NEONATAL SERAL DIAGNOSIS METHOD

Abstract

The present invention relates to a method for determining immunoglobulin G allotypes using a proteomic approach. This method, in particular, allows for the differentiation of maternal and newborn immunoglobulins in a blood sample taken during the first months of the child's life. The invention also relates to the use of this method in the early diagnosis of vertically transmitted diseases in newborns. The invention provides peptides that discriminate G3m allotypes and a kit containing these peptides.

Description

Neonatal Serological Diagnostic Method - Field of Invention: The present invention relates to a method for determining immunoglobulin allotypes in a blood sample, enabling, in particular, the distinct identification and quantification of maternal and newborn immunoglobulins. The invention also relates to the use of this method as a serological diagnostic tool in the field of vertically transmitted diseases. Background of the Invention: Biological diagnosis in newborns of certain vertically transmitted diseases is difficult when antigen detection methods are unreliable. Indeed, serological tests are not feasible in infants due to the presence, up to 6 to 9 months of age, of maternal antibodies transmitted during pregnancy (Simister et al., J. Reprod. Immun., 1997, 37: 1-23; Williams et al., Arch. Dis. Childhood, 1969, 44: 511-514), which prevents the assessment of the proportion of antibodies generated by the child. Presumptive treatments, which are not without toxicity or adverse effects, are therefore administered from birth to newborns suspected of having a congenital disease.These treatments are continued until the child is at least six months old and until a serological test can be performed to measure only the child's antibodies, once the maternally transmitted antibodies have been completely eliminated. This is the case, for example, with Chagas disease, also known as American trypanosomiasis, a parasitic infection caused by Trypanosoma cruzi, which is prevalent in the tropical regions of South and Central America. In some newborns, congenital Chagas disease can be diagnosed by microscopic examination of the parasite in a blood sample and / or by amplification of portions of parasite genes by PCR (Schijman et al., J. Antimicrob. Chemother., 2003, 52: 441-449). However, these techniques encounter obstacles that prevent a definitive diagnostic conclusion.These include excessively high sensitivity thresholds for microscopic detection, which can lead to false-negative diagnoses, or PCR amplification of DNA from fragments of soluble parasitic genetic material (rather than from viable parasites) that have crossed the placental barrier, which can also lead to false-positive diagnoses. Therefore, serological testing is necessary 6 to 9 months after birth to rule out the presence of an undetected infection (Chippaux et al., Tropical Med. International Health, 2010, 15: 87-93). Due to limitations in available diagnostic methods, benznidazole is administered to the newborn immediately after birth as a presumptive treatment. Although early administration improves both the efficacy and tolerability of benznidazole, this substance is toxic, induces numerous side effects, and cannot be administered without close medical supervision.Moreover, such treatment represents a significant cost for families affected by this disease occurring in low-income countries. A similar problem arises in the case of toxoplasmosis, a parasitic infection caused by the protozoan Toxoplasma gondii. Although it is observed in all regions of the globe, infection with this parasite is more prevalent in parts of Europe, the Caribbean, and South America than in Asia, the United States, or Australia. Congenital infection can be diagnosed by PCR, and a qualitative Western blot test is used to compare the IgM and IgG immunological profiles of the mother and child, without any assessment of immunoglobulin levels (Pinon et al., J. Clin. Microbiol., 2001, 39: 2267-2271). However, this test requires maternal plasma collected very shortly after delivery, and its use is therefore limited to the perinatal period. Conversely, enzyme immunoassay (EIA) testing for differences in maternal and infant antibody avidity can only be performed approximately 6 months after birth. To compensate for these limitations, an effective but aggressive treatment with pyrimethamine and sulfonamides is administered to the newborn as a presumptive measure. However, this treatment can have dramatic side effects without close medical supervision. Malaria is another example of a parasitic disease where an understanding of the mechanisms by which infants acquire specific natural immunity could help inform public health interventions to protect at-risk groups and better target future malaria vaccination strategies.Malaria is caused by a parasite of the genus Plasmodium, of which Plasmodium falciparum is the most common and pathogenic species in humans and the one responsible for fatal cases. Malaria affects approximately one hundred countries worldwide, particularly in impoverished tropical areas of Africa, Asia, and Latin America. It primarily affects children under five and pregnant women, who are especially vulnerable because the placenta provides a target where parasites can accumulate. In pregnant women, malaria infection can cause a range of harmful effects: miscarriage, premature birth, low birth weight, congenital transmission, and neonatal death.In all these cases of vertically transmitted infectious diseases, it is therefore crucial to develop new strategies enabling a precise and definitive diagnosis of the congenital disease in the first months of a newborn's life. Early biological diagnosis of these infections would provide invaluable support in deciding whether to initiate or shorten drug treatments, benefiting not only the child's health but also helping to combat these diseases and their consequences. Summary of the Invention: In general, the present invention relates to a method for distinguishing maternal and newborn immunoglobulins in a blood sample taken during the first months of the child's life. This method is particularly applicable to the diagnosis of vertically transmitted diseases. More specifically, the inventors have shown that it is possible to use immunoglobulin allotypes to distinctly identify and quantify maternal and infant immunoglobulins. They have developed a technique for the differential detection and quantification, in newborn plasma, of maternal and infant immunoglobulin G (IgG) using a proteomic approach that leverages the individual characteristics of IgG3, which represents the most polymorphic subclass of IgG.The inventors identified, among the peptides obtained by a series of enzymatic digestions performed on the purified Fc fragment of IgG3, a list of peptides indicating the G3m allotypic identity. The method was validated, in particular, on blood samples from a Beninese mother and her baby, and the results were confirmed by the prior determination of the polymorphism of IgG allotypes using an immunohematological method that allows the definition of the child's IgG3 alleles from determinations made in the child and in its biological parents (see Examples). Accordingly, a first aspect of the present invention relates to an in vitro method for detecting antibodies in a newborn, comprising the steps of: determining, by mass spectrometry, the IgG3 allotypes present in a blood sample from the newborn, the newborn blood sample comprising newborn IgG3 and maternal IgG3 transmitted during pregnancy; comparing the IgG3 allotypes present in the infant's blood sample with the IgG3 allotypes present in a maternal blood sample; and detecting, and optionally quantifying, the newborn IgG3. In the methods of the invention, the newborn blood sample is a sample taken between birth and the ninth month of the newborn's life. In some embodiments, the blood sample is a plasma sample.Preferably, a method according to the invention further comprises steps of: isolating the IgG3 present in the newborn blood sample; subjecting the isolated IgG3 to proteolysis or enzymatic digestion to obtain a mixture of IgG3 proteotypic peptides; and detecting, by mass spectrometry, among the mixture of IgG3 proteotypic peptides, the presence of at least one G3m allotype discriminating peptide in order to determine the IgG3 allotypes present in the newborn blood sample. The step of isolating the IgG3 present in the newborn sample can be carried out by any suitable method. In some embodiments, the IgG3 is isolated by affinity chromatography (for example, by affinity chromatography performed on a Protein A column followed by affinity chromatography performed on a Protein G column).In other embodiments, IgG3 is isolated by liquid chromatography, in particular by rapid protein liquid chromatography (FPLC). The step of subjecting the isolated IgG3 to proteolysis or enzymatic digestion can be carried out by any suitable method15 leading to a mixture of proteotypic IgG3 peptides. For example, in some preferred embodiments, the enzymatic digestion includes digestion in the presence of AspN endoproteinase and digestion in the presence of trypsin, or digestion in the presence of AspN endoproteinase and trypsin, and the discriminating peptide(s)20 of allotype G3m for determining the allotypes of IgG3 present in the newborn blood sample belong to the group consisting of peptides with SEQ ID NO: 1 to 32 sequences shown in Table 4.In one method according to the invention, mass spectrometry analysis is performed using a tandem mass spectrometry technique, in particular 25 MALDI-TOF / TOF or ESI-LTQ Orbitrap. The IgG3 allotypes present in the maternal blood sample can be determined by any suitable method. In some embodiments of the invention, the IgG3 allotypes present in the maternal blood sample are determined by an immunohematological method, in particular a hemagglutination inhibition method. In other embodiments, the IgG3 allotypes present in the maternal blood sample are determined by a method of the invention. The methods described here for the separate identification and quantification of maternal and infant immunoglobulins have applications in the field of diagnosis of