FR2942042A1 - Differentiating two samples of human serum/plasma comprises comparing concentration of class G immunoglobulins directed against at least three different antigens selected for their ability to induce a long-term acquired humoral immunity - Google Patents

Abstract

Method for differentiating two samples of human serum or plasma, comprises: comparing the concentration of class G immunoglobulins (IgG) directed against at least three different antigens selected for their ability to induce a long-term acquired humoral immunity, in the samples, where the concentration between those two different samples for at least one IgG, which is directed against a single antigen, is indicating that both samples obtained from two different blood samples. Independent claims are included for: (1) a method for determining a serological impression of a human serum or plasma sample, comprising determining the concentration of immunoglobulin G (IgG) directed against at least three different antigens selected for their ability to induce long-term acquired humoral immunity, where the serological footprint is defined by the concentrations of IgG against the at least three selected different antigens; and (2) a kit or cabinet to differentiate serum or plasma samples or for determining a serological fingerprint of a human serum or plasma sample, comprising Epstein-Barr nuclear antigen 1 (EBNA-1) protein of Epstein-Barr virus, protein pertussis toxin (PT) of B. pertussis virus and/or protein OMP2 C. pneumoniae virus, or a peptide comprising or constituted of SEQ ID NO: 1 (Ser-Ser-Ser-Gly-Ser-Pro-Pro-Arg-Arg-Pro-Pro-Pro-Gly-Arg-Arg-Pro-Phe-Phe-His-Pro-Val), a peptide comprising SEQ ID NO: 2 (Leu-Thr-Trp-Leu-Ala-Ile-Leu-Ala-Val-Thr-Ala-Pro-Val-Thr-Ser-Pro-Ala-Trp-Ala-Asp-Asp) and/or a peptide comprising SEQ ID NO: 3 (Met-Thr-Ala-Lys-Lys-Val-Arg-Leu-Val-Arg-Arg-Asn-Lys-Gln-Pro-Val-Glu-Gln-Lys-Ser), each of the peptides can be conjugated with one biotin molecule, and optionally, an anti-human IgG antibody.

Description

The present invention relates to a method of differentiating at least two samples of serum or human plasma.

The Biological Resource Centers (BRCs) are specialized resource centers for the acquisition (acquisition), conservation, validation, study and distribution of organism collections and non-renewable biological samples (tissues, fragments tissues, serums, plasmas, etc.). Serum or plasma samples, kept by the Biological Resource Centers, are often intended for serological or proteomic analysis, in the context of comparative studies in case / control subjects or exposed / unexposed subjects. It is important, for the scientific validity of these studies, that the compared samples supposed to be different do not actually come from the same blood sample.

However, it may happen that, due to handling errors occurring for example during the collection of samples, their aliquoting, recording or storage, the serum or plasma samples to be compared, which are supposed to be different, come in fact from the same sample. thus leading to erroneous results.

In this context, it is therefore necessary to introduce a quality control of the serum or plasma samples to be compared, so as to ensure that these samples come from different blood samples. However, there is currently no method to perform this check, which is fast, reliable and can be used routinely.

The Applicant then aims to provide a method of differentiation of two samples of serum or human plasma. The Applicant has discovered that each human serum or plasma has a serological imprint. This serological imprint can be determined by measuring the concentration of polyclonal Class G (IgG) immunoglobulins directed against at least three different antigens selected for their ability to induce long-lasting acquired humoral immunity in humans. The Applicant has also demonstrated that the measurement of the concentration of IgG

directed against only three antigens as defined above was sufficient to determine a serological imprint. The Applicant has then developed a method for differentiating at least two samples of human serum or plasma, comprising a step of comparing said serological imprint between the samples. More particularly, the Applicant has shown that two different serum or human plasma samples have between them at least one different IgG (polyclonal) concentration for the same antigen chosen from EBNA-1 proteins of the Epstein-Barr virus (EBV). ), B. pertussis PT and C. pneumoniae OMP2.

The subject of the present invention is therefore a method of differentiating two samples of serum or human plasma, said method comprising a step of comparing the concentration, in the samples, of class G immunoglobulins (IgG) directed against at least three different antigens selected for their ability to induce a long-lasting acquired humoral immunity, a different concentration between said two samples for at least one IgG directed against the same antigen meaning that the two samples come from two different blood samples. The concentration of said IgG can be determined by any method known to those skilled in the art, such as the following methods: ELISA (Enzyme Linked Immunosorbent Assay), EIA (Enzyme Immunoassay), RIA (Radio Immunoassay), immunofluorescence, multiplex (Bio-Plex, Luminex), Western blot or protein chip, preferably by the ELISA method. Preferably, the concentration of IgG directed against an antigen corresponds to the average value of at least three measurements (triplicate).

More preferably, the concentration of said immunoglobulins is determined spectrophotometrically to measure an optical density. Class Ig immunoglobulin directed against an antigen is an IgG that is capable of binding to an antigen or peptide fragment of that antigen comprising an epitope.

Long-term acquired humoral immunity or long-lasting acquired humoral immune response is defined as a humoral immunity specific for an antigen that has induced it and is characterized by the formation of

memory B cells (bearing IgG membranes specific for said antigen) in subjects. These memory B cells may remain in the body for at least 10 years, or even for life, after the first contact with said antigen by infection or vaccination.

