Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
  • G01N33/56905—Protozoa
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
  • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
  • G01N2333/44—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from protozoa
  • G01N2333/45—Toxoplasma
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2469/00—Immunoassays for the detection of microorganisms
  • G01N2469/20—Detection of antibodies in sample from host which are directed against antigens from microorganisms

Definitions

• the present invention provides a method for early in vitro diagnosis of toxoplasmosis infection in humans, as well as a diagnostic kit.
• Toxoplasmosis an infection caused by the protozoan parasite Toxoplasma gondii, affects at least one third of the world's human population. There is only one genus and one species of the parasite Toxoplasma gondii, but parasite isolates are currently classified into 12 haplogroups I to XII. Strains mostly found in Europe and North America are of type II. The parasite most often infects warm-blooded animals, including humans, but its definitive host is a feline.
• toxoplasmosis The prevalence of toxoplasmosis varies from country to country; it is between 60% and 80% in certain areas of South America. In Western Europe, the prevalence of toxoplasmosis varies from 50 to 70%; it varies from 20 to 50% in Southern Europe as well as in the humid regions of Africa; it is less than 30% in the Scandinavian countries and the United Kingdom, 25 to 30% in the Central European countries and is very low in South East Asia and North America (Pappas et al. 2009).
• CNR 201 1 Three hundred fetuses with congenital toxoplasmosis (CNR 201 1), 30 of which will develop serious sequelae, are identified each year in France.
• T. gondii infection it is therefore important to be able to diagnose T. gondii infection early in order to put in place the appropriate treatments: combination of pyrimethamine-sulfadiazine and folic acid in patients with congenital or immunodepressed toxoplasmosis, and spiramycin in pregnant woman.
• the current diagnosis of toxoplasmosis relies mainly on the detection, from the serum of patients, of isotype-specific antibodies G and M directed against the entire parasite.
• the immunoglobulin M (IgM) levels which are in principle markers of a recent infection, then of serum immunoglobulin G (IgG) rise within two weeks after infection. If IgM is a sign of a recent infection (they appear in a few days, the peak is reached in 2 - 3 months and then decrease), they can persist for several months, even years, and are therefore not reliable indicators of infection. recent seroconversion.
• IgM immunoglobulin M
• IgG serum immunoglobulin G
• TORCH targets pathogens that can pass the placental barrier
• T - Toxoplasma gondii O - Other infections (Coxsackievirus, Syphilis, Varicella - Zoster Virus, HIV, Parvovirus B19), R - Rubella, C - Cytomegalovirus, H - Herpes simplex virus).
• Toxoplasma gondii Several antigens of Toxoplasma gondii have been identified and classified into different families according to their cellular localization:
• Antigens of the dense granules of toxoplasm GRA2, GRA3, GRA4, GRA5, GRA6, GRA7, GRA8 .
• Rhoptries antigens secretory organelles specific to Apicomplexa
• ROP1 secretory organelles specific to Apicomplexa
• Abbott's patent application EP 1 082 343 describes a diagnostic composition comprising the antigens p29 (GRA7), p30 (SAG1) and p35 (GRA8), or p29 (GRA7), p35 (GRA8) and p66 (ROP1).
• the antigens GRA2 / p28 (Mercier et al., 1993, FR 2 692 282) and GRA6 / p32 (Lecordier et al., 1993, FR 2 702 497) are antigens derived from dense granules, secreted by T. gondii and playing an essential role in intracellular parasitism. They are major components of the dense granules (secretory organelles specific to Apicomplexa) and the vacuole in which the parasite multiplies within the infected cell. These highly immunogenic proteins are good candidates for diagnostic applications.
• the recombinant GRA2 protein purified from a bacterial production system, was tested in an ELISA test whose specificity was 96.4% and the sensitivity of 95.8% to 100% for acute infections, lower for chronic infections (Golkar ef al., 2007)
• the recombinant GRA6 protein was tested in an ELISA test which shows much better specificity for the sera of patients with acute infection than for those with chronic infection (Golkar et al., 2008).
• the recombinant GRA2 protein has been associated with the ROP1 antigen (P66); these two antigens are detected more frequently by the sera of patients with acute infection than by the sera of patients with chronic infection, thus allowing a distinction between the two clinical cases (Holec-Gasior et al., 2009).
• the recombinant GRA6 protein was combined with GRA1, another dense granule antigen, in an ELISA assay. This test, designed to differentiate patients with a recent infection profile from those with chronic infection, was found to be insufficiently sensitive for this application (Ferrandiz et al., 2004).
