Cassette for expressing a Toxoplasma gondii P30 protein

The present invention provides a reagent for the detection or monitoring of a Toxoplasma gondii infection, which includes as a reactive substance a truncated Toxoplasma gondii P30 protein in which all of the hydrophobic C-terminal region of the native protein starting with the amino acid positioned after the amino acid 299 (SEQ ID NO: 1) has been deleted and all of the region of the native protein having the sequence starting with amino acid 31 and ending with the amino acid 299 (SEQ ID NO: 1) is contained. The present invention further describes methods of screening for anti-Toxoplasma antibodies in a biological sample

EXAMPLES The constructs described below were prepared according to the general genetic engineering and molecular cloning techniques detailed in Maniatis et al. (1989, Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.). All the cloning steps using bacterial plasmids are carried out by passage through the Escherichia coli ( E. coli ) 5K strain. As for the steps involving an M13-type bacteriophage, they are performed in E. coli JM101. The lithium acetate technique (Ito et al., 1983, J. Bacteriol., 153, 163-168) is used to introduce the different vectors into the Schizosaccharomyces pombe and Saccharomyces cerevisiae strains. However, any other standard technique can also be used. Moreover, as regards the gene encoding the P30 protein, the position of the nucleotides corresponds to that specified in Burg et al. (1988, supra). 
 Example 1 
 Production of a P30 protein in Saccharomyces cerevisiae 1. Construction of the expression vector pTG3747. The sequences encoding the Toxoplasma gondii P30 protein are isolated from the prior art plasmid comprising the corresponding gene (P30 gene) stretching at least from nucleotides 334 to 1632. This starting plasmid is designated hereinafter pbM 89. A DNA fragment encoding a mature P30 protein, free of the natural signal peptide and having a stop codon in the C-terminal part just upstream of nucleotide 1258, is generated by PCR (Polymerase Chain Reaction). The vector pbM89 and the primers 1367 carrying in 5′ a HindIII site (SEQ ID NO: 2) and 1366 carrying in 5′ a SalI site (SEQ ID NO: 3). In parallel, an SphI-HindIII fragment which comprises the promoter of the MF&agr; gene followed by sequences encoding the pre-pro peptide of the same gene is isolated from the vector M13TG3868. The vector M13TG3868 is derived from the vector M13TG3841, which is described in International Application Publication W090/13646, into which an HindIII site has been introduced in 3′ of the pro MF&agr; sequence. Such a modification is within the capability of a person skilled in the art. This SphI-HindIII fragment as well as the HindIII and SalI-digested amplification product are cloned between the SphI and SalI sites of the vector pTG4812. The latter is derived from the vector pTG3828, which is described in European Application EPA 396 436, into which the KEX2 gene encoding an endoprotease capable of cleaving the pre and then pro MF&agr; sequences (sequence indicated in this same European document) has been introduced. pTG3747 is obtained which contains, especially, the MF&agr; promoter directing the expression of a fragment comprising the pre-pro MF&agr; sequences followed by the sequences encoding the mature P30. The selectable gene which allows the isolation of the clones consists of the Saccharomyces cerevisiae URA3 gene. Of course, it is within the capability of a person skilled in the art to reclone the P30 gene, previously amplified by PCR, starting with the DNA extracted from Toxoplasma gondii, into appropriate plasmids for the production of new constructs. 2. Production of the P30 protein by Saccharomyces cerevisiae pTG3747 is introduced into a yeast strain of the Saccharomyces cerevisiae species, such as the TGY 73.4 strain of the MF&agr;, pra1, prb1, prc1, csp1, ura3, his3, pep4-3 genotype. The nontransformed strain develops in a rich medium, for example the YPG medium (1% yeast extract, 1% DIFCO bactopeptone, 2% glucose). After transformation with the vector pTG3747, the Ura&plus;prototrophs are selected on a minimum medium, for example the YNBG medium (containing 0.7% yeast nitrogen bases (Yeast Nitrogen Base DIFCO), 0.5% casamino acids and 1% of glucose) . The colonies selected are analyzed in the following manner. After 24 h of preculture in minimum medium, the cells are diluted so as to have an OD 600nm of about 1 and the culture is continued under the same conditions for about 72 h. Cells and supernatant are separated by centrifugation. An aliquot of culture supernatant is precipitated either with trichloroacetic acid (0.3M final) in the case of a recognition with an antibody of animal origin or with PEG-4000 (20% final) in the case of a recognition with an antibody of human origin. The precipitation pellet is taken up in a deposition buffer (about 1/200 of the starting volume) not comprising a reducing agent (-mercaptoethanol or DTT). The analysis is performed by Western blotting after an electrophoretic migration on a 12% SDS-PAGE gel, followed by a transfer onto a nitrocellulose membrane (0.45 &mgr;m, Schleicher Schüll). The latter is incubated with a positive anti-Toxoplasma rabbit polyclonal serum, a positive human antitoxo antiserum or the monoclonal antibody 1E1E7 which recognizes an epitope of the P30 protein (Fortier et al., 1991, Eur. J. Microbiol. Infect. Dis., 10, 38-40). The reaction is revealed by an anti-rabbit, anti-human or anti-mouse conjugate labeled with alkaline phosphatase (Immunoresearch). When the culture supernatants of the TGY 73.4 strain are analyzed, a recombinant P30 protein is detected which exists in the form of a heterogeneous material, probably because of the proteolytic cleavages by the KEX2 enzyme or a high heterogeneity in the glycosylation, but which is nevertheless recognized in Western blotting by positive human anti-Toxoplasma antisera. 