An antigen having the capacity to induce long-term acquired humoral immunity is understood to mean an antigen that is capable of eliciting a long-lasting acquired humoral immune response in a human. Those skilled in the art know many antigens capable of inducing long-term acquired humoral immunity in humans, such as those inducing the production of long-lasting infectious or vaccine antibodies. Among the long-term infectious antibodies are those directed against EBNA-1 protein expressed by Epstein-Barr virus, heat shock proteins (HSP) such as HSP60 (for example that of E. coli; accession number AAS75782.1 or GI: 45686198 in the GenBank database, or that of Chlamydia trachomatis, Yi et al., Infect Immun., 1993, 61: 1117-1120), HSP70 and HSP90 (see for review Zügel and Kaufman, Clin Microbiol, Rev., 1999, 12: 19-39), OMP2 or MOMP (Major Outer Membrane Protein) proteins, accession number AAB24363.1 or GI: 260973 in the GenBank database. ) expressed by Chlamydophila pneumoniae (Cunningham and Ward, FEMS Microbiol Lett., 2003, 227: 73-79) or lipopolysaccharides (O antigens) of Gram-negative bacteria (see for review Helander et al., Lipopolysaccharides, Encyclopedia of Life Sciences, 2001, John Wiley & Sons, Ltd.). Among the long-lasting vaccine antibodies are those directed against tetanus toxin (tetanospasmine) produced by Clostridium tetani (eg accession number PO4958.2 or GI: 135624 in the GenBank database), capsid protein of the polio virus (eg, accession number ABW24064.1 or GI: 158252122 in the GenBank database) or PT protein, pertactin (eg, accession number P14283.3 or GI: 464364 in the base GenBank data) or phytohemagglutinin (FHA, eg accession number CAD12824.1 or GI: 17154501 in the GenBank database) produced by Bordetella pertussis (Guiso et al., Vaccine, 2007, 25: 1390-1397) .

According to an advantageous embodiment of the invention, said antigens come from three microorganisms of different kinds. Microorganism means a virus, a bacterium, a parasite, a fungus or a yeast, preferably a virus or a bacterium.

According to yet another advantageous embodiment of the invention, said antigens are selected from Epstein-Barr virus (EBV) antigens, Bordetella pertussis antigens and Chlamydophila pneumoniae antigens. According to a preferred embodiment of this embodiment of the invention, said three antigens are the EBNA-1 protein of Epstein-Barr virus (EBV), the B. pertussis PT protein and the C. OMP2 protein. pneumoniae. Epstein-Barr virus, also known as EBV (Epstein-Barr virus) or HHV-4 (Human herpesvirus 4) is responsible for infectious mononucleosis (IM) and is involved in some cancers in humans. The majority of the adult world population has been infected with this virus, and about 90-100% of the infected individuals are infected for life because of the particular properties of this virus; indeed, the hallmark of an EBV infection is the ability of this virus to establish a non-productive and quiescent infection in B cells, which usually persists without symptoms throughout the life of the infected subject (latent infection). The genomic persistence of the virus (type I latency) is related to the expression of viral proteins, including the nuclear latency protein EBNA-1 (Epstein Barr Nuclear Antigen 1, accession number P03211 or GI: 119110 in the base of GenBank data). Anti-EBNA-1 class G (IgG) immunoglobulins appear during primary infection and persist throughout life in the majority of infected individuals; these IgG may disappear in immunocompromised persons in particular (Henle et al., Proc Natl Acad Sci US A, 1987, 84: 570-574, Ascherio et al., JAMA 2001, 286: 3083-3088). . The EBNA-1 protein has an immunodominant epitope of Ser-Ser-Ser-Gly-Ser-Pro-Pro-Arg-Arg-Pro-Pro-Pro-Gly-Arg-Arg-Pro-Phe-Phe-Hi sequence. Pro-Val (SEQ ID NO: 1) (amino acids 389 to 409 of the accession number protein sequence P03211 defined above) (Fachiroh et al., J. Clin Microbiol., 2006; 44: 1459-1467, Blake et al., J. Immunol., 2000, 165: 7078-87).