vertically transmitted diseases, such as viral, bacterial and parasitic infectious diseases.Consequently, in another aspect, the invention relates to an in vitro method for diagnosing an infectious disease caused by a pathogen in a newborn. The method comprises steps consisting of: determining, by mass spectrometry, the pathogen-specific IgG3 allotypes present in a blood sample from the newborn, the newborn blood sample comprising newborn IgG3 and maternal IgG3 transmitted during pregnancy; comparing the pathogen-specific IgG3 allotypes present in the infant's blood sample with the IgG3 allotypes present in a maternal blood sample; and detecting, and optionally quantifying, the pathogen-specific IgG3 in the newborn. In the diagnostic methods of the invention, the newborn blood sample is a sample taken between birth and the ninth month of the newborn's life.In some embodiments, the blood sample is a plasma sample. Preferably, a diagnostic method according to the invention further comprises steps of: isolating pathogen-specific IgG3s present in the newborn blood sample; subjecting the isolated pathogen-specific IgG3s to proteolysis or enzymatic digestion to obtain a mixture of IgG3 proteotypic peptides; and detecting, by mass spectrometry, among the mixture of IgG3 proteotypic peptides, the presence of at least one G3m allotype discriminating peptide in order to determine the allotypes of pathogen-specific IgG3s present in the newborn blood sample.Preferably, the G3m allotype discriminating peptide(s) used to determine the pathogen-specific IgG3 allotypes present in the newborn blood sample belong to the group consisting of peptides with SEQ ID NO: 1-32 sequences shown in Table 2, and the enzymatic digestion of the pathogen-specific IgG3 is carried out in the presence of AspN and trypsin. In some embodiments, the infectious disease diagnosed by a method according to the invention is a viral infectious disease, and the isolated IgG3s are specific to the virus causing the infectious disease. In other embodiments, the infectious disease diagnosed by a method according to the invention is a bacterial infectious disease, and the isolated IgG3s are specific to the bacterium causing the infectious disease.15 In still other embodiments, the infectious disease diagnosed by a method according to the invention is a parasitic infectious disease, and the isolated IgG3s are specific to the parasite causing the infectious disease. For example, when the parasitic infectious disease is malaria, the isolated IgG3s are specific to the parasite Plasmodium falciparum; when the parasitic disease is Chagas disease, the isolated IgG3s are specific to the parasite Trypanosoma cruzi; or when the parasitic disease is toxoplasmosis, the isolated IgG3s are specific to the parasite Toxoplasma gondii. The step of isolating the pathogen-specific IgG3s present in the newborn sample can be carried out by any suitable method.In some preferred embodiments, total IgG3 is first isolated from the newborn blood sample, for example by affinity chromatography or rapid protein liquid chromatography; and then pathogen-specific IgG3 is isolated from total IgG3. Isolation of pathogen-specific IgG3 can be achieved by any suitable method, for example, via the formation of an immune complex resulting from the contact of total IgG3 with a pathogen-specific antigenic preparation. The remaining steps of a diagnostic method of the invention can be performed as in a method carried out on total IgG3. In particular, the IgG3 allotypes present in the mother's blood sample can be determined by any suitable method using either total IgG3 or pathogen-specific IgG3. In some embodiments of the invention, the determination is performed by an immunohematological method, notably a hemagglutination inhibition method. In other embodiments, the determination is performed by a method of the invention. Indeed, as those skilled in the art will recognize, a method according to the invention can be used to determine the polymorphism of IgG allotypes in an individual at any stage of life, that is, without any consideration of the problems related to the diagnosis of vertically transmitted diseases in newborns.Consequently, in another aspect, the present invention relates to a method for determining the IgG allotypes of an individual, comprising a step of determining, by mass spectrometry, the IgG3 allotypes present in a blood sample of the individual. In some preferred embodiments, this method further comprises steps of isolating the IgG3 present in the individual's blood sample; subjecting the isolated IgG3 to enzymatic digestion to obtain a mixture of IgG3 proteotypic peptides; and detecting, by mass spectrometry, among the mixture of IgG3 proteotypic peptides, the presence of at least one G3m allotype discriminating peptide in order to determine the individual's IgG3 allotypes. Preferably, the enzymatic digestion comprises digestion in the presence of AspN endoproteinase and digestion in the presence of trypsin, or alternatively, digestion in the presence of AspN endoproteinase and trypsin. The G3m allotype discriminating peptide(s) used to determine the IgG3 allotypes present in the individual's blood sample belong to the group consisting of peptides with SEQ ID NO: 1 to 32 sequences. In another aspect, the present invention relates to the use of a plurality of G3m allotype discriminating peptides to calibrate a mass spectrometer used in a method of the invention. Preferably, the plurality of G3m allotype discriminating peptides is selected from the group consisting of peptides with SEQ ID NO: 1 to 32 sequences and any combination thereof.According to a related aspect, the present invention relates to a kit for determining the polymorphism of IgG3 allotypes of an individual, and a kit for the diagnosis, in the newborn, of an infectious disease caused by a pathogen, each kit comprising a plurality of G3m allotype discriminating peptides chosen from the group consisting of peptides of SEQ ID NO: 1 to 32 sequences and any combination of these peptides, where the peptides are intended for the calibration of a mass spectrometer used in a method of the invention. A more detailed description of some preferred embodiments of the invention is given below. Detailed Description of the Invention In general, the present invention relates to a method for determining the polymorphism of IgG allotypes in an individual. This method makes it possible, in particular, to distinguish and / or quantify maternal and newborn immunoglobulins in a blood sample taken during the first months of the child's life. The method is applicable in the field of diagnosing vertically transmitted diseases in newborns. I - Methods for Detecting Immunoglobulins in Newborns As mentioned above, the presence in newborn plasma of maternal antibodies, which are actively transported prenatally via the placenta, prevents serological diagnosis in infants. Maternal antibody transport during pregnancy is limited to immunoglobulin G (IgG1, IgG2, IgG3, and IgG4). These immunoglobulins persist in the newborn during the first 6 to 9 months of life. The methods of the invention are based on the use of immunoglobulin G allotypes to distinctly identify and quantify maternal and newborn immunoglobulins in a blood sample from the child. In the context of the present invention, the terms "newborn" and "infant" are used interchangeably. These terms refer to a human being during the first months of life.The term "first months of life," as used here, refers to the period from birth to the ninth month (inclusive) after birth. In the context of the present invention, the terms "immunoglobulin allotype" and "Gm allotype" are used interchangeably. Gm allotypes (Lefranc and Lefranc, "The Human IgG Subclasses: Molecular analysis of structure, function and regulation," Pergamon Press, Oxford, 1990, pp. 43-78) are antigenic determinants located on the heavy chains of three of the four IgG subclasses (IgG1, IgG2, IgG3, and IgG4) that allow for the differentiation of individuals of the same species. At the molecular level, they correspond to the substitution of one or more amino acids in the polypeptide sequence of immunoglobulins. Polymorphic Gm allotypes are coded by alleles of very closely related genes and inherited through particular combinations called haplotypes.The 11 recognized Gm haplotypes and their frequencies vary greatly from one population to another, thus defining four major groups: "African", "Asian", "European" and "European and Asian" (see Table 1). Table 1: List of Gm haplotypes found in human populations. European population Gm haplotypes: Gm 5, 10, 11, 13, 14, 26, 27, 3, ... Gm 5, 10, 11, 13, 14, 26, 27, 3, 23 European and Asian Gm 21,26,27,28;1,17;.. Gm 21,26,27,28;1,2,17;.. African Gm 5,10,11,13,14,26,27;1,17;.. Gm 5,6,11,24,26;1,17;.. Gm 5,6,10,11,14,26,27;1,17;.. Gm 10,11,13,15,27;1,17;.. Asian Gm 10,11,13,15,16,27;1,17;.. Gm 5,10,11,13,14,26,27;1,3;.. Gm 5,10,11,13,14,26,27;1,3;23 The Gm allotypes of the gamma 1, gamma 2, and gamma 3 chains are listed in the order of the position of the subclass genes on the IGHG gene (G3, G1, and G2), with semicolons to separate subclasses and commas to separate allotypes. The symbol " " indicates the absence of the G2m allotype. The Gm system consists of 18 allotypes (see Table 2). The G3m allotypes (IgG3 allotypes) are the most polymorphic and are found on the CH2 (n=6) and CH3 (n=7) domains of the gamma3 heavy chains. Consequently, in some preferred embodiments, a method according to the invention includes the determination, by mass spectrometry, of the IgG3 allotypes present in a blood sample from the newborn.However, as a person skilled in the art will recognize, it is also conceivable to develop a detection method according to the invention comprising the determination, by mass spectrometry, of the allotypes of IgG1 or IgG2 present in a blood sample from the newborn. Alternatively, it is also conceivable to develop a detection method according to the invention comprising the determination, by mass spectrometry, of the allotypes of IgG3 and IgG1, or of the allotypes of IgG3 and IgG2, or even of the allotypes of IgG1, IgG2, and IgG3, present in a blood sample from the newborn. Table 2: Localization of G3 allotypes on the three