• the invention relates to a method for detecting the presence of anti-Toxoplasma gondii antibodies in human or animal serum, comprising contacting said serum with a composition comprising recombinant GRA2 and GRA6 proteins. More particularly, the invention relates to a direct or indirect ELISA (Enzyme-Linked ImmunoSorbent Assay) test which allows the early detection in human serum of specific antibodies directed against the antigenic combination GRA2 and GRA6.
• ELISA Enzyme-Linked ImmunoSorbent Assay
• the first advantage of this test is its precocity because it can detect a very recent infection in the serum of the individuals tested.
• the second major advantage of this test is that it makes it possible to distinguish more rapidly, on serums of newborns taken at different times after birth, the presence of antibodies, which has the advantage of excluding or confirming rapidly. the diagnosis of congenital toxoplasmosis in the newborn.
• the invention relates to a method for detecting the presence of anti-Toxoplasma gondii antibodies in human or animal serum, comprising contacting said serum with a composition comprising recombinant GRA2 and GRA6 proteins.
• ImmunoSorbent Assay that applies this method, allows its use to confirm recent seroconversion in pregnant women and for the diagnosis of congenital toxoplasmosis in neonates.
• the invention relates to a method for identifying the presence or absence of anti-Toxoplasma gondii antibodies in human or animal serum, comprising contacting said pre-collected serum with antigens capable of binding with said antibodies.
• -Toxoplasma gondii and the finding of a binding or an absence of binding of antibodies with said antigens characterized in that said antigens comprise the combination of two recombinant proteins GRA2 and GRA6.
• An antibody is a complex protein produced and used by the immune system to specifically detect and neutralize pathogens.
• the antibodies are glycoproteins of the immunoglobulin superfamily. They are formed of 4 polypeptide chains: 2 heavy chains and 2 light chains which are connected to each other by a variable number of disulfide bridges. These chains form a Y structure. Each light chain consists of a constant domain and a variable domain; heavy chains are composed of a variable fragment and 3 or 4 constant fragments according to the isotype. For a given antibody, the two heavy chains are identical, likewise for the two light chains.
• Antibodies have the ability to recognize and specifically bind to an antigen, this specificity being conferred by the presence of variable domains.
• anti-Toxoplasma gondii antibody any antibody capable of binding to an antigen derived from this parasite Toxoplasma gondii. This designation refers to polyclonal and monoclonal antibodies.
• Pre-collected serum means any collection of human or animal blood, taken according to techniques well known to those skilled in the art, in particular by means of a syringe, and centrifuged to eliminate blood cells and coagulation factors. .
• the serum will preferably be kept cold to limit the degradation of the proteins. Serum may also be referred to herein as the "sample”.
• antigen designates, according to the invention, any protein derived from Toxoplasma gondii or any recombinant protein synthesized from DNA derived from Toxoplasma gondii, and capable of inducing an immune response in an infected subject.
• An antigen generally has several different epitopes that are as many antibody binding sites.
• binding of antibodies to said antigens refers to the interaction between an antigen and an antibody specific for that antigen and more specifically, the immune complex resulting from the combination of an immunogenic epitope of the antigen with a specifically directed antibody. against this epitope.
• Both GRA2 and GRA6 proteins refer to proteins with high antigenic potency of Toxoplasma gondii, as previously described.
• recombinant proteins refers to proteins produced in genetically modified cells, in particular by introduction of an expression vector carrying a gene of interest. This gene encoding a protein of interest is expressed by the producing species (bacteria, mammalian cells in culture, etc.). Preferably, the recombinant proteins will be produced in a bacterial system and in particular in Escherichia coli. After purification, they will be used to 'capture' antibodies potentially present in human or animal serum.
• the antigens present in the method according to the invention comprise at least the recombinant proteins GRA2 and GRA6 but may also comprise other recombinant proteins such as GRA1, GRA3, GRA4, GRA5, GRA7, GRA8, etc.
• the method is characterized in that the antigens consist of the combination of the two recombinant proteins GRA2 and GRA6 only.
• the GRA2 and GRA6 proteins can be present in all proportions and in particular, their molar ratio GRA2 / GRA6 can be 10/90, 20/80, 30/70, 40/60, 50/50, 60/40, 70/30 , 80/20, or 90/10. According to a preferred aspect of the invention, the molar ratio GRA2 / GRA6 is between 40/60 and 60/40. According to a preferred aspect of the invention, the molar ratio GRA2 / GRA6 is 50/50.
• the method described above can be carried out according to all types of immunological assays known to those skilled in the art, such as radioimmunoassays which use radioactive compounds associated with antigens or enzyme immunoassays which use coupled enzymes. antigens.