Bordetella pertussis is a Gram-negative coccobacillus, responsible for pertussis in humans. In the body, the bacterium attaches, via adhesins, to the ciliated epithelium of the respiratory system. It then multiplies and synthesizes several toxins, including the PT protein (pertussis toxin) (accession number PO4977 or GI: 136005 in the GenBank database). The majority of the world's population is vaccinated against B. pertussis. Pertussis vaccines generally consist of either whole bacteria (including PT toxin) inactivated (cell vaccine) or a mixture of antigens including recombinant PT toxin (acellular vaccine). Anti-PT IgG persist for many years after illness or vaccination. The PT protein has an immunodominant epitope of Leu-Thr-Trp-Leu-Ala-Ile-Leu-Ala-Val-Thr-Ala-Pro-Val-Thr-Ser-Pro-Ala-Trp-Al a-Asp-sequence. Asp (SEQ ID NO: 2) (amino acids 16 to 36 of the sequence of the accession number protein PO4977 defined above) (Raupach et al., Microb Pathog., 1994; 17: 213-226; Locht et al., Infect Immun., 1987, 55: 2546-2553). The bacterium Chlamydophila pneumoniae (formerly Chlamydia pneumoniae) is responsible, in humans, for respiratory infections, which vary from asymptomatic forms to bronchitis and pneumonia. It evolves in three antigenically distinct forms: the elementary body (EC), extracellular form of dissemination of infection, the reticulated body (CR), intracellular form of multiplication and the aberrant body (CA), a form of responsible persistence of the chronic infection. The elementary body consists of a cytoplasmic membrane and a wall close to that of Gram-negative bacteria. The outer wall contains several proteins rich in cysteine residues, including the Major Outer Membrane Protein (MOMP) with a molecular weight of about 40 kDa and the OMP2 protein (accession number P23700 or GI: 129112 in the GenBank database). MOMP and OMP2 are markers of infection with this bacterium. The majority of the global adult population has been in contact with this bacterium and the acquired immunity is of very long duration (Cunningham and Ward, 2003, cited above, Halme et al., Scand J. Immunol., 1998, 47: 517-520). The OMP2 protein contains the peptide fragment of Met-Thr-Ala-Lys-Lys-V-Al-Arg-Leu-Val-Arg-Arg-Asn-Lys-Gln-Pro-Val-Glu-Gln-Lys-Ser sequence.

(SEQ ID NO: 3), itself containing a immonureate moiety (amino acids 2-16, Cunningham and Ward, 2003, cited supra). This fragment (SEQ ID NO: 3) has a hydrophobicity index of -2.5 determined according to the Kyte-Doolittle method (Kyte and Doolittle, J. Mol Biol., 1982, 157: 105-32); it can therefore be considered as an immonodominant epitope (Margalit et al., J. Immunol., 1987, 138: 2213-2229). The potential epitopes of the peptide antigens can be identified by any method known to those skilled in the art, in particular the Pepscan technique (Geysen et al., Proc Natl Acad Sci USA, 1984, 81, 3998-4002, Geysen et al. al., J. Immunol Methods, 1987, 102, 259-274). According to a preferred embodiment of the invention, said IgG bind an epitope chosen from the peptide of sequence SEQ ID NO: 1, the peptide of sequence SEQ ID NO: 2 and the peptide of sequence SEQ ID NO: 3, as defined above.

According to another embodiment of the invention, the antigens or the peptide fragments as defined above can be conjugated, optionally via a peptide spacer, to biotin to facilitate their attachment on a support. solid, for example on a multi-well plate sensitized to streptavidin when the comparison of the concentrations of antibodies is carried out using the ELISA method. By way of non-limiting example of a peptide spacer, mention may be made of the dipeptide of Gly-Gly sequence. According to an advantageous embodiment of the invention, the serum or plasma samples are chosen from the group comprising samples of: serum or non-hemolyzed plasma, non-lactating serum or plasma, serum or non-lipemic plasma serum or non-dehydrated plasma, and plasma removed from an anticoagulant, such as EDTA, citrate or heparin, preferably EDTA.

According to another preferred embodiment of the invention, the method comprises the following steps: (a) fixing the peptides of sequence SEQ ID NO: 1, 2 and 3, on an appropriate support, in particular a microplate, each of said peptides capable of being conjugated to a biotin molecule as defined above; (b) optionally diluting the serum or plasma samples to be tested between 1/20 to 1/200, preferably to 1 / 40th in serum / antiserum dilution buffer for tests using the peptides of SEQ ID sequences NO: 1 and 2, and between 1/200 to 1/1000, preferably 1 / 400th in said buffer for the test with the peptide of sequence SEQ ID NO: 3; (c) contacting a fraction of the diluted samples (eg 100 microliters) with each of the three peptides respectively; (d) adding an appropriately labeled anti-human IgG antibody; (e) determining the amount of antigen / IgG / anti-IgG antibody complexes formed in order to determine the concentration in peptide-binding IgG samples of SEQ ID NO: 1, 2 or 3. (f) compare the concentrations of IgG between the samples. According to an advantageous arrangement of this embodiment of the invention, said support is washed between steps (c) and (d), and / or (d) and (e). The term "anti-IgG antibody" is intended to mean a whole antibody or an antibody fragment (for example an Fab fragment) capable of binding to any human IgG. According to another advantageous arrangement of this embodiment of the invention, the labeled anti-IgG antibody is an anti-IgG antibody conjugated to an enzyme chosen from acetylcholinesterase, beta-galactosidase and glucose-6. phosphate dehydrogenase, glucose oxidase, lysozyme, malate dehydrogenase, peroxidase and alkaline phosphatase, preferably alkaline phosphatase. The methods of conjugation between an antibody are well known to those skilled in the art. Some are commercially available.