immunoglobulin G subclasses. Localization Allotype IgG Subclass Constant Domain IgG1 CH3 Glm(1) CH3 Glm(2) CH1 Glm(3) CH1 Glm(17) IgG2 CH2 G2m(23) IgG3 CH2 G3m(5) ​​CH3 G3m(6) CH3 G3m(10) CH3 G3m(11) CH3 G3m(13) CH2 G3m(14) CH2 G3m(15) CH2 G3m(16) CH2 G3m(21) CH3 G3m(24) CH2 G3m(26) CH3 G3m(27) CH3 G3m(28) Peptides Discriminating Allotypes The methods of the invention are based on the detection, and optionally the quantification, in the sample of newborn, of Gm allotype discriminating peptides. In particular, in some preferred embodiments, a method according to the invention comprises the detection of one or more G3m allotype discriminating peptides. The terms "G3m allotype discriminating peptide", "G3m allotype marker peptide" and "G3m allotype specific peptide" are used interchangeably herein.They refer to a proteotypic peptide that is indicative (or a signature) of the identity of a G3m allotype. A "proteotypic peptide" is a peptide generated by the enzymatic digestion of a given protein in a reproducible and protein-specific manner and that can be detected by mass spectrometry. As described in the Examples, a list of G3m allotype discriminating peptides was established by theoretical cleavage of the IgG3 heavy chain by a combination of AspN endoproteinase and trypsin and identification of IGHG3-specific proteotypic peptides that discriminate against alleles.Ces peptides sont : TKPWEEQYNSTFR (SEQ ID NO: 1), TKPREEQYNSTFR (SEQ ID NO: 2), LREEQYNSTFR (SEQ ID NO: 3), DGVEVHNAKTKPWEEQYNSTFR (SEQ ID NO: 4), EEQYNSTFRVVSVLTVLHQ (SEQ ID NO: 5), EEQYNSTFRVVSVLTVVHQ (SEQ ID NO: 6), TKPWEEQYNSTFRVVSVLTVLHQ (SEQ ID NO: 7), GFYPSDIAVEWESSGQPENNYK (SEQ ID NO: 8), GFYPSDIAMEWESSGQPENNYK (SEQ ID NO: 9), GFYPSDIAVEWESSGQPENNYNTTPPML (SEQ ID NO: 10), GFYPSDIAVEWESSGQPNNNYNTTPPML (SEQ ID NO: 11), GFYPSDIAVEWESSGQPENNYNTTPPVL (SEQ ID NO: 12), GFYPSDIAVEWESNGQPENNYNTTPPML (SEQ ID NO: 13), DIAVEWESSGQPENNYK (SEQ ID NO: 14), DIAMEWESSGQPENNYK (SEQ ID NO: 15), DIAVEWESSGQPENNYNTTPPML (SEQ ID NO: 16), DIAVEWESSGQPNNNYNTTPPML (SEQ ID NO: 17), DIAVEWESSGQPENNYNTTPPVL (SEQ ID NO: 18), DIAVEWESNGQPENNYNTTPPML (SEQ ID NO: 19), SRWQQGNIFSC[deg.]SVMHEALHNHYTQK (SEQ ID NO: 20), SRWQQGNIFSC[deg.]SVMHEALHNR (SEQ ID NO: 21), SRWQEGNVFSC[deg.]SVMHEALHNR (SEQ ID NO: 22), SRWQEGNIFSC[deg.]SVMHEALHNR (SEQ ID NO: 23), WQQGNIFSC[deg.]SVMHEALHNHYTQK (SEQ ID NO: 24), WQQGNIFSC[deg.]SVMHEALHNR (SEQ ID NO: 25), WQEGNVFSC[deg.]SVMHEALHNR (SEQ ID NO: 26), WQEGNIFSC[deg.]SVMHEALHNR (SEQ ID NO: 27), WQQGNIFSC[deg.]SVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 28), WQQGNIFSC[deg.]SVMHEALHNRFTQK (SEQ ID NO: 29), WQEGNVFSC[deg.]SVMHEALHNRFTQK (SEQ ID NO: 30), WQEGNIFSC[deg.]SVMHEALHNRFTQK (SEQ ID NO: 31), and WQQGNIFSC[deg.]SVMHEALHNRYTQK (SEQ ID NO: 32), where Cc represents a carbamidomethylated cysteine. The present invention therefore relates to a plurality of G3m allotype discriminating peptides selected from the group consisting of the peptides listed below or any combination thereof. The present invention also relates to the use of these peptides in a method according to the invention. As a person skilled in the art will recognize, a different list of IgG3 allotype discriminating peptides could be established by a method similar to that used by the inventors but based on enzymatic cleavage using a different enzyme or combination of enzymes. In embodiments where a method according to the invention is used to determine IgG1 or IgG2 allotypes, a list of IgG1 or IgG2 allotype discriminating peptides could be obtained by a similar process. Sample Preparation The methods of the invention are performed using a blood sample from the newborn. Generally, a plasma sample is obtained from the blood sample. Methods for obtaining plasma from human blood are known in art.As mentioned above, a newborn blood (or plasma) sample comprises newborn IgG and maternal IgG transmitted in utero. "Newborn IgG" includes fetal IgG (i.e., IgG synthesized by the fetus during pregnancy) and IgG synthesized by the newborn after birth. In some embodiments, the methods according to the invention are performed on a single subclass of immunoglobulins among IgG1, IgG2, and IgG3. In some preferred embodiments of the invention, the methods are performed on IgG3 present in the newborn blood or plasma sample. Generally, prior to mass spectrometry analysis, the IgG3 present in the blood or plasma sample is isolated and then subjected to enzymatic digestion.The IgG3 isolation step can be performed by any suitable method; the isolation method used is not a critical or limiting factor. For example, IgG3 can be isolated by affinity chromatography. As described in the examples below, it is possible, for instance, to isolate IgG3 from a plasma sample by performing Protein A column affinity chromatography followed by Protein G column affinity chromatography. Protein A and Protein G columns are commercially available, and those skilled in the art know how to select the appropriate elution conditions to obtain purified or substantially purified IgG3. Alternatively or additionally, IgG3 can be isolated by liquid chromatography, for example, by fast protein liquid chromatography. The fast protein liquid chromatography or FPLC system (Sheehan et al.FPLC (Meth. Mol. Biol., 2004, 244: 253-258) was developed by Pharmacia (now GE Healthcare) in 1982 to separate or purify proteins or other macromolecules from complex mixtures. FPLC instruments (AKTAFPLC™ and BioLogic Duoflow™) are currently marketed by GE Healthcare and Bio-Rad Laboratories. The columns used in FPLC can separate macromolecules based on their size, charge distribution, hydrophobicity, or affinity. A separation method using the FPLC technique can standardize IgG3 purification and optimize the amount of purified IgG3 recovered. After isolation, IgG3s are processed to obtain a mixture of IgG3 proteolytic peptides comprising IgG3 proteotypic peptides (i.e., peptides generated by enzymatic digestion in a reproducible and specific manner with respect to immunoglobulin G3). The terms "proteolysis" and "enzymatic digestion" are used interchangeably here. They refer to the cleavage of a protein into several fragments (peptides) by the action of an enzyme or a combination of enzymes. As those skilled in the art will recognize, the enzymatic digestion process used must be based on the theoretical enzymatic cleavage conditions used for the identification of IgG3 allotype discriminating peptides.In some preferred embodiments, the proteolysis of IgG3 isolated from the newborn blood sample will therefore be carried out by digestion in the presence of AspN endoproteinase and by digestion in the presence of trypsin, or by digestion in the presence of AspN endoproteinase and trypsin. Preferably, before digestion, IgG3s are subjected to reduction, for example in the presence of dithiothreitol (DTT) which reduces the disulfide bonds of immunoglobulins into thiols and thus separates the heavy chains from the light chains, then to protection of the thiol groups of the heavy chain of IgG3, for example by alkylation in the presence of chloroacetamide. Mass Spectrometry Analysis. The determination of IgG allotypes according to a method of the invention is carried out by mass spectrometry. Mass spectrometry is a physical analytical technique that allows the detection and identification of molecules of interest by measuring their mass, characterizing their chemical structure, and possibly quantifying them. Its principle lies in the separation in the gas phase of charged molecules (ions) according to their mass / charge ratio ( / z). In a method according to the invention, the mass spectrometry analysis can be carried out by any suitable mass spectrometry technique, allowing the detection of peptides in a complex mixture and the deduction of their sequence. The sample comprising the mixture of IgG3 proteolytic peptides can be introduced directly into the mass spectrometer in liquid or solid form (e.g., deposition on a MALDI plate) or by coupling to a separation method (e.g.Liquid chromatography, capillary electrophoresis). After introduction into the mass spectrometer, the peptides in the mixture are vaporized and ionized (the ionization source can be used either in positive mode to study positive ions or in negative mode to study negative ions). Various ionization methods exist, the most suitable in the context of the invention being electrospray ionization (ESI) and matrix-assisted laser desorption / ionization (MALDI). The ions are then separated by an analyzer according to their mass-to-charge ratio (m / z). Preferably, the analyzer is a high-resolution analyzer capable of accurately measuring the monoisotopic mass of an ion and deducing its composition after fragmentation.A high-resolution analyzer can be a time-of-flight (TOF) analyzer, a Fourier transform ion cyclotron resonance (FTICR) analyzer, a linear quadrupole trap (LTQ-Orbitrap), or a quadrupole filter (QqQ). These analyzers can be coupled together to perform tandem mass spectrometry (MS / MS) experiments. In tandem mass spectrometry, a first analyzer separates ions, a collision cell fragments the ions, and a second analyzer separates the fragmented ions. Some analyzers, such as the LTQ-Orbitrap, combine several analyzers in one, allowing for both ion fragmentation and direct analysis of the fragments. In some preferred embodiments of the invention, the IgG3 proteolytic peptide mixture is analyzed by a tandem mass spectrometry method, for example MALDI in MS / MS mode, in particular MALDI-TOF / TOF or electron spray in MS / MS mode (ESI-LTQ-Orbitrap).In certain preferred embodiments, the mass spectrometer is coupled to a nano-liquid chromatography (nLC) system. Mass measurements of the peptides and their respective fragments allow for sequence determination (manually or using specialized software). Comparison with Maternal IgG Allotypes: Mass spectrometry analysis aims to detect, among the mixture of IgG3 proteolytic peptides, one or more discriminating peptides of the G3m allotype, and thus determine the IgG3 allotypes present in the newborn blood sample (see Table 4). However, since the newborn sample contains both newborn IgG3 and maternal IgG3 transmitted during pregnancy, it is necessary to compare the allotypes determined for the