• the method for detecting the presence or absence of anti-Toxoplasma gondii antibody in a serum also called an antibody assay method, can be carried out in solution or on a solid support.
• the invention relates in particular to a method characterized in that the antigens are fixed on a support.
• This support will preferably be an ELISA test plate.
• the enzyme-linked immunosorbent assay which is an enzyme-linked enzyme immunoabsorption assay, that is to say a solid enzyme immunoassay, is conventionally used in immunology. to detect and assay an antibody or antigen in a sample.
• This test is characterized in that the assay is coupled to an enzyme catalyzed reaction that releases a colored component followed by spectroscopy.
• the ELISA is generally based on the use of two antibodies: one of these is specific for the antigen, while the other reacts with the immune complexes (antigen-antibody) and is coupled to an enzyme. This secondary antibody, responsible for the name of the technique, can also cause the emission of a signal by a chromogenic or fluorogenic substrate.
• ELISA techniques are known to those skilled in the art, such as direct ELISA, indirect ELISA, sandwich ELISA or competitive ELISA.
• the enzyme acts as an enhancer: even if few antibodies conjugated to the enzyme would be attached, the enzyme would catalyze the formation of many signals, making this test very sensitive but also increasing the number of false positives. It is therefore necessary to provide "control" wells.
• the finding of a binding or an absence of antibody binding with said antigens comprises the application of reagents for detecting the antibodies attached to the recombinant proteins.
• these reagents make it possible to determine the amount of antibody present in the serum.
• the serum is from a pregnant woman.
• the serum is from a newborn.
• the method of the invention may be advantageously used to diagnose congenital toxoplasmosis early.
• the term "early” indicates that the diagnosis can be made very early in the first months of life of the child.
• the method according to the invention can also be characterized in that it is carried out in addition to another diagnostic test for toxoplasmosis.
• this test is preferably a test of "second intention", having a very good sensitivity vis-à-vis the sera of recent infection.
• This diagnostic test will preferably be proposed in special situations where recent seroconversion is suspected, as a complementary test to routine diagnostic tests.
• the present invention also relates to a kit for identifying the presence or absence of aniloxoplasma gondii antibody in human or animal serum comprising:
• Reagents for detecting antibodies that bind to recombinant proteins are provided.
• the recombinant proteins GRA2 and GRA6 will be in a molar ratio of between 40/60 and 60/40 in this kit.
• GRA2 21 -185) (II) denoted GRA2 (II), or
• the recombinant proteins GRA2 and GRA6 are fused with the peptide pUET and thus purified after synthesis; for greater accuracy of the test, the absorbance values obtained with GRA2 (II), GRA6 (II) or the mixture are deduced from the absorbance value of the pUET peptide.
• the solid support undergoes three washes with a conventional buffer PBS + 0.05% Tween 20 (PBS-T) on the scrubber.
• PBS-T 0.05% Tween 20
• Non-specific sites are blocked with bovine serum albumin (BSA) at a concentration of 1% for one hour at 37 ° C.
• BSA bovine serum albumin
• the support is washed three times in PBS-T buffer.
• the support is washed three times in PBS-T buffer.
• the support is washed three times in PBS-T buffer.
• FIG. 1 Summary analysis of 259 negative sera, making it possible to determine the positivity threshold (at 2 standard deviations and 3 standard deviations) of each of the 3 GRA ELISAs, as well as the specificity of each of the 3 tests: (A) GRA (II); (B) GRA6 Nt (It; (C) GRA2 + GRA6
• FIG. 1 Summary analysis of 253 chronic sera (infections older than 12 months: IgG-positive sera by routine but negative IgM tests) to show that GRA ELISA tests are not suitable for first-line diagnosis; (A) ELISA GRA2 + GRA6; (B) Vidas IgG.
• Figure 6 summary table of the sensitivity of the ELISA GRA tests.
• the number of samples found positive in the GRA ELISA test is represented in relation to the number of samples found positive in the VIDAS IgG test ("against VIDAS IgG” columns) and with respect to all two IgG tests ("columns” compared to IgG tests (VIDAS + IF) ").
• o 253 sera diagnosed as positive for old toxoplasmosis that is, more than 12 months old (ie sera for which positive IgG levels were detected but no IgM).
• the method according to the invention is a test whose sensitivity for sera of recent infection and the specificity are particularly good.
• the sensitivity of a test is its ability to give a positive result when the disease (here, Toxoplasma gondii infection) is present.
• the specificity of the test is, it, the ability of the test to give a negative result when the disease is not present.