According to yet another particular provision of this embodiment of the invention, the amounts of antigen / IgG / anti-IgG antibody complexes are determined by spectrophotometry (measurement of the optical density). According to one provision of this embodiment of the invention, the labeled anti-human IgG antibody is conjugated to alkaline phosphatase. In this case, the amount of antigen / IgG / anti-IgG antibody complexes formed can be determined using a suitable substrate, preferably pNPP (para-nitrophenyl phosphate) and by density measurement. optical (OD) at 405 nm. The method of differentiation of two serum or plasma samples according to the present invention also applies to the differentiation of serum or plasma samples in a sample bank by comparing said samples in pairs according to the method defined above. The present invention also relates to a method for determining a serological imprint of a sample of serum or human plasma, comprising a step of determining the concentration in the sample of class G immunoglobulins (IgG) directed against at least three different antigens selected for their ability to induce long-lasting acquired humoral immunity as defined above; the serological imprint being defined by the respective concentrations of IgG against said at least three different selected antigens. The concentrations are determined by a method as defined above. The present invention also relates to a kit or kit for differentiating samples from human serum or plasma or to determining a serological imprint of a sample of human serum or plasma, comprising: an EBNA-1 protein of the Epstein-Barr virus (EBV), a B. pertussis PT protein and / or a C. pneumoniae OMP2 protein, as defined above, or a peptide comprising or consisting of the sequence SEQ ID NO: 1, a peptide comprising or consisting of of the sequence SEQ ID NO: 2 and / or a peptide comprising or consisting of the sequence SEQ ID NO: 3, each of said peptides being capable of being conjugated to a biotin molecule,

and optionally an anti-human IgG antibody. The subject of the present invention is also the use of: - an Epstein-Barr virus (EBV) EBNA-1 protein, a B. pertussis PT protein and / or a C. OMP2 protein. pneumoniae, as defined above, or a peptide comprising or consisting of the sequence SEQ ID NO: 1, a peptide comprising or consisting of the sequence SEQ ID NO: 2 and / or a peptide comprising or consisting of the sequence SEQ ID NO: 3, each of said peptides being capable of being conjugated to a biotin molecule, for differentiating between samples of human serum or plasma, or for determining a serological imprint of a sample of human serum or plasma. In addition to the foregoing, the invention further comprises other provisions which will emerge from the description which follows, which refers to the validation of the method according to the present invention.

EXAMPLE: VALIDATION OF THE DIFFERENTIATION METHOD OF TWO SAMPLES OF SERUM OR HUMAN PLASMA The method described below is not used to make a quantitative result, but only a qualitative result to ensure that two samples of plasma or serum come from different samples.

This method is based on the detection of three specific serological markers (antigens) by measuring the optical density (OD) at 405 nm using the ELISA technique. Consequently, the following parameters have no influence on the results of the method and have not been validated: accuracy, slope, linearity, sensitivity, correlation with a reference method. The only parameters that are critical for the results of this method are fidelity (intra-assay repeatability and inter-assay) and specificity. Validation of the fidelity of the method was performed according to the validation procedure proposed by the Clinical and Laboratory Standards Institute (CLSI, EP10-A3 Preliminary Evaluation of Quantitative Clinical Laboratory Measurement Procedures, Approved Guideline, Third Edition, 68p). The risk of erroneously concluding on the identity of two samples when they belong to different samples (specificity

of the method), as well as the possibility of testing samples of both serum and human plasma have been validated. 1. Materials and Methods: Serum and human plasma samples were provided by the Biological Resource Center of the University Hospital Center of Amiens (France). Three immunodominant peptides (epitopes) of antigens capable of inducing long-lasting immunity in humans have been used: the peptide of sequence SEQ ID NO: 1, fragment of the EBNA-1 protein of the Epstein Barr virus; the peptide of sequence SEQ ID NO: 2, fragment of Bordetella pertussis PT toxin, the peptide of sequence SEQ ID NO: 3, a fragment of the OMP2 protein of Chlamydophila pneumoniae.

These peptides were each conjugated to a biotin molecule in the N-terminal position via a peptide spacer of Gly-Gly sequence, respectively giving the serological markers PEPEB (Biotinyl-Gly-Gly-SEQ ID NO: 1). , PEPBP (Biotinyl-Gly-Gly-SEQ ID NO: 2) and PEPCP (Biotinyl-Gly-Gly-SEQ ID NO: 3). The coupling method has been described in the article by Geahlen et al., 1992 (Anal Biochem., 1992, 202: 68-70). The concentrations of IgG binding to the peptides of sequence SEQ ID NO: 1, 2 or 3 respectively were measured using the ELISA method, which is well known to those skilled in the art, according to the following method: peptide conjugates (PEPEB, PEPBP and PEPCP) to streptavidin-sensitized multiwell plate; bringing the serum or plasma samples (100 l), diluted in serum / anti-serum dilution buffer (1X PBS, 4% NaCl, 0.1% Tween 20), into contact with each of said conjugates; incubate for 1 hr 5 min at 37 ° C 2 ° C with gentle agitation; wash three times with washing buffer (0.1X PBS, 0.1% Tween 20);