IgG3 present in the newborn blood sample with the allotypes of the mother's IgG3 in order to deduce the allotypes of the newborn's IgG3.The allotypes of IgG3 present in a maternal blood sample can be determined by any suitable method. In some embodiments of the invention, the determination is carried out using an immunohematological method, in particular a hemagglutination inhibition method (Lefranc et al., Acta Anthropogenetica, 1976, 1: 34-45; Dugoujon et al., Vox Sanguinis, 1989, 57: 133-136; Field and Dugoujon, Gen. Epidemiol., 1989, 6: 31-33). In other embodiments, the allotypes of IgG3 present in the mother's blood sample are determined by a method of the invention (see below). II - Diagnostic Methods for Vertically Transmitted Diseases The methods of the invention for distinguishing between maternal and newborn IgG in a blood sample taken during the first months of the child's life are applicable to the diagnosis of vertically transmitted diseases. The term "vertically transmitted disease" refers to any disease transmitted from mother to child during pregnancy, particularly infectious diseases. Infectious diseases that can, a priori, be diagnosed by a method according to the invention include bacterial, viral, and parasitic infections. The diagnostic methods according to the invention are particularly useful when methods for detecting viral, parasitic, or bacterial antigens are unreliable.In some embodiments, a method according to the invention is used to diagnose, in a newborn, a parasitic infectious disease, in particular a parasitic disease caused by a protozoan. Examples of parasitic diseases that can be diagnosed by a method of the invention include, without limitation, malaria, toxoplasmosis, Chagas disease, sleeping sickness (also known as African trypanosomiasis), caused by the flagellated protozoan Trypanosoma brucei ssp. gambiense or Trypanosoma brucei ssp. rhodesiense, babesiosis (a rare disease related to malaria caused by a protozoan of the genus Babesia, B. divergens, B. microti), and leishmaniasis (resulting from infection by flagellated protozoa of the genus Leishmania). In some preferred embodiments, a method of the invention is used to diagnose malaria, toxoplasmosis, Chagas disease, or sleeping sickness. In other embodiments, a method according to the invention is used to diagnose a bacterial infectious disease in a newborn.Examples of congenital bacterial fetal diseases that can be diagnosed by a method of the invention include, without limitation, infections caused by group B streptococcus (streptococcus), Listeria monocytogenes (listeriosis), Treponema pallidum (syphilis), Chlamydia trachomatis (chlamydia), and mycoplasma infections. In other embodiments, a method according to the invention is used to diagnose a viral infectious disease in a newborn. Examples of congenital viral fetal diseases that can be diagnosed by a method of the invention include, without limitation, rubella, chickenpox, and infections caused by parvovirus B19 (erythema infectiosum), cytomegalovirus (cytomegalic inclusion disease), and herpes simplex virus. When a method of the invention is used to diagnose an infectious disease, it is performed, as described above, but on the pathogen-specific IgG3s (parasite, virus, or bacterium) responsible for the disease to be diagnosed, and not on total IgG3s. The terms "parasite-specific IgG3s," "virus-specific IgG3s," and "bacterial-specific IgG3s" refer to the immunoglobulin G3s produced by the human immune system in response to the parasitic, viral, and bacterial pathogens, respectively. The diagnostic methods according to the invention therefore require a step of isolating the pathogen-specific IgG3s present in the blood sample of the newborn to be tested. Isolation of pathogen-specific IgG3 can be achieved by any suitable method known in the art. For example, such isolation can be accomplished by forming an immune complex (i.e., an antigen-antibody complex) resulting from the contact of purified IgG3 with a specific antigenic preparation. After washing to remove any IgG3 not bound to the antigen (i.e., not complexed and therefore not pathogen-specific), dissociation of the immune complexes can be performed to recover the pathogen-specific IgG3, for example, by applying an acidic buffer solution. In the diagnostic methods for Chagas disease according to the invention, the antigenic preparation used to isolate pathogen-specific IgG3s can be a parasitic extract of Trypanosoma cruzi obtained by lysis of an exponential culture of epimastigotes in fetal calf serum (Flechas et al., BMC Infectious Diseases, 2009, 9: 186).In diagnostic methods for toxoplasmosis, the antigenic preparation used to isolate pathogen-specific IgG3 may be a soluble parasite extract of Toxoplasma gondii obtained from an in vitro culture of tachyzoites in murine cells (Fatoohi et al., Clin. Diag. Lab. Immunol., 2001, 9: 704-707). In diagnostic methods for malaria, the antigenic preparation used to isolate pathogen-specific IgG3 may be a recombinant AMAl (Apical Membrane Protein 1) protein from the asexual blood stages of Plasmodium falciparum (Nebie et al., Infection and Immunity, 2008, 76: 759-766). Alternatively, the antigenic preparation can be a parasitic extract of P. falciparum obtained from an in vitro culture of a parasitic line enriched with more than 50% schizonts (Fievet et al., Am. J. Trop. Med. Hyg., 1995, 53: 612-617).A diagnostic method according to the invention therefore proceeds as described above, except that it is carried out on the specific IgG3s of the pathogen responsible for the disease to be diagnosed and not on the total IgG3s. In some cases, particularly when low levels of pathogen-specific IgG3 are obtained, a selective and sensitive mass spectrometry technique called Multiple Reaction Monitoring (MRM), also known as Selected Reaction Monitoring (SRM), can be used. This mass spectrometry mode exhibits dual selectivity, selecting both the parent ion and the fragment ion produced. "Pathogen-specific newborn IgG3" isolated from the blood sample includes maternally inherited pathogen-specific IgG3 transmitted during pregnancy and, if there has been congenital transmission, pathogen-specific IgG3 synthesized by the fetus. It may also contain pathogen-specific IgG3 synthesized by the newborn in response to postnatal exposure to the pathogen.In a diagnostic method according to the invention, pathogen-specific IgG3 allotypes isolated from a newborn blood sample and determined by mass spectrometry are compared to maternal IgG3 allotypes. As described above, the determination of maternal IgG3 allotypes can be performed using a conventional immunohematological method or a method of the invention. The determination of maternal G3m allotypes can be carried out on total IgG3 or on pathogen-specific IgG3, both methods yielding the same results since the polymorphisms are located on the constant domains of the gamma3 heavy chains. A definitive diagnosis of the congenital disease can be based solely on a diagnostic method according to the invention.Alternatively, a clinical diagnosis can be established based on the results of a method according to the invention combined with the results of other diagnostic methods (such as, for example, microscopic observation of the parasite in a blood sample from the newborn or amplification of portions of the pathogen's genes by PCR). Armed with a definitive diagnosis, the attending physician can make an informed decision about initiating drug treatment for the newborn, shortening a course of drug treatment administered presumptively, or continuing such treatment. III - Methods for Determining G3m Allotype Polymorphism As those skilled in the art will recognize, a method according to the invention can be used to determine the polymorphism of IgG allotypes in an individual at any stage of life, that is, without any consideration of the problems related to the diagnosis of vertically transmitted diseases in newborns. This is all the more important given that the classical method for determining Gm allotypes, which is a serological method of hemagglutination inhibition using O Rh+ erythrocytes coupled to anti-Rh antibodies of known Gm allotypes and to monospecific anti-allotype antibodies, will soon no longer be available. Indeed, the collections of monospecific anti-allotype and anti-RhD sera that were obtained in the past from pregnant women and blood donations are practically exhausted. The present invention therefore also relates to a method for determining the IgG allotypes of an individual, comprising a step of determining, by mass spectrometry, the IgG3 allotypes present in a blood sample from an individual. Preferably, the method comprises: isolating the IgG3 present in the blood sample; proteolyzing the isolated IgG3 to obtain a mixture of IgG3 proteolytic peptides; and detecting, by mass spectrometry, among the mixture of IgG3 proteolytic peptides, the presence of at least one G3m allotype discriminating peptide in order to determine the individual's IgG3 allotypes. In some preferred embodiments, the G3m allotype discriminating peptides belong to the group consisting of peptides with SEQ ID NO: 1 to 32; and proteolysis of IgG3 is carried out in the presence of AspN endoproteinase and trypsin.The term "individual," as used in the context of the present invention, refers to a human being who has passed the infancy stage, that is, a human being older than 6 or 9 months (when maternally transmitted immunoglobulins have been completely eliminated). The isolation and proteolysis steps of IgG3 can be carried out as described above. The description of the invention focuses on the determination of IgG3 allotypes, but, as indicated above, it is also conceivable to develop a method according to the invention comprising the determination, by mass spectrometry, of the IgG1 or IgG2 allotypes of an individual. Alternatively, it is also possible to develop a method according to the invention comprising the determination, by mass spectrometry, of the IgG3 and IgG1 allotypes of an individual, or of the IgG3 and IgG2 allotypes of an individual, or even of the IgG1, IgG2, and IgG3 allotypes of an individual. IV - Kits The present invention also relates to kits comprising materials useful for carrying