• Example 1 Summary analysis of 259 negatives, making it possible to determine the positivity threshold of each of the 3 GRA ELISA tests (with 2 standard deviations and with 3 standard deviations) as well as the specificity of each of the 3 tests.
• IgG IFI (Indirect Immunofluorescence) test threshold 8 IU / ml
• the specificity of the ELISA GRA test is calculated by dividing the number of sera found negative in the GRA ELISA test by the number of negative sera passed in the ELISA test and the whole multiplied by 100.
• the sensitivity of the 3 GRA ELISA tests, on all the sera tested, is very variable, depending on whether one considers old infection sera (low sensitivity, 86.56% for the GRA2 + GRA6 Nt test).
• recent seroconversions or sera from infants suspected of congenital toxoplasmosis (sensitivity greater than 100% for the GRA2 + GRA6 Nt (II) test because it detects more serum than routine tests (Cf.
• the GRA2 + GRA6 Nt (II) ELISA test has a very good sensitivity with respect to sera of very recent infection, which justifies proposing this diagnostic test in particular situations where recent seroconversion is suspected as a complementary test to routine diagnostic tests (see Figure 6).
• FIGS. 2A and 2B show the analysis of 253 old infection sera, using two different tests:
• This kit uses the "ELFA” assay principle combining the ELISA method with a final fluorescence blue reading.
• the threshold of positivity of the kit is 4-8 IU / mL. It can detect:
• the GRA2 + GRA6 Nt (II) ELISA detects 86.56% of old infections whereas the Biomérieux kit detects 99.6%.
• the dark background colors of the cells represent the absorbance values above the positivity threshold (3 ET).
• the light colors represent the absorbance values located between the positivity threshold (2 AND) and the positivity threshold (3 AND).
• the light colors represent the values located in the equivocal zone, when it exists.
• the GRA2 + GRA6 Nt (II) test is therefore more sensitive and makes it possible to detect the infection earlier than the ELFA IgG VIDAS ,.
• the GRA2 + GRA6 NT (II) test actually allows you to "recover":
• the GRA2 + GRA6 test therefore makes it possible to identify sera judged to be "negative” in IgG by another test but that are actually "positive” for Toxoplasma gondii infection and for which the diagnosis was based solely on IgM detection. .
• a ninth column indicates the diagnosis formulated by the practitioners, without taking into account the results obtained afterwards with the ELISA tests GRA2 (II), GRA6 Nt (II) and GRA2 + GRA6 Nt (ll).
• the GRA2 + GRA6 Nt (II) IgG ELISA test is revealed positive even before IgM is detected.
• patient 3 underwent four samples at 1-month intervals.
• the test serum 1 according to the Biomerieux kit, the serum was considered negative for the presence of IgG anl ⁇ -Toxoplasma gondii.
• the GRA2 + GRA6 Nt (II) test indicated the presence of anti-GRA IgG antibodies.
• patient 1 underwent four samples at 1-month intervals.
• the serum was considered negative for the presence of IgG anl ⁇ -Toxoplasma gondii.
• the values were still below the detection threshold for the Biomérieux test, but they indicated the presence of anti-GRA IgG antibodies when using the GRA2 + GRA6 Nt (II) test.
• the GRA2 + GRA6 Nt (II) IgG ELISA test is negative at the same time as the IgM IFI if we consider the positivity threshold at 3 ET but it remains positive at 2 ET. There is therefore a significant decrease in the amount of IgG directed against GRA2 (II) and GRA6 Nt (II).
• the GRA2 + GRA6 NT (II) IgG ELISA is more rapidly negative than the Vidas IgA ELFA: Patients 4, 5, 8, 12, 13, 16, 20 (7/10 TC - at 3 ET and 9 / 10 to 2 ET)).
• Patient # 8 was diagnosed positive for IgG at birth (Sample # 37) and therefore had to be followed while already negative by the GRA2 + GRA6 Nt (II) ELISA.
• the IgGs detected by the routine tests (VIDAS IgG and IF IgG) were thus residual IgG of the mother, passed in the child's blood during delivery. These IgG are eliminated naturally after a few weeks.
• patient no. 12 was diagnosed positive for anti-Toxoplasma gondii IgG by routine tests up to sample # 64 whereas he was diagnosed negative from sample # 63 by the GRA2 + ELISA test.
• GRA6 Nt (II) was diagnosed positive for anti-Toxoplasma gondii IgG by routine tests up to sample # 64 whereas he was diagnosed negative from sample # 63 by the GRA2 + ELISA test.

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• 2012
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