports grafted with antigen groups having similarity with blood groups A and B, respectively. 10. Method according to any of claims 5 to 9, wherein the immunoaffinity chromatography of step b) is carried out by means of a mixture of two medium supports, a first chromatographic medium support grafted with antigen groups having similarity with blood group A and a second chromatographic medium support grafted with antigen groups having similarity with blood group B. 11. Method according to any of claims 5 or 10, wherein the oligosaccharide groups arc trisaccharides corresponding to epitopes of blood groups A and B. 12. Method according to claim 11, wherein the trisaccharides corresponding to the epitope of blood group A have the structure N-acetylgalactosamine (GalNAc) - Galactose (Gal) - Fucose (Fuc), and those corresponding to the epitope of blood group B have the structure Galactose-Galactose-Fucose. 13. Method according to any of claims 5 to 12, wherein the viral inactivation step is conducted with a solvent-detergent. 14. Method according to any of claims 5 to 13, wherein the mixture of medium supports grafted with groups having antigenic similarity with blood group A and blood group B has a respective proportion comprised between 25/75 and 75/25 (v/v), preferably 50/50 (v/v) of each of said medium supports. 15. Method according to any of claims 5 to 14, comprising the steps of concentration by ultrafiltration, and sterilizing filtration. 16. Method according to any of claims 5 to 15, wherein filtration to remove viruses is conducted by nanofiltration. 17. Method according to any of claims 5 to 16 which, after step e) comprise: a step to add stabilizers for the storage of said IgG concentrate. 18. Method according to any one of claims 5 to 17, wherein the IgG concentrate is an IgG concentrate for therapeutic use. 19. Method according to any of claims 5 to 18, wherein the IgG concentrate has respective contents of anti-A and anti-B antibodies conforming to a negative result for the Coombs in vitro indirect test. 25 20. Method according to any of claims 5 to 19, winerein tlie IgG concentrate has a content of anti-A antibodies of no more than 23 ng/mg IgG, and a content of antl-B antibodies of no more than 20 ng/mg IgG. 21. Method according to any of claims 5 to 20, wherein the IgG concentrate has a residual content of polyreactive IgGs of between 0.01% and 0.1%, in particular between 0.07 and 0.1%, relative to the total IgG content. 22. Assay method of anti-A and anti-B antibodies in a concentrate of immunoglobulin G such as defined by any of claims 1 to 4 comprising the steps consisting of: a) preparing and calibrating a suspension of red blood cells of blood group A or B, b) contacting said red blood cells with diluted samples of lg(i solutions, and incubating the mixture obtained for a predetermined time. c) incubating said red blood cells in the presence of an anti-IgG antibody labelled with a fluorochrome, and d) subjecting the suspension of red blood cells obtained at step c) to flow cytometry. 23. Assay method for anti-A and anti-B antibodies in a concentrate of immunoglobulin G such as defined under any of claims 1 to 4, comprising the steps consisting of: a) radiolabelling a suspension of papain-treated red blood cells chosen from among blood groups A, B, AB and 0, b) previously counted, with a suitable radioactive marker, c) contacting the radiolabelled red blood cells with samples of IgG concentrates of predetermined volume, d) adding a volume identical to the volume in step b) of normal scrum from blood group AB, e) incubating the mixture obtained at step c) for a predetermined lime, and f) measuring the radioactivity of the incubated solution thus obtained. Dated this on the 04'n Day of July 2008 /^ . Q.ARUN KUMAR OF M&S PARTNERS AGENT FOR THE APPLICANT 26

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