out a method according to the invention. In particular, the present invention relates to a kit for determining the polymorphism of IgG3 allotypes in an individual and a kit for diagnosing, in a newborn, a vertically transmitted disease, in particular a congenital parasitic disease. Generally, a kit comprises a plurality of G3m allotype discriminating peptides belonging to the group consisting of peptides with SEQ ID NO: 1 to 32 and any combination thereof. These peptides will advantageously be used for calibrating the mass spectrometer used. Optionally, the kit may further include instructions for performing the calibration. "Calibration" means any procedure for adjusting or calibrating the mass spectrometer used in a method according to the invention. In some embodiments, the kit includes all the G3m allotype discriminating peptides (SEQ ID NO: 1-32) and can therefore be used and marketed worldwide. In other embodiments, the kit includes only the G3m allotype discriminating peptides necessary for determining an "African," "Asian," or "European" haplotype for geographically localized use and marketing. The G3m allotype discriminating peptides useful for determining an "African" haplotype are the peptides with sequences SEQ ID NO: 2, SEQ ID NO: 5-6, SEQ ID NO: 8-10, SEQ ID NO: 14-16, SEQ ID NO: 18, SEQ ID NO: 20-31, and any combination thereof.The G3m allotype discriminating peptides useful for determining an "Asian" type haplotype are the peptides with sequence SEQ ID NO: 1-10, SEQ ID NO: 12-16, SEQ ID NO: 19-21, SEQ ID NO: 24-25, SEQ ID NO: 28-29, SEQ ID NO: 32, and any combination of these peptides. The G3m allotype discriminating peptides useful for determining a "European" haplotype are the peptides with sequences SEQ ID NO: 2-3, SEQ ID NO: 5-6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 13-14, SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 25, SEQ ID NO: 29, SEQ ID NO: 32, and any combination of these peptides. The kit may include reagents or solutions for preparing calibration samples. The various kit components may be supplied in solid form (e.g., lyophilized) or in liquid form. A kit may optionally include a container for each of the reagents or solutions, and / or containers (test tubes, flasks, etc.) for preparing the calibration samples. The kit may also include reagents for preparing the blood sample to be analyzed (e.g.reagents necessary for the isolation of IgG3, and / or reagents necessary for the enzymatic digestion of IgG3, etc...). These reagents may be contained in containers included in the kit. The kit may also include instructions in the form prescribed by a government agency regulating the sale and use of medical or pharmaceutical products. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as that commonly understood by an ordinary specialist in the field to which this invention relates. Likewise, all publications, patent applications, patents, and other references mentioned herein are incorporated by reference. The following examples and figures are presented to illustrate some embodiments of the procedures described above and should in no way be considered a limitation to the scope of the invention. Examples The following examples describe some embodiments of the present invention. However, it is understood that the examples are presented for illustrative purposes only and do not in any way limit the scope of the invention. Methods Used 1. Definition of the Theoretical List of G3m Allotype Discriminant Peptides The list of proteotypic peptides was established based on a comparison of the peptide sequences of four IGHG proteins. The sequences were virtually cleaved by AspN endoproteinase and trypsin with or without a missing enzymatic cleavage. All peptides obtained by theoretical enzymatic digestion were compared to determine those that were specific to IGHG3 and discriminant with respect to alleles. A list was defined comprising 32 proteolytic peptides suitable for analysis using a MALDI-TOF / TOF technique or an Orbitrap technique. 2. Plasma Samples Used in Purification and Digestion Protocols The samples analyzed in this study came from two distinct geographical regions: Europe and Africa.European blood samples were obtained from healthy adult volunteers of French origin. Five (5) milliliters of blood were collected in EDTA Vacutainer tubes. After centrifugation, 1 mL of fresh plasma was purified, and aliquoted fractions were prepared from the remaining plasma and frozen at -20°C for later use. African plasma samples were obtained during two studies conducted in Benin by the inventors' team. The first study, carried out in 2006–2007, involved 155 children, primarily from the Fon ethnic group (Migot-Nabias et al., J. Infect. Dis., 2008, 198: 1892–1895). Blood samples were collected in 5 mL EDTA Vacutainer tubes, and after centrifugation, plasma samples were prepared and frozen at -20°C. In the second study, a cohort of 627 newborns from a semi-rural region of southern Benin and their mothers was established and followed from 2007 to 2010 (Le Port et al., in preparation). At birth, a blood sample from the mother, as well as cord blood, was collected in EDTA Vacutainer tubes. Next, a blood sample was collected from each child every three months until the age of 18 months. The plasma samples were stored at -80°C.In this study, samples from the mother at the time of birth and from her child (cord blood and plasma samples obtained at 3, 6, and 9 months postpartum) were used. 3. Serological Determination of Gm Immunoglobulin Allotypes. The Gm allotypes of plasma specimens were analyzed by the standard qualitative hemagglutination inhibition method (Dugoujon et al., Vox Sanguinis, 1989, 57: 133-136; Field and Dugoujon, Gen. Epidem., 1989, 6: 31-33). Briefly, human blood group O Rh+ erythrocytes were coated with anti-Rh antibodies against known Gm allotypes, plasma samples, and monospecific anti-allotype antibodies. After sedimentation, plasma specimens containing IgG of a particular allotype inhibit hemagglutination by the anti-allotype antibody, while plasma specimens tested negative for the relevant IgG do not cause inhibition. 4.Purification of Total IgG3: Plasma from freshly collected blood samples was directly purified, and frozen plasma samples were used after centrifugation to remove fibrin aggregates. A volume of 250 g L of plasma is sufficient to detect and quantify the peptides of purified total IgG3. A Protein A column (HiTrap Protein A HP, GE Healthcare) was used for the first purification step, performed according to the manufacturer's instructions. This column is used to retain the interfering immunoglobulins IgG1, IgG2, and IgG4 while allowing IgG3 to pass into the filtrate. Retention of IgG1, IgG2, and IgG4 in the Protein A column requires low ionic strength and a pH of 7. The filtrate fraction resulting from Protein A column chromatography and containing IgG3 must be cleared of other serum proteins.The sample was then passed through a Protein G column (Sepharose HP SpinTrap, GE Healthcare), which exhibits a high affinity for the Fc fragments of immunoglobulins regardless of class. Other serum proteins that were not retained were eliminated in the filtrate (Figure 1). IgG3 retention in the column was achieved at neutral pH, and IgG3 was eluted by lowering the pH. The eluate was neutralized to preserve the quality of the labile immunoglobulins in an acidic environment. The quality of the purification was assessed by ELISA against the different IgG classes. 5. Reduction, Alkylation, and Enzymatic Digestion. The reduction, alkylation, and enzymatic digestion reactions were performed either in solution or on 12% acrylamide gel strips corresponding to the IgG heavy chains after electrophoretic migration of the purified samples under reducing conditions.Several enzymes were tested, alone or in combination, including papain, G1uC, PNGase, AspN, and trypsin. The objective was to select the enzyme(s) that could isolate the greatest number of discriminating (i.e., allotype-specific) peptide sequences within a mass range accessible to high-resolution mass spectrometers. A combination of AspN and trypsin at a final concentration of 10 ng / µL provided the best results. IgG samples were analyzed using SDS-PAGE gels. Fifteen (15) µL of a 2X (volume / volume) (3-mercapthoethanol / Laemmli buffer mixture were added to 15 µL of purified samples. After being boiled at 100°C for 5 minutes, the samples were migrated onto a 12% SDS-PAGE gel stained with Coomassie Blue. The 60 kDa bands corresponding to the IgG heavy chain were clipped.The gel samples were decolorized with 50% acetonitrile, reduced with 20 mM DTT for 30 minutes at 56°C, and alkylated with 25 mM chloroacetamide for 30 minutes at room temperature to protect the protein thiol groups. After adding 1 volume of acetonitrile to dehydrate the gel and removing the supernatant, 1 g of AspN (10 ng / mL, Roche) was added to the gel. The samples were incubated overnight at 30°C or for 3 hours at 37°C. Further digestion was performed with 2 g of freshly activated trypsin (10 ng / mL, Modified Sequencing Grade from Promega). The supernatants were collected and replaced with 10 g L of formic acid (4%) for 5 minutes at room temperature. After a 30-second sonication step, the supernatant was collected and replaced with 50% acetonitrile to dehydrate the gel and extract the peptides.At each step, the supernatants were collected and completely dehydrated under vacuum. The samples were resuspended in a mixture of 10% acetonitrile and 0.1% trifluoroacetic acid (TFA). IgG samples were used in solution. DTT was added to a final concentration of 10 mM to 37 g L of purified sample in solution to reduce disulfide bonds for 30 minutes at 56°C. Chloroacetamide was added at a concentration of 25 mM. AspN (1 µL, 10 ng / mL) was added to the reaction mixture and incubated for 3 hours at 37°C or overnight at 30°C. Freshly activated trypsin (2 µL, 10 ng / µL) was then added under the same incubation conditions. After the reaction, trifluoroacetic acid (TFA, 20%, 1 µL) was added to stop the enzymatic digestion. 