 Example 2 
 Production of a P30 protein in Schizosaccharomyces pombe 1. Construction of the expression vector pTG8610 (constitutive expression in an auxotrophic strain) The plasmid pEVp11 (Russell and Nurse, 1986, Cell, 45, 145-153) contains the promoter region of the adh gene of Schizosaccharomyces pombe in the form of a 700 bp SphI-EcoRI fragment, the EcoRI site being situated in the 5′ region of the adh gene, 59 bp upstream of the initiator ATG. It is digested with the enzymes EcoRI and HindIII and a synthetic fragment resulting from the reassociation of the single-stranded oligonucleotides OTG2781 and OTG2782 (described in SEQ ID NO: 4 and NO: 5 respectively) is introduced with the aim of completing the adh promoter region up to 11 bp upstream of the initiator ATG and of creating appropriate restriction sites which facilitate the subsequent cloning steps. pTG1702 is generated. A 0.92 kb HpaI-ClaI fragment is isolated from pCVH3 (Van Huffel et al., 1992, Eur J. Biochem., 205, 33-43) and then treated with the Klenow fragment of DNA polymerase. This fragment comprises the last codons of the Schizosaccharomyces pombe arg3 gene, followed by the sequence for termination of transcription. It is inserted into pTG1702 digested with HindIII and whose ends have been made blunt by treatment with the Klenow fragment of DNA polymerase, in order to give pTG1746. pTG1746 is digested with XbaI and treated with the Klenow fragment of DNA polymerase before being digested with BamHI. The fragment comprising the sequences for termination of transcription is introduced into the vector pDW230 digested with EcoRI, subjected to a treatment with the Klenow fragment of DNA polymerase and then digested with BamHI. pTG1751 is obtained. The vector pDW230 is similar to pDW232 which is described in the literature (Weilguny et al., 1991, Gene, 99, 47-54). They are both derived from pGEM3 into which a replication origin which is functional in Schizosaccharomyces pombe (ars1 origin) as well as the Schizosaccharomyces pombe ura4 gene has been inserted as selectable marker, the only difference being that the introduction of the fragment carrying the ars1 origin at the level of the NaeI site of pGEM3, giving rise to pDW230, caused a deletion of this site up to the base 2862. The vector pTG1754 was generated by ligation of the SphI-BamHI fragments of pTG1751 (comprising the sequence for termination of transcription arg3) and of pTG1702 (comprising the adh promoter). In parallel, the sequences encoding P30, provided with its own secretory signal, are obtained in the following manner: the P30 gene is modified by mutagenesis, so as to generate a consensus initiation sequence for yeast at the level of the second initiator ATG (TAAAAA ATG TCT) and a stop codon at the same position as above. The PCR uses the vector pbM89 as template and the oligonucleotides 722 (SEQ ID NO: 6) and 723 (SEQ ID NO: 7) carrying a BglII site at their 5′ end. The BglII-treated amplification product is cloned into the vector pTG2886 linearized with the enzyme BglII. This plasmid is described in European Publication EPA 396 436.1. The recombinant clones are analyzed by HindIII-BamHI enzymatic digestion in order to determine the orientation of the insert relative to the promoter. A clone having the appropriate orientation is designated pTG2886-P30. After digestion with BglII, the fragment containing the sequences encoding the P30 protein, equipped with its own secretory signal, is inserted into the vector pTG1754 previously digested with BamHI. pTG 3733 is obtained. The sequence encoding the natural secretory signal of the P30 protein (residues &plus;1 to &plus;30 of SEQ ID NO: 1) is replaced by the sequence of the Schizosaccharomyces pombe pho1 gene encoding the phosphatase secretory signal. For this, the vector pTG2886-P30 is digested with BamHI and a synthetic fragment having the same protruding ends, carrying an internal BglII site in 5′ and resulting from the reassociation of the oligonucleotides OTG4096 and OTG4097 (SEQ ID NO: 8 and 9), is introduced. The BglII fragment carrying the sequence encoding the pho1secretory signal followed by the mature P30 (residues &plus;31 to &plus;299 of SEQ ID NO: 1) is isolated from the vector obtained in the preceding step. It is inserted into the BamHI site of the vector pTG1754. The transformants having the correct orientation relative to the adh promoter are selected by EcoRI-BamHI digestion. pTG8610 is generated. 