add (100 μl) to each well an anti-human IgG antibody coupled to alkaline phosphatase (A3187, Sigma-Aldrich), capable of binding to the previously formed antigen / IgG complexes; incubate for 1 h 5 min at 37 ° C 2 ° C with gentle agitation; wash three times with the wash buffer; add (100 μl) to each well the substrate (1 mg / ml) (para-nitrophenyl phosphate (pNPP) in Tris / MgCl 2 buffer); incubate, 20 min for the PEPEB marker tests and 30 min for the PEPBP and PEPCP markers, in the dark at room temperature; add (25 μl) of stop solution (4N NaOH); - read the optical density at 405 nm using a microplate reader (Thermo Labsystems Multiscan) within half an hour following the addition of the stop solution (make the blank on the wells incubated with serum / antiserum dilution buffer alone). 2. Results: a) Fidelity study Three sera, SER REF 1, SER REF 2 and SER REF 3, from three different individuals were aliquoted per 100 μl and stored at -80 ° C. The dilutions of the sera samples and the incubation times with pNPP to which the three reference sera make it possible to obtain optical densities (OD) at 405 nrn "low", "intermediate" and "high" respectively, and this for each of the three serological markers, were defined. The results are presented in the following Tables I, II and III: Table I: OD at 405 nm obtained according to the dilutions for the PEPEB serological marker. SER REF dilution time (min) OD at 405 nm expected 1 1/2560 20 0.11-0.17 1 1/320 20 0.76-0.95 _ 1 1/80 20 1.92-2.16 Table II: OD at 405 nm obtained as a function of the dilutions for the PEPBP serological marker. SER REF dilution time (min) OD at 405 nm expected 3,160 30,11-0,17 1,80 30 0,81-0,91 1 20 30 1,98-2,16 Table III: OD at 405 nm obtained as a function of the dilutions for the PEPCP serological marker. SER REF dilution time (min) OD at 405 nm expected 2,160 30 0,04-0,08 3,80 30 0,14-0,22 3 20 30 0,36-0,66 To determine the accuracy of the method the "low", "intermediate" and "high" optical densities obtained from the serum reference batches were measured for 5 days, with respect to the three serological markers PEPEB, PEPBP and PEPCP, in the following order of reference sera (SER REF), without modification or interruption: 2, 3, 1, 2, 2, 1, 1, 3, 3 and 2. The results for each serological marker are presented in Tables IV , V and VI: Table IV: Calculation of Fidelity (CV) for serological marker assay PEPEP DO Low DO High Intermediate DO Variance Daily Mean (VM) 0.001 0.019 0.040 Pooled Variance Intra-assay (VW) 0.001 0.006 0.006 inter-test adjusted variance (DV) = (VM) - 0.000 0.004 0.011 (VW) / 3 combined variance VT = VW + VD 0.001 0.010 0.017 combined standard deviation (S) ) = iVT 0.031 0.101 to 0.132 large average (0) 0.137 0.856 2.044 CV% = (S) / O x 100% 0.228 0.118 0.064 Table V: Calculation of Fidelity (CV) for the serum marker test PEPBP DO low DO DO high intermediate variance of daily means (VM) 0.001 0.002 0.006 pooled variance intra-test (VW) 0.001 0.003 0.004 adjusted variance inter-trial (VD) = (VM) - 0.000 0.000 0.001 (VW) / 3 combined variance VT = VW + VD 0.001 0.003 0.005 combined standard deviation (S) = JVT 0.029 0.053 0.069 large average (0) 0.137 0.856 2.044 CV% = (S) / O x 100% 0.213 0.062 0.034 Table VI: Calculation of Fidelity (CV) for the Serum marker test PEPCP DO low DO DO high intermediate variance of daily means (VM) 0.000 0.001 0.004 pooled variance intra-assay (VW) 0.000 0.003 0.032 inter-assay adjusted variance (RV) = (VM) - 0.000 -0.001 - 0.009 (VW) / 3 combined variance VT = VW + VD 0.000 0.002 0.023 combined standard deviation (S) = ~ VT 0.020 0.044 0.150 large average ( 0) 0.059 0.184 0.508 CV% = (S) / O x 100% 0.344 0.236 0.295 These results show that the intra- and inter-assay fidelity is controlled over the entire validity range ("low", "intermediate" and "low" ODs). high ") of the method. b) Study of the specificity For the study of the specificity, 91 samples of different human sera were tested. These correspond to (912-91) / 2 possible binary (or pairwise) combinations of two serum samples, that is, 4095 different combinations. Optical density (OD) at 405 nm was measured for each of the three serological markers (PEPEB, PEPBP and PEPCP). Serum samples were diluted 1:40 for PEPBP and PEPCP serologic markers, and 1 / 400th for the PEPEB serine marker assay. The tests were performed in triplicate. The results are shown in Table VII below, where: PEPEB 1, 2 and 3 respectively represent the 3 values of the OD for the serological PEPEB marker test; - PEPBP 1, 2 and 3 represent respectively the 3 values of the OD for the test with the serological marker PEPBP;