6. MALDI mass spectrometry (MALDI-MS) and nanochromatography analyses were performed.The peptides from the samples obtained after enzymatic digestion were concentrated and separated by HPLC (Ultimate3000, Dionex). Briefly, 10 gL of eluate were injected into a C18 pre-column (Acclaim pepmapl00 C18, 5 µm particles, 100 Å pores, 300 µm internal diameter, and 5 mm length). The peptides were then eluted (300 nL / min) to the analytical column (Cl8pepmapl00, 3 µm particles, 100 Å pores, 75 µm internal diameter, and 15 cm length) with a gradient from 7% solvent B (80% acetonitrile, 20% solvent A) at the time of peptide introduction into the analytical column to 20% solvent B after 7 minutes, and from 20% solvent B to 60% solvent B after 58 minutes. The fractions were mixed in a 1:9 ratio with 3 mg / mL of HCCA (Laser Biolabs) in 70% acetonitrile (Carlo Erba), 0.1% TFA (Pierce), and 3 µmol / spot of Glu-fibrinopeptide.A total of 192 fractions were collected and analyzed using a 4800 MALDI TOF / TOF (ABI) analyzer. MALDL Spectrometry: Mass spectra were acquired and processed using 4000 Series Explorer software (ABSciex, version 3.5.28193 build 1011) in positive reflectron mode at a constant laser fluence with low-mass ion filtering and delayed extraction. External calibration of the plate was performed using four calibration points distributed across the plate. Additionally, internal calibration using glufibrinopeptide achieved a measurement accuracy of less than 10 ppm. For each fraction, 10 series of 50 spectra were recorded in the 700–4000 kDa range at a laser frequency of 200 Hz. For each sample, the 500 raw spectra were summed and processed to obtain monoisotopic values ​​from isotopic bulks with a minimum signal-to-noise ratio of 20.MALDI-MS / MS Spectrometry: In each mass spectrum, the 8 most abundant peaks were selected for fragmentation, starting with the least abundant. Neighboring precursors at a resolution of 200 were excluded. For each precursor, 1000 MS / MS spectra were summed in increments of 50. The spectra were processed as follows: baseline subtraction, Stavitsky-Golay smoothing with 3 points on the peak, and a polynomial order of 4. The peak lists reflect the monoisotopic values ​​of the isotope clusters with a minimum signal-to-noise ratio of 22. The MS / MS-generated peak lists were then submitted to an internal Mascot (Matrix Science) search engine, version 2.2, to identify peptides (see below). LTQ-ORBITRAP MS and MS / MS Spectrometry.The analyses were performed by a rapid separation liquid chromatography system (Ultimate 3000 RSLC, Dionex) coupled to a mass spectrometer (LTQ-Orbitrap Velos, Thermo Fisher Scientific). Briefly, the peptides obtained by enzymatic digestion were loaded onto a reversed-phase pre-column (C18, 3 µm particles, 100 Å pores, 75 µm internal diameter, and 2 cm length) with a loading solvent containing 98% water, 2% acetonitrile, and 0.1% trifluoroacetic acid at 5 µL / minute. The peptides were separated on a reversed-phase analytical column (C18, 2 [micro]m particles, 100 Å pores, 75 [micro]m internal diameter, and 15 cm length) with a 45-minute gradient from 100% solvent A (5% acetonitrile, 0.1% AF and 95% water) to 40% solvent B (80% acetonitrile, 0.085% formic acid and 20% water). The mass spectrometer (LTQ-Orbitrap) acquired data throughout the elution process and operated as follows: MS scans were acquired with the Orbitrap, followed by up to 10 LTQ MS / MS CID spectra on the most abundant precursors detected in the MS scans. The latency exclusion was set to 24 seconds for pre-fragmented precursors. The mass spectrometer settings used were as follows: MS (AGC: 1x10⁶, resolution: 3x10⁴, m / z 400–2000, maximum ion injection time: 1000 ms); MS / MS (AGC: 1x10⁴, maximum ion injection time: 200 ms, minimum signal threshold: 2000, isolation width: 2 Da). Fragmentation was permitted for precursors with a charge state of 2 or 3. Spectral Processing. The software used to extract peak lists and generate MGF (Mascot Generic File) data was Proteome Discoverer 1.2 with a signal-to-noise ratio of 3. Database Search. The MS / MS spectra obtained by the two mass spectrometers were submitted to an internal Mascot (Matrix Science) search engine, version 2.2, to identify peptides. The database used was the internally developed "Parasitoswissprot" database (resulting from the concatenation of the IMGT IgHG Immunoglobulin-variants database with the Plasmodium fasta "plasmoDB" database and the SwissProt fasta database, totaling 529,942 sequences and 189,364,547 residues). The mass tolerance for precursors was set at 20 ppm for MALDI and 3 ppm for Orbitrap.For the fragments, the concentration was set at 0.45 Da, methionine oxidation was partially allowed, and carbamidione methylation was considered complete. The search was not restricted to a single species. The defined enzymatic cleavage specificities were a combination of those of trypsin and AspN. A filter was applied to the search to reduce the probability of false positives to less than 5% (minimum Mascot score of 25 for peptides). Semi-quantitative information was obtained without labeling. Progenesis software (Version 3.0; Nonlinear Dynamics Ltd) was used to quantify the variation of the discriminant peptides based on retention time, m / z ratio, and peak intensity (peak area) of the samples. Progenesis processes the raw data files from the Orbitrap in two steps: alignment followed by normalization.The data file that yielded the greatest number of features (1:1 ratio) was used as a reference to align the retention time with the other measurements. Experimental variations were corrected by calculating the robust distribution of all ratios (log(ratio)). The chromatographic peaks (events) were converted into a list of intensities and retention times using the raw data files. The data were filtered, retaining events according to the following criteria: mass-to-charge ratio between 300 and 1700, retention time from 8 to 25 minutes, and charge state from 2 to 4. A matrix of all samples, consisting of all masses corresponding to the peak intensities of each sample, was generated.MGF (Mascot Generic Files) were exported from Progenesis and imported into Mascot software; a query was then performed on the Parasitoswissprot database (taxonomy: mammals, enzyme: AspN+trypsin, fixed modification: carbamodylmethylation, variable modification: oxidation, number of allowed missing cuts: up to 2, peptide tolerance: + / - 5 ppm, MS / MS tolerance: + / - 0.45 Da, peptide charge: 2+, 3+, or 4+). The resulting XML files were imported into Progenesis to assign peptides to events. Normalized peptide abundances were analyzed using Excel. Figure Legends Figure 1 shows the acrylamide gel migration of purification fractions obtained from the passage of a plasma sample through a Protein A column followed by a Protein G column. AF: Successive filtrate fractions from the Protein A column purification containing IgG3 and plasma proteins; AE: Elution fractions from the Protein A column purification containing IgG1, IgG2, and IgG4; GE: Elution fractions from the Protein G column purification containing IgG3; GW: Wash fractions from the Protein G column purification containing plasma proteins. Figure 2 is a list of the 32 IGHG3 allele discriminating peptides observable after theoretical cleavage of the IgG3 heavy chain by an AspN / trypsin combination, and whose mass was determined by MALDI and ESI-ORBITRAP.Amino acids in bold are involved in discriminating between the IGHG3 alleles that make up the G3m alleles. Peptides are represented by showing enzymatic cleavage sites with a dot (".") and the amino acids before and after the enzymatic cleavages. Only one cleavage defect is tolerated. Carbamodethylated cysteines are shown by "C[deg.]". The m / z masses (Mox) are the masses calculated taking into account the possibility of methionine (M) oxidation. The m / z masses determined by the ESI-ORBITRAP are the masses of bi-, tri-, and quadru-charged peptides. Figure 3 is a MALDI-TOF spectrum of a tryptic digestion of purified IgG3 heavy chain from a plasma sample of an individual of European origin. The assigned tryptic peptides corresponding to the labeled masses (*) are indicated in red (green* for propionamid cysteines). The selected precursor with a m / z of 2314.06 was subjected to CID fragmentation in MALDI MS / MS mode. The fragmentation spectrum yielded a Mascot result, unambiguously confirming the presence of the IGHG3 347-365 peptide: R.WQQGNIFSCSMHEALHNR.F, whose mass allows for the discrimination of amino acid variations (in red). Figure 4 is a graph showing the relative abundance of discriminating peptides of two G3m alleles as a function of different volume ratios of the two isoforms. Figure 5 is a graph showing the relative abundance of the WQQGNIFSCSVMHEALHNR peptide in field samples. CI = maternal blood, CO = umbilical cord blood, M = infant blood at 3, 6, and 9 months; three technical replicates of the experiment. Results 1. Serological determination of Gm allotypes of immunoglobulins from plasma samples. The French individual has a Gm5,10,11,13,14,26,27;3 phenotype comprising the G3m5,10,11,13,14,26,27 and Glm3 alleles, which are commonly associated with Gm haplotypes in individuals of European origin. Two children from Benin from a previous study (Migot-Nabias et al., J. Infect. Dis., 2008, 198: 1892-1895), homozygous for the alleles G3m5,10,11,13,14,26,27 (individual AS20, phenotype Gm5,10,11,13,14,26,27;1,17) and G3m5,6,11,24,26 (individual NP47, phenotype Gm5,6,11,24,26;1,17) which are commonly expressed in sub-Saharan populations were selected. Plasma samples from a mother and her child were selected from a multidisciplinary study on malaria carried out in Benin in 2007-2010. Serological determination concluded a Gm5,6,10,11,13,14,24,26,27,28;1,17 phenotype for the mother (individual A170M) and a Gm5,10,11,13,14,26,27;1,17 phenotype for the child (serological determination carried out on the plasma of A170M15 collected at the age of 15 months). Serological determination of the father (individual A170P, Gm5,10,11,13,14,15,26,27,28;1,17) allowed us to deduce that the mother was G3m heterozygous (G3m5,10,11,13,14,26,27 / G3m5,6,11,24,26,28) and her child, homozygous for G3m5,10,11,13,14,26,27. 