2. Construction of the expression vector pTG8630 encoding a nonglycosylated P30 protein (constitutive expression in an auxotrophic strain) The BamHI-SmaI fragment, containing the P30 sequences, is isolated from pTG8610 and introduced between the same sites of M13TG130 (Kieny et al., 1983, Gene, 26, 91-99) in order to mutate the glycosylation site. M13TG8620 is generated. The mutagenesis is carried out with the aid of a commercial kit according to the recommendations of the supplier (for example Amersham) and using the oligonucleotide OTG5829 (SEQ ID NO: 10). The aim of the mutagenesis is to substitute the asparagine residue in position 241 by a glutamine, while creating an HindIII site, thereby facilitating the selection of the mutants. M13TG8622 is obtained. The expression vector pTG8630 is prepared by inserting, between the SphI-PstI sites of the vector pTG8610, a first SphI-DraII fragment obtained from pTG8610 and a second DraII-PstI fragment purified from M13TG8622. 3 . Construction of the expression vector pTG8644 (thiamine-regulable expression in an auxotrophic strain) A synthetic DNA fragment derived from the reassociation of the oligonucleotides OTG2872 and OTG2873 (SEQ ID NO: 11 and NO: 12) was inserted between the BamHI and SacI sites of the vector pTG1702 (Example 2.1). pTG1716 is generated which comprises the sequence encoding the Schizosaccharomyces pombe major acid phosphatase (pho1) secretory signal downstream of the adh promoter region. pTG1716 is modified by inserting, between the MluI and HindIII sites, a DNA sequence encoding the Lys47 variant of hirudin (HV2 Lys47). pTG1722 is obtained. The SphI-SacI fragment comprising the adh promoter region, followed by the sequence encoding the pho1 secretory signal and HV2 Lys47 is isolated from pTG1722. It is then subcloned between the same sites of pTG1751. pTG1757 is obtained therefrom. Moreover, the promoter region of the Schizosaccharomyces pombe pho4 gene is obtained by PCR from the clone pSp4B (Yang and Schweingruber, 1990, Current Genet., 18, 269-272) and with the aid of the primers OTG3569, comprising an SphI site (SEQ ID NO: 13) and OTG3239 equipped with a BamHI site (SEQ ID NO: 14). The SphI-BamHI fragment thus generated is substituted for the SphI-BamHI fragment carrying the adh promoter region of pTG1757 in order to give pTG2734. A 642 bp fragment containing the 5′ flanking seqences of the Schizosaccharomyces pombe pho4 gene, which are situated upstream of the TATA box, is isolated by PCR from the vector pTG2734. The primers OTG3569 (SEQ ID NO: 13) and OTG3210 (SEQ ID NO: 15) are used. The SphI-NcoI PCR fragment thus obtained is introduced into pTG1757 digested with the same enzymes in order to give pTG2735. Finally, the EcoRI fragment isolated from pTG8610 and containing the sequence encoding the P30 protein followed by the arg3 terminator is introduced into the vector pTG2735 digested with EcoRI. The vector pTG8644 is obtained in which the sequences encoding the secretory signal pho1 and the P30 protein (residues &plus;31 to &plus;299 of SEQ ID NO: 1) are under the control of a hybrid promoter consisting of the sequences of the pho4 gene which are responsible for the regulation by thiamine, placed upstream of the TATA box of the adh gene. 4. Construction of the expression vector pTG8638 (constitutive expression in a wild-type strain). The aim is to generate expression vectors such as those of Examples 2.1, 2.2 or 2.3 containing, in addition, a gene allowing selection in a wild-type strain not exhibiting auxotrophy, for example a gene for resistance to an antibiotic, such as the neo (Neomycin) gene which confers resistance to G418. The neo gene is derived from the vector Tn5 (Berck et al., 1982, Gene, 19, 327-336). The latter is modified by conventional mutagenesis techniques in order to create restriction sites which facilitate subsequent cloning steps, such as a BamHI site at the 5′ (in position 138) and 3′ (in position 1280) ends of the neo gene. The BamHI fragment is treated with the Klenow DNA polymerase before being introduced into the EcoRI site of pDW230 whose ends have been made blunt by treating with Klenow. pTG1796 is obtained. The neo gene is placed under the control of the CMV (cytomegalovirus) IE1 promoter extending from nucleotides −727 to &plus;78 (Boshart et al., 1985, Cell, 41, 521-530). A BamHI site is introduced in 5′ of the promoter and a HindIII site in 3′. The BamHI-HindIII fragment, treated with Klenow DNA polymerase, is inserted upstream of the neo gene into the BamHI site made blunt by treating pTG1796 with Klenow. pTG3777 is generated into which it will be possible to introduce the cassettes for the expression of the P30 protein which are described above. The SphI-NdeI fragment containing the expression cassette “adh promoter-P30-term arg3” is isolated from pTG8610 and then treated with T4 DNA polymerase. It is cloned into the HindIII site, treated with T4 DNA polymerase, of pTG3777in order to give the vector pTG8638. 