- PEPCP 1, 2 and 3 respectively represent the 3 values of the OD for the test with the serological marker PEPCP; MY PEPEB, PEPBP and PEPCP represent the mean value of the OD for each of the 3 serological markers; - PEPEB +, PEPBP + and PEPCP + deviation represent the upper confidence interval of each average, taking into account the CV% corresponding to each peptide (PEPEB, PEPBP or PEPCP) and for each concentration; - Difference PEPEB-, PEPBP- and PEPCP- represent the lower confidence interval of each average, taking into account the CV% corresponding to each peptide (PEPEB, PEPBP or PEPCP) and for each concentration. SERUM PEPE PEP PEPE MY Ecart PEPB PEPB PEPB MY Ecart Ecart PEPC PEP PEP MY Ecart Difference BI EB2 B3 PEPE PEPE PEPE P2 P3 PEPB PEPB PEPBP PI CP2 CP3 PEP PEP PEPC BB + B- P P + - CP CP + P-9979 0 , 04 0.00 0.00 0.01 0.02 0.01 0.07 0.07 0.09 0.08 0.10 0.06 0.03 0.01 0.01 0.02 0.02 0.01 9673 0.02 0.02 0.02 0.02 0.03 0.02 0.26 0.23 0.24 0.24 0.30 0.18 0.06 0.04 0.04 0 , 0.06 0.03 0.08 0.03 0.03 0.03 0.03 0.02 0.02 0.00 0.02 0.30 0.30 08 0.08 0.07 0.09 0.05 14952 0.07 0.01 0.02 0.03 0.04 0.03 0.29 0.33 0.31 0.31 0.33 0.29 0.07 0.09 0.02 0.06 0.08 0.04 14986 0.04 0.04 0.04 0.04 0.05 0.03 0.28 0.32 0.26 0.29 0 , 36 0.22 2.00 2.01 2.26 2.09 2.72 1.46 14992 0.04 0.04 0.04 0.04 0.05 0.03 0.28 0.29 0, 35 0.31 0.32 0.29 0.15 0.24 0.17 0.19 0.24 0.13 14958 0.06 0.03 0.03 0.04 0.05 0.03 0.52 0.47 0.54 0.51 0.54 0.48 0.05 0.10 0.11 0.09 0.12 0.06 14964 0.03 0.06 0.04 0.04 0.05 0 , 03 0.55 0.54 0.51 0.53 0.56 0.51 0.10 0.09 0.26 0.15 0.20 011 26262 0.04 0.05 0.04 0.04 0 , 05 0.03 0.61 0, 66 0.57 0.61 0.64 0.58 0.12 0.17 0.20 0.16 0.21 0.11 15007 0.04 0.05 0.05 0.05 0.06 0.04 0.53 0.51 0.50 0.51 0.54 0.49 0.55 0.53 0.44 0.51 0.66 0.35 32331 0.06 0.05 0.05 0.05 0 , 07 0.04 0.54 0.54 0.47 0.52 0.54 0.49 0.26 0.20 0.23 0.23 0.30 0.16 37038 0.01 0.13 0.021 0 , 0.07 0.04 0.58 0.52 0.61 0.57 0.60 0.54 0.13 0.10 0.09 0.11 0.14 0.07 14984 0.07 0, 05 0.08 0.07 0.08 0.05 0.51 0.49 0.46 0.49 0.51 0.46 0.78 0.54 0.29 0.54 0.70 0.38 14967 0.08 0.07 0.06 0.07 0.09 0.05 0.66 0.59 0.71 0.65 0.69 0.62 0.13 0.18 0.16 0.16 0, 0.11 40363 0.02 0.02 0.19 0.08 0.10 0.06 0.55 0.51 0.56 0.54 0.57 0.51 0.14 0.14 0.21 0.16 0.21 0.11 10901 0.08 0.10 0.09 0.09 0.11 0.07 0.30 0.29 0.31 0.30 0.32 0.29 0.07 0 , 07 0.10 0.08 0.11 0.05 14936 0.08 0.10 0.11 0.10 0.12 0.07 0.75 0.72 0.76 0.74 0.78 0, 71 0.05 0.09 0.09 0.08 0.10 0.05 15003 0.18 0.06 0.07 0.10 0.13 0.08 0.41 0.58 0.50 0.50 0.52 0.47 0.39 0.28 0.36 0.34 0.45 0.24 14915 0.13 0.15 0.13 0.14 0.17 0.10 0.76 0.70 0 , 60 0.70 0.74 0.67 0.18 0.18 0.20 0 , 19 0.24 0.13 25239 0.16 0.15 0.15 0.15 0.19 0.12 0.43 0.51 0.46 0.47 0.49 0.44 0.13 0, 15 0.19 0.16 0.20 0.11 14941 0.17 0.16 0.16 0.16 0.20 0.12 0.53 0.51 0.49 0.51 0.54 0.48 0.80 0.83 0.83 0.82 1.07 0.57 10902 0.17 0.17 0.20 0.18 0.23 0.14 0.50 0.60 0.57 0.56 0 , 58 0.53 0.17 0.22 0.23 0.21 0.27 0.14 40518 0.20 0.18 0.16 0.18 0.23 0.14 0.62 0.59 0, 57 0.59 0.62 0.56 0.24 0.41 0.20 0.28 0.37 0.20 13546 0.18 0.18 0.18 0.18 0.23 0.14 0.71 0.80 0.78 0.76 0.80 0.73 0.14 0.22 0.24 0.20 0.26 0.14 25843 0.18 0.18 0.18 0.18 0.23 0 , 14 0.92 0.94 0.86 0.91 0.95 0.86 0.38 0.37 0.39 0.38 0.49 0.27 31924 0.19 0.18 0.18 0, 18 0.23 0.14 0.58 0.56 0.56 0.57 0.60 0.54 0.17 0.11 0.15 0.14 0.19 0.10 14916 0.19 0.20 0.21 0.20 0.25 0.15 0.45 0.45 0.50 0.47 0.49 0.44 0.27 0.31 0.25 0.28 0.36 0.19 13698 0 , 0.20 0.19 0.21 0.27 0.16 0.61 0.76 0.66 0.68 0.71 0.64 0.25 0.16 0.16 0.19 0.25 0.13 10378 0.21 0.24 0.25 0.23 0.29 0.18 0.30 0.37 0.35 0.34 0.36 0.32 0.17 0.09 0.09 0 , 12 0.15 0.08 14913 0.24 0.25 0.22 0.24 0.30 0.18 0.75 0.79 0.75 0.76 0.80 0.73 0.32 0.35 0.34 0.34 0.44 0.24 61: 0 9 to 0 8Z ' 0 8Z`0 5Z`0 0 to 0 590 l The 0 89`0 L9'0 690 89`0 001 1 l `1 904 I 90: 1 90: 1 50: 1 0L Z 86`0 L6`0 58 `0 55`0 ZO` l 8L`0 6L`0 5L`0 18`0 117`0 917`0 17`0 1717`0 17`0 17`0 77`0 601 0` 1 170` l 170` 1 ZO` 1 _9_17671_ LO` 1 Z0` 1 S 1 to 1 68`0 Z0 'I Z8 I 511 00` l 06`0 86`0 l 1 to 1 66171 ZI`I L6`0 6` to 56`0 0`l 9b01Z 61 7 0L ZZS l OZ`0 L `0 6Z`0` 0 5Z`0 8Z`0 Ob`0 1717 `0 Z17`0 117`0 017`0 ZZ`0 Zb`0 Z` 0 0 `0 SE'0 1 0 Sb`0 05`0 Lb`0 17`0 Lb`0 Z5`0 OZ`0 9E`0 8Z`0 ZZ`0 SZ`OL` 0 59`0 ZL`0 89 `0 L5`0 ZL'0 9L`0 Z8`OZ`0 Z17`0` 0 `0 I` 0 Z `0 9L`0 1 78`0 08`0 ZL`0 Z8`0 98`0 Z8` 0 ll 96`0 Z6`O L60 00 l 81`0 `0 SZ`0 Z`0 Z`0 8Z`0 LS`0 9`0 09`0 59`0 95`0 65`0 8L`0 N / A `l Z6`0 Z6`0 06`0 6`0 Z l` 0 Z`0 81` 0 81'0 51'0 OZ`0 8940 I ZL'0 5L`0 1L'0 69'0 5L`0 ZO '1 88'0 8'0 L0'1 5L`0 VVA 0 `0 17L'0 00'1 0'0 LO'0 50`0 170`0 0`0 80`0 6V0 I 0` 0 0 ` 0 0 to 0 L8'0 178`0 178'0 6'0 018L 1 LO'0 171'0 11'0 Z1'0 0 1'0 01'0 l` 0 I `0 I` 0 17 `0 17` 0 1 CO 96'0 8'0 18`0 68'0 08'0 St.