2.Purification of IgG3 from a Plasma Sample from a French Individual. All fractions resulting from affinity chromatography on Protein A and Protein G columns were migrated onto a 12% SDS-PAGE gel (Figure 1) and then tested by ELISA (Enzyme-Linked Immunosorbent Assay) to measure the quantities of each immunoglobulin subclass throughout the purification process. On the unreduced gel, the AF fraction (3 wells) consists of IgG3 with a band at approximately 160 kDa and several other plasma proteins, represented by the other bands. In the AE fraction, IgG1, IgG2, IgG4, and other plasma proteins are eluted. The GE fraction (2 wells) contains plasma protein contamination. Washing these fractions (3 wells) removes most of the contaminating proteins.On the reduced gel, the first eluate GE 1 from the Protein G column is contaminated with plasma proteins, but IgG is predominant. The wash fractions (3 wells) show a 60 kDa band. It is possible that some immunoglobulins were lost during the wash steps. An ELISA assay on all fractions confirmed these 30 observations (Table 3): IgG3 is released in fractions AE and GF; contamination by other IgG subclasses occurred in fraction GE; eluates GE1 and GE2 are composed of IgG1 (22.7%), IgG2 (2%), IgG3 (29%), and IgG4 (46.3%). Purification by Protein A column chromatography eluted 95% IgG3. Table 3: ELISA assay of IgG subclasses contained in purification fractions from the passage of a plasma sample through Protein-A and then Protein-G columns. Fractions IgGl (AU) IgG2 (AU) IgG3 (AU) IgG4 (AU) AE1 91.3 103.8 89.5 0 AE2 133.0 106.6 214.8 972.6 AE3 113.3 106.5 152.7 640.3 AE4 90.9 64.8 0 0 AE5 74.9 31.2 0 0 GF1 65.8 57.0 101.1 0 GF2 0 0 93.7 0 GF3 0 0 100.5 0 GF4 0 57.8 99.2 0 GF5 0 0 90.4 0 GF6 0 99.5 30.9 0 GF7 0 0 0 0 GE1 135.8 0 0 0 GE2 0 11.8 173.5 276.5 GE3 0 22.6 100 0 GE4 0 0 0 0 Results expressed in Arbitrary Units (AU); AE: Elution fractions from column purification of Protein A containing IgG1, IgG2, IgG4; GF: Filtrate fractions from column purification of Protein G containing plasma proteins; GE: Elution fractions from column purification of Protein G containing IgG3.In conclusion, the Protein A and Protein G columns allow for the purification of the majority of IgG3. Mass spectrometry results show that contamination by IgG subclasses interferes with the measurement of IgG3 peptides. However, the list of discriminating peptides presented in Table 4 below is specifically representative of IgG3 among other IgG subclasses and compensates for this limitation. Table 4: List of the 32 IGHG3 allele-specific peptides observable after theoretical cleavage by an AspN / trypsin combination of the IgG3 heavy chain. Proteotypic peptides Alleles IGHG3 CH2 region TKPWEEQYNSTFR *18, *19 TKPREEQYNSTFR LREEQYNSTFR DGVEVHNAKTKPWEEQYNSTFR EEQYNSTFRVVSVLTVLHQ EEQYNSTFRVVSVLTVVHQ TKPWEEQYNSTFRVVSVLTVLHQ *01 to *10, *13, *17 *14 to *16 *13 to *17 *18, *19 *01 to *08, *10, *09 *18, *19 CH3 region GFYPSDIAVEWESSGQPENNYK *06, *07 *09 to *13 GFYPSDIAMEWESSGQPENNYK *17, *18, *19 GFYPSDIAVEWESSGQPENNYNTTPPML *01, *04, *05, GFYPSDIAVEWESSGQPNNNYNTTPPML *02 GFYPSDIAVEWESSGQPENNYNTTPPVL GFYPSDIAVEWESNGQPENNYNTTPPML *03 *08, *14 to *16 DIAVEWESSGQPENNYK *06, *07 *09 to *13 DIAMEWESSGQPENNYK *17, *18, *19 DIAVEWESSGQPENNYNTTPPML *01, *04, *05, DIAVEWESSGQPNNNYNTTPPML *02 DIAVEWESSGQPENNYNTTPPVL DIAVEWESNGQPENNYNTTPPML *03 *08, *14 to *16 SRWQQGNIFSC[deg.]SVMHEALHNHYTQK *17 to *19 *12, *14 to *16 SRWQQGNIFSC[deg.]SVMHEALHNR *01, *02, *06 to SRWQEGNVFSC[deg.]SVMHEALHNR *03 SRWQEGNIFSC[deg.]SVMHEALHNR *13 WQQGNIFSC[deg.]SVMHEALHNHYTQK *17 to *19 *12, *14 to *16 WQQGNIFSC[deg.]SVMHEALHNR *01, *02, *06 to WQEGNVFSC[deg.]SVMHEALHNR *03 WQEGNIFSC[deg.]SVMHEALHNR *13 WQQGNIFSC[deg.]SVMHEALHNHYTQKSLSLSPGK *17 to *19 *12 WQQGNIFSC[deg.]SVMHEALHNRFTQK *01, *02, *06 to WQEGNVFSC[deg.]SVMHEALHNRFTQK *03 WQEGNIFSC[deg.]SVMHEALHNRFTQK *13 WQQGNIFSC[deg.]SVMHEALHNRYTQK *14 to *16 In Table 4, the amino acids in bold are involved in discriminating between the IGHG3 alleles that make up the G3m alleles; and Cc represents a carbamidine-methylated cysteine. 3. List of G3m allotype discriminating peptides The 32 proteolytic peptides suitable for analysis using a MALDI-TOF / TOF or Orbitrap technique, theoretically identified as described above, are presented in Table 4 with the IGHG3 alleles they discriminate against, and Table 5 shows a correspondence between the IGHG3 alleles and the G3m alleles.Table 5: Correspondence between IGHG3 alleles and G3m alleles. IGHG3 Alleles G3m Alleles Nomenclature Simplified Complete Nomenclature IGHG3*01, IGHG3*05, G3m5,10,11,13,14,26,27 G3m5* IGHG3*06, IGHG3*07, IGHG3*09, IGHG3*10, IGHG3*11, IGHG3*12 IGHG3*13 G3m5,6,10,11,14,26,27 G3m6* IGHG3*03 G3m5,6,11,24,26 G3m24* IGHG3*17 G3m10,11,13,15,27 G3m15* IGHG3*18, IGHG3*19 G3m10,11,13,15,16,27 G3m16* IGHG3 * 14, IGHG3 *15, G3m21,26,27,28 G3m21 * IGHG3 *16 Figure 2 presents the 32 peptides, also showing the enzymatic cleavage sites as well as the amino acids preceding and following the enzymatic cleavages. Figure 2 also lists the corresponding masses of these peptides determined by MALDI-MS and ESI-MS. 4.MALDI-TOF / TOF Spectrum of the Digested Heavy Chain of Purified IgG3 from a Plasma Sample of an Individual of French Origin. As indicated above, serological testing concluded that the French individual is homozygous for the G3m5,10,11,13,14,26,27 allele. Total IgG3 from a blood sample of this individual was isolated, and the IgG3 heavy chain was subjected to enzymatic digestion in the presence of trypsin and AspN. A MALDI-TOF / TOF analysis according to the invention detected the WQQGNIFSCSVMHEALHNR peptide with an m / z ratio of 2314.06 (Figure 3). In addition to the latter, another discriminating peptide of the G3m5,10,11,13,14,26,27 allele was identified by ESI-LTQ-Orbitrap: DIAVEWESSGQPENNYNTTPPML (with methionine oxidation) at m / z-870.05. The same spectra were obtained whether the sample was prepared from freshly collected whole blood plasma or from plasma obtained after freezing and thawing. 5.Sensitivity of the G3m Allotype Determination Method by Mass Spectrometry. Experiments were performed on a series of controlled mixtures of plasma samples from two individuals from Benin who had been determined by a serological method to be homozygous for the G3m5,10,11,13,14,26,27 and G3m5,6,11,24,26 alleles, respectively. The results of the mass spectrometry determination according to the invention show the presence of the peptide sequence WQQGNIFSCSVMHEALHNR for the first individual and the peptide sequence WQEGNVFSCSVMHEALHNR for the second individual. The mixtures with volume / volume ratios as shown in Figure 4 were prepared before reduction, alkylation, and enzymatic digestion. There is a tendency towards a progressive decrease of the discriminatory peptide 1 representative of the G3m5,10,11,13,14,26,27 allele and conversely a progressive increase of the discriminatory peptide 2 representative of the G3m5,6,11,24,26 allele.In the 1:0 and 0:1 mixtures, the "missing" peptide is measured when it is not expected, which is explained by the fact that the background signal is measured by the software. Furthermore, the resulting amount of peptides for these two volume ratios is lower than expected. In fact, this result is based on only one technical replicate considering only one form (+3) of the relevant peptides, the other two forms (+2, and M-oxidized 2+ and 3+) not being considered. Nevertheless, peptides are detected at least at the 1:16 volume ratio, indicating good sensitivity of the detection method. 6. Application of the proteomic approach to plasma samples from a mother and her child. Experiments were performed on purified total IgG3 from plasma samples obtained from a Beninese mother (individual A170) and her infant from birth to 9 months of age (Figure 5).As indicated above, serological testing showed that the mother was heterozygous (G3m5,10,11,13,14,26,27 / G3m5,6,11,24,26,28) and that the child was homozygous for G3m5,10,11,13,14,26,27. Mass spectrometry analysis according to the invention revealed the presence of the peptide sequence WQQGNIFSCSVMHEALHNR in both the mother and the child. The results shown in Figure 5 demonstrate an increase in the amount of the WQQGNIFSCSVMHEALHNR peptide between birth (sample CO) and 3 months of age (sample M3). This increase can be explained by the appearance of newly synthesized IgG3. A decrease in the quantity of this peptide at 6 months of age (sample M6) corresponds to the loss of maternal IgG3 transmitted during pregnancy and partially containing this peptide. At 9 months (sample M9), only newly synthesized IgG3 by the child is observed. 7.Discriminatory peptides of G3m alleles observed by mass spectrometry. Mass spectrometry analysis of plasma samples obtained from the individual of French origin, the two Beninese children, and the mother and her Beninese child allowed the observation of three peptides, namely: DIAVEWESSGQPENNYNTTPPML, WQQGNIFSC[deg.]SVMHEALHNR, and WQEGNVFSC[deg.]SVMHEALHNR, which are all included in the list of theoretical peptides presented in Table 4. The IGHG3 alleles that they allow us to distinguish correspond to the G3m alleles as follows (Lefranc et al., "Human Gm, Km and Am allotypes and their molecular characterization: a remarkable demonstration of polymorphism", In: 30 Methods in Molecular Biology, Eds. B. Trait and F. Christiansen, in press): DIAVEWESSGQPENNYNTTPPML (SEQ ID NO: 16) is discriminant of alleles IGHG3*01, *04, *05, *09 to *13. WQQGNIFSC[deg.]SVMHEALHNR (SEQ ID NO: 25) is discriminant of the alleles IGHG3*01, *02, *06 to *12, *14 to *16; and WQEGNVFSC[deg.]SVMHEALHNR (SEQ ID NO: 26) is discriminant of the allele IGHG3 *03, where IGHG3*01, *05, *06, *07, *09, *10, *11, *12 corresponds to the allele G3m5,10,11,13,14,26,27; IGHG3*13 corresponds to the allele G3m5,6,10,11,14,26,27; IGHG3*3 corresponds to the G3m5,6,11,24,26 allele, and IGHG3*14,*15,*16 corresponds to the G3m21,26,27,28 allele. Restrictions to the use of this method may arise in the rare cases where the mother and child are either homozygous or heterozygous for the same G3m alleles. G3m polymorphism is not as widespread in certain population groups distributed worldwide (Dugoujon et al., Am. J. Phys. Anthrop., 2004, 125: 175-192). In these cases, the use of the Glm polymorphism (4 IGHG1 alleles) will be necessary to ensure the ability to distinguish the IGHG alleles of the mother and child.