5. Production of the P30 protein in an auxotrophic Schizosaccharomyces pombe strain. The expression vectors of Examples 2.1, 2.2 and 2.3 are introduced into a mutant Schizosaccharomyces pombe strain, for example the D18 strain which is auxotrophic for uracil and which is available at the AFRC under the reference 2036. The same strain transformed in parallel with the vector pTG1754 is used as negative control. Of course, any other strain having this type of auxotrophy may be suitable. The strains for which the expression of P30 is constitutive, namely those transformed by the vectors pTG8610 and pTG8630, are cultured at 30° C. in a Kappeli synthetic medium optimized for yeast (Fiechter et al., 1981, Adv., Microbiol. Physiol. 22, 123-183) and supplemented with 2% glucose and a mixture of vitamins. The strains transformed by the vector pTG8644 are precultured in the same supplemented Kappeli medium. However, the mixture of vitamins comprises especially thiamine at a final concentration of 0.002 g/l (thi &plus; medium). When the cultures reach an OD (optical density) at 600 nm of between 1 and 2, they are diluted to an OD of about 0.05, either in thi &plus; medium or in a Kappeli medium supplemented with 2% glucose and a mixture of vitamins free of thiamine (thi − medium). The culture is continued at 30° C. up to the end of the exponential phase. Aliquots of each of the cultures are collected regularly during the exponential phase as well as at the end of the growth phase. The culture supernatants are analyzed by Western blotting, as described in Example 1.2. The yeasts transformed by pTG8610 or pTG3733 synthesize and secrete a recombinant P30 which is recognized by any of the three antibodies mentioned above (rabbit antiserum, human antiserum, which are toxopositive, and 1E1E7 antibody). The recombinant P30 is provided in the form of a homogeneous material with a slightly diffused appearance, with an apparent molecular mass (AMM) of about 35 kDa. A more intense signal is observed in the culture supernatant of pTG8610, which indicates that the efficiency of the pho1 secretory signal is greater for the secretion of the P30 protein in Schizosaccharomyces pombe than that of the natural P30 signal. It can be noted that, under reducing conditions (the loading buffer contains a reducing agent), the antibodies no longer recognize the recombinant P30, indicating that only conformational epitopes are involved in the recognition in Western blotting, as is observed with the native P30. Likewise, a production of recombinant P30 protein is detected in the culture supernatants of Schizosaccharomyces pombe transformed by pTG8644, this occurring when the culture is carried out in the absence of thiamine. It is revealed by the human antisera and has an AMM of 35 kDa. On the other hand, no product is visualized by these same P30 protein-specific antibodies when the culture is carried out in the presence of thiamine. The analysis of the supernatants of yeasts transformed by pTG8630, carried out using a toxo-positive rabbit antiserum, reveals a predominant band of about 28 kDa under nonreducing conditions, that is to say about 5 kDa smaller than that observed for the product of expression of pTG8610 or pTG8644. This difference in molecular mass is explained by the fact that the protein is a nonglycosylable protein since it is mutated at the level of the N-glycosylation site. 6. Production of the P30 protein in a wild-type Schizosaccharomyces pombe strain. Various wild-type strains of Schizosaccharomyces pombe are available at the AFRC. There may be mentioned, by way of example, the strains 20 286 and 26 760 in which the vector pTG8638 is transformed. The transformants are selected because of their resistance to G418 (concentration of 0.2 to 1 mg/ml). After culture in a selective liquid medium (YPG medium containing neomycin), the culture supernatants are analyzed by Western blotting with the aid of human antisera. A predominant band is detected at the position expected for the glycosylated recombinant P30. 