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SZ`I 61`1 Z` l 171 '! 12`1 _ 61812 69'0 _ 917`0 9S'0 01'1 1Z`l SI 'l S `I 1S`1 09'0 1706 1 S8'0 98'0 6L'0 50` l L l` l 11'1 Z1'1 91'1 SO`1 ZSSSZ Lb'0 1 S`0 S17'0 SO'l 91`1 01'1 SO` l Z0'1 1Z` 1 886171: L_'0 69`0 179'0 170'1 171`1 60'1 11'1 Z 1 'l 170` 1 S 169Z 20 Of the 4095 possible combinations, only 22 identifications of two samples were erroneous (the method did not differentiate two different samples for these 22 identifications). The discrimination power and the specificity of the method according to the present invention is therefore 99.5%. c) Validation of the method with plasma samples The use of human plasma samples was validated by testing 10 plasma samples recovered on EDTA from the Center of Biological Resources (CRN) of the University Hospital Center (CHU). Amiens (France). Plasma samples were diluted 1:40 for PEPBP and PEPCP peptide conjugate tests, and 1 / 400th for the PEPEB peptide conjugate test. Each sample was tested in triplicate. Table VIII below shows the means and standard deviations of the results (Optical Density) obtained: PEPEB samples PEPBP PEPCP 72528 0.55 +/- 0.1 0.36 +/- 0.006 0.13 + 1-0.006 72549 2.34 + / 60.01 0.81 + 1-0.13 0.29 +/- 0.02 72547 0.03 +/- 0.01 0.44 +/- 0.05 0.25 + 1 -0.06 P 1 0.73 +/- 0.05 0.26 +/- 0.01 0.21 +/- 0.05 P2 0.61 +/- 0.03 0.42 +/- 0 , 03 0.24 +/- 0.05 P3 1.22 +/- 0.17 0.26 +/- 0.006 0.15 +/- 0.07 P4 2.85 + 1-0.11 0.41 +/- 0.01 0.23 +/- 0.006 P5 0.47 +/- 0.09 1.12 +/- 0.12 0.25 +/- 0.03 P6 2.03 + 1-0, 0.49 +/- 0.05 0.09 +/- 0.01 P7 0.2 + 1-0.02 0.24 +/- 0.03 0.15 +/- 0.07 The samples present all of them have a different concentration (Optical Density) for at least one serological marker. These results show that the implementation of the method according to the present invention makes it possible to differentiate at least two samples of human plasma. d) Conclusion The precision of the method is sufficient to render it an excellent specificity for the desired purpose: the differentiation of two samples of serum or human plasma. Both the plasma samples and the serum samples are suitable for the method according to the present invention.