Claims

Demands 1. An in vitro method for determining the allotypes of immunoglobulin G3 (IgG3) in an individual, comprising a step consisting of: isolate the IgG3 present in the individual's blood sample; subject the isolated IgG3s to enzymatic digestion to obtain a mixture of IgG3 proteotypical peptides; and detect by mass spectrometry, among the mixture of proteotypic IgG3 peptides, the presence of at least one discriminating peptide of allotype G3m in order to determine the allotypes of IgG3 present in the individual's blood sample.

2. Method according to claim 1, characterized in that the discriminating peptide(s) of allotype G3m belong / belong to the group consisting of peptides of sequence SEQ ID NO: 1 to 32.

3. Method according to claim 1 or claim 2, characterized in that the mass spectrometry analysis is performed by a tandem mass spectrometry technique, in particular MALDI-TOF / TOF or ESI-LTQ Orbitrap.

4. Method according to any one of claims 1 to 3, characterized in that the step of isolating the IgG3 present in the individual's blood sample comprises Protein A column affinity chromatography followed by Protein G column affinity chromatography.

5. Method according to any one of claims 1 to 4, characterized in that the step of subjecting the isolated IgG3 to enzymatic digestion to obtain a mixture of proteotypic IgG3 peptides comprises enzymatic digestion in the presence of endoproteinase AspN and trysin.

6. In vitro method for detecting antibodies in a newborn comprising steps consisting of: isolate the IgG3 present in a blood sample from the newborn, the newborn's blood sample including IgG3 from the newborn and maternal IgG3 transmitted to the newborn during pregnancy; subject the isolated IgG3s to enzymatic digestion to obtain a mixture of proteotypical IgG3 peptides; detect by mass spectrometry, among the mixture of proteotypic IgG3 peptides, the presence of at least one discriminating peptide of allotype G3m in order to determine the allotypes of IgG3 present in the blood sample of the newborn; compare the IgG3 allotypes present in the child's blood sample with the IgG3 allotypes present in a blood sample from the mother; and detect, and possibly quantify, the newborn's IgG3.

7. An in vitro method for diagnosing, in a newborn, an infectious disease caused by a pathogen, the method comprising steps consisting of: isolate pathogen-specific IgG3s present in the newborn blood sample, the newborn blood sample comprising pathogen-specific IgG3s from the newborn and pathogen-specific maternal IgG3s transmitted to the newborn during pregnancy; subject the isolated pathogen-specific IgG3s to enzymatic digestion to obtain a mixture of IgG3 proteotypical peptides; detect, by mass spectrometry, among the mixture of proteotypic IgG3 peptides, the presence of at least one discriminating peptide of allotype G3m in order to determine the pathogen-specific IgG3 allotypes present in the newborn blood sample; compare the pathogen-specific IgG3 allotypes present in the child's blood sample with the IgG3 allotypes present in a maternal blood sample; and detect, and possibly quantify, the IgG3 specific to the newborn pathogen.

8. Method according to claim 6 or claim 7, characterized in that the discriminating peptide(s) of allotype G3m belong / belong to the group consisting of peptides of sequence SEQ ID NO: 1 to 32.

9. Method according to any one of claims 6 to 8, characterized in that T mass spectrometry analysis is performed by a tandem mass spectrometry technique, in particular MALDI-TOF / TOF or ESI-LTQ Orbitrap.

10. Method according to any one of claims 6 to 9, characterized in that the newborn blood sample is a blood sample taken between birth and the 9th month of the newborn's life.

11. Method according to any one of claims 6 to 10, characterized in that the newborn blood sample is a plasma sample.

12. Method according to any one of claims 6 to 11, characterized in that the allotypes of IgG3 present in the mother's blood sample are determined by an immunohematology method, in particular a hemagglutination inhibition method.

13. Method according to any one of claims 6 to 11, characterized in that the allotypes of IgG3 present in the mother's blood sample are determined by a method according to any one of claims 1 to 5.

14. Method according to any one of claims 6 to 13, characterized in that the step of isolating the IgG3 present in the newborn blood sample comprises Protein A column affinity chromatography followed by Protein G column affinity chromatography.

15. Method according to any one of claims 6 to 14, characterized in that the step of subjecting the isolated IgG3 to enzymatic digestion to obtain a mixture of proteotypic IgG3 peptides comprises enzymatic digestion in the presence of endoproteinase AspN and trysin.

16. Method according to any one of claims 7 to 15, characterized in that the infectious disease is a viral infection, a bacterial infection or a parasitic infection.

17. Method according to any one of claims 16, characterized in that the parasitic infectious disease is malaria caused by the pathogen Plasmodium falciparum or Chagas disease caused by the parasite Trypanosoma cruzi or toxoplasmosis caused by the pathogen Toxoplasma gondii.

18. Kit for determining the IgG3 allotypes of an individual by a method according to any one of claims 1 to 5 or of a newborn by a method according to any one of claims 6 and 8-15, the kit comprising a plurality of G3m allotype discriminating peptides belonging to the group consisting of peptides of sequence SEQ ID NO: 1 to 32, wherein the peptides are intended to be used to calibrate a mass spectrometer.

19. Kit for the diagnosis of a vertically transmitted infectious disease in a newborn by a method according to any one of claims 7 and 8-17, the kit comprising a plurality of discriminating peptides of allotype G3m belonging to the group consisting of peptides of sequence SEQ ID NO: 1 to 32, wherein the peptides are intended to be used for calibrating a mass spectrometer 20. Use of a plurality of G3m allotype discriminant peptides belonging to the group consisting of peptides of sequence SEQ ID NO: 1 to 32 to calibrate a mass spectrometer used in a method according to any one of claims 1 to 17.

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