7. Construction of pTG9643 (thiamine-regulable production of a nonglycosylated P30 protein). The EcoRI fragment isolated from pTG8630 (Example 2.2) and containing the sequence encoding the nonglycosylated P30 followed by the arg3 terminator was introduced into the vector pTG2735 (Example 2.3) digested with EcoRI. pTG9643 is generated. The fusion protein can be produced based on the same technology as that used in Example 2.5. 8. Construction of pTG8667 (thiamine-regulable production of a glycosylated P30 protein fused with a poly His tail at the C-terminus). The vector pTG8644 (Example 2.3) is digested with PstI and SmaI and a synthetic fragment is introduced which is derived from the reassociation of oligonucleotides OTG6438 and 6439 (SEQ ID NO: 16 and 17). pTG8667 is generated which comprises the pho4/adh hybrid promoter, the sequences encoding the pho1 secretory signal and the P30 protein followed by 6 histidine residues forming a poly His tail. The fusion protein can be produced based on the same technology as that used in Example 2.5. 9. Construction of pTG9618 (thiamine-regulable production of a nonglycosylated P30 protein fused with a poly His tail at the C-terminus). The BamH1-Pst1 fragment of pTG8667 (containing the glycosylated P30 sequence) is replaced by an equivalent fragment isolated from pTG8630 (nonglycosylated P30). pTG9618 is thus generated. The fusion protein can be produced based on the same technology as that used in Example 2.5. 10. Construction of pTG8221 (thiamine-regulable production of a nonglycosylated P30 protein fused with a poly His tail at the N-terminus). The plasmid pTG9643 is digested with BamH1 and a synthetic DNA fragment obtained from the reassociation of the oligonucleotides OTG10055 and 10094 (SEQ ID NO: 18 and 19) is cloned at the level of this site. pTG8221 is obtained in which there are 8 histidine residues preceding the P30 protein. 
 Example 3 
 Production of a P30 protein in a baculovirus The DNA fragment encoding the P30 protein equipped with its signal peptide is isolated from pTG2886-P30 after digestion with BglII and inserted into a baculovirus vector; for example the vector pACMP1, digested with BamHI (Hill-Perkins and Possee, 1990, J. Gen. Virol., 71, 971-976) or the vector described by Guaniro et al. (1986, J. Virol., 57, 563), in order to give pTG3729 and pTG3731 respectively. The recombinants are generated as described by J. M. VLAK et al. in Proceedings of the Baculovirus and Recombinant Protein Production Workshop, “Baculovirus and recombinant protein production processes”, March 29-April 1, Interlaken, Switzerland. The supernatants are centrifuged for 10 minutes at 1,000 rpm (revolutions per minute) and stored at −20° C. before being analyzed on 12% SDS-PAGE, followed by Western blotting and by radioimmunoprecipitation. For the latter technique, the cell culture is labeled with 300 &mgr;Ci of tran 35 S-label (trade name: ICN Biomedical France ref. 51006) at 1 hour 30 min or 26 hours after the infection. The supernatant is harvested 48 hours after the infection. The immunoprecipitation is performed according to the following procedure. The culture supernatants are centrifuged at 2,000 rpm for 20 min, diluted one half in 2x NET buffer (0.1% NP40, 1 mM EDTA, 50 mM Tris-HCl pH 8.0, 150 mM NaCl, 0.25% gelatin, 0.02% NaN 3 , 2% aprotinin, 2 mM PMSF) and then immunoprecipitated with either toxo-positive human sera or with 1E1E7 monoclonal antibody for 2 hours at 4° C., and the final addition of protein A Sepharose (Pharmacia). The precipitate is washed successively with the buffers 1x NET, Tris-NP40, 1x PBS (8mM Na 2 HPO 4 .12H 2 O, 2 mM KH 2 PO 4 , 150 mM NaCl) and 0.1x PBS. The analysis is carried out after electrophoretic migration on a 12% SDS-PAGE gel and autoradiography. The recombinant P30 is observed in the culture supernatant of the cells infected with the recombinant baculoviruses. When the culture supernatants are analyzed by Western blotting under nonreducing conditions, both the toxo-positive human sera and the rabbit antiserum recognize a doublet composed of a predominant band with an AMM of about 28 kDa and a minor band with an AMM of about 29 kDa. No significant difference is observed between the recombinant P30 proteins produced by the recombinant viruses. On the other hand, no product is detected under conditions reduced by DTT. 