Claims (13)

REVENDICATIONS1. A method of differentiating two samples of serum or human plasma, characterized in that it comprises a step of comparing the concentration, in the samples, of class G immunoglobulins (IgG) directed against at least three different antigens selected for their ability to induce a long-lasting acquired humoral immunity, a different concentration between said two samples for at least one IgG directed against the same antigen meaning that the two samples come from two different blood samples. 2. Method according to claim 1, characterized in that said IgG are selected from the group consisting of IgG directed against EBNA-1 protein expressed by the Epstein-Barr virus, heat shock proteins, OMP2 proteins or MOMP expressed by Chlamydophila pneumoniae, lipopolysaccharides of gram-negative bacteria, tetanus toxin produced by Clostridium tetani, capsid protein of poliovirus or PT protein, pertactin or phytohemagglutinin produced by Bordetella pertussis. 3. Method according to claim 1 or claim 2, characterized in that said antigens come from three microorganisms of different kinds. 4. The method of claim 2 or claim 3 characterized in that said antigens are selected from the antigens of Epstein-Barr virus, Bordetella pertussis and Chlamydophila pneumoniae. 5. Method according to claim 4, characterized in that said antigens are the EBNA-1 protein of the Epstein-Barr virus, the B. pertussis PT protein and the C. pneumoniae OMP2 protein. 6. Method according to claim 5, characterized in that said IgG bind a peptide selected from the peptide of sequence SEQ ID NO: 1, the peptide of sequence SEQ ID NO: 2, and the peptide of sequence SEQ ID NO: 3. 7. A method for determining a serological imprint of a sample of human serum or plasma, characterized in that it comprises a step of determining the concentration, in the sample, of class G immunoglobulins (IgG) directed against at least three different antigens selected for their ability to induce long-lasting acquired humoral immunity, as defined in any one of claims 3 to 6; the serological imprint being defined by the respective concentrations of IgG against said at least three different selected antigens. 8. Method according to any one of claims 1 to 7, characterized in that the concentration of said IgG is determined using a method chosen from ELISA methods (Enzyme Linked Immunosorbent Assay), EIA (Enzyme Immunoassay), RIA (Radio Immunoassay), immunofluorescence, multiplex, Western blot or protein chip. 9. A method of differentiating two serum or plasma samples according to any one of claims 1 to 6 and 8, characterized in that it comprises the following steps: (a) fixing the peptides of SEQ ID NO: 1, 2 and 3, on a suitable support, in particular a microplate, each of said peptides being capable of being conjugated to a biotin molecule; (b) optionally diluting the serum or plasma samples to be tested; (c) contacting a fraction of the diluted samples or samples with each of the three peptides respectively; (d) adding an appropriately labeled anti-human IgG antibody; e) determining the amount of antigen / IgG / anti-IgG antibody complexes formed so as to determine the concentration in the peptide-binding IgG samples of SEQ ID NO: 1, 2 or 3. (f) compare the concentrations of IgG between samples. 10. Method according to claim 10, characterized in that said labeled anti-human IgG antibody is an anti-human IgG antibody conjugated to alkaline phosphatase and that the amount of antigen / IgG / anti-IgG antibody complexes formed is determined by using para-nitrophenyl phosphate by measuring the optical density at 405 nm. 11. Method according to any one of claims 1 to 10, characterized in that the serum or plasma samples are chosen from the group comprising samples of serum or non-hemolysed plasma, non-lactating serum or plasma, serum or plasma. non-lipemic, serum or non-dehydrated plasma, and plasma taken from an anticoagulant. 30 12. A kit or kit for differentiating serum or plasma samples or for determining a serological imprint of a human serum or plasma sample comprising: - Epstein-Barr virus (EBV) EBNA-1 protein, the PT protein of B. pertussis and / or the OMP2 protein of C. pneumoniae, or a peptide comprising or consisting of the sequence SEQ ID NO: 1, a peptide comprising or consisting of the sequence SEQ ID NO: 2 and / or a peptide comprising or consisting of the sequence SEQ ID NO: 3, each of said peptides being capable of being conjugated to a biotin molecule, and optionally an anti-human IgG antibody. 13. Use of (i) EBNA-1 protein of Epstein-Barr virus (EBV), B. pertussis PT protein and / or C. pneumoniae OMP2 protein, or (ii) a peptide comprising or consisting of the sequence SEQ ID NO: 1, a peptide comprising or consisting of the sequence SEQ ID NO: 2 and / or a peptide comprising or consisting of the sequence SEQ ID NO: 3, each of said peptides which can be conjugated to a biotin molecule, to differentiate serum or human plasma samples, or to determine a serological imprint of a sample of human serum or plasma.