 Example 4 
 Purification of the P30 protein produced in Schizosaccharomyces pombe 1) Purification by ion-exchange chromatography (column technique). The culture supernatants (volume 10 liters) obtained as described in Example 2 are reduced 20 to 50 times by concentrating on hollow fibers or on tangential ultrafiltration cassettes (final volume: 500 to 200 ml) (molecular weight cut-off 10,000 daltons), and dialyzed by this same operation in a 50 mM Tris buffer, pH 8.5 approximately without NaCl or an equivalent buffer, which will be used in the next step. The second step consists of a conventional ion-exchange chromatography, for example on a column of 100 ml of the gel DEAE TRISACRYL (DEAE: diethylaminoethyl) or Hyper D, (marketed by the company Biosepra) or DEAETSK (Merck) which makes it possible to recover, by a gradient with an ionic strength of 0 to 1M NaCl, the eluates which are tested on an SDS-PAGE gel and analyzed by Western blotting. The analysis makes it possible to reveal the recombinant P30 band of about 30 kD. In order to perfect the isolation of this recombinant protein, an affinity chromatography step can be performed on a chromatographic support covalently coupled to the anti-P30 1E1E7 monoclonal antibody. After specific elution by varying the pH (pH 2.5 or pH 11.5), the P30 protein peak obtained is of sufficient purity for this protein to be used in diagnostic kits. When the P30 protein is fused with a polyhistidine tail, at both the N- and C-termini, a metal chelate chromatography step may be included. The supports which are suitable for this technology are known to persons skilled in the art and are commercially available. There may be mentioned, as examples, chelatine Sepharose (Pharmacia) to be charged with metal ions and nickel chelate (NINTA Agarose, Quiagen). 
 Example 5 
 Reactivity towards antibodies. The reactivity of the recombinant P30 was tested against 151 known sera of which 95 were given as positive sera and 56 as negative sera, using the VIDAS apparatus (trademark) marketed by the company bioMérieux SA. 1) Reference test: VIDAS TOXO IgG The test automated on the Vidas system allows the quantitative measurement of anti-Toxoplasma IgGs in a human serum. The principle of the assay combines the immunoenzymatic method with a final fluorescence detection (ELFA). All the steps of the reactions are controlled by the instrument. Onto a disposable cone made of K resin (butadiene-styrene copolymer), which serves both as solid phase and as pipetting system, is adsorbed a preparation comprising the Toxoplasma gondii membrane and cytoplasmic antigens, this preparation being obtained by sonicating whole Toxoplasma cells. The other reagents necessary for the diagnostic test are pre-distributed into a cartridge combined with the cone. The human serum incubation phase allows the attachment of the anti-Toxoplasma IgGs if they are present; then anti-human IgG globulin (Boehringer ref: 1272 896) labeled with alkaline phosphatase (Boehringer ref; 556 602) is added in order to reveal the presence of these anti-Toxoplasma IgGs which may be present. The result or titre is expressed in IU/ml (International Units) relative to a calibration curve stored in the Vidas. The results are presented in the table below (see Vidas-Titers column). 2) Microplate test for the recombinant P30 protein The P30 (5 ng/well) to be tested is attached in the wells of a microtiter plate (Polylabo, ref: 13159). The same human sera which were tested on the VIDAS apparatus as described above were incubated for 1 hour at 37° C., after which a peroxidase-labeled anti-human IgG globulin (ref. 815462) was added in order to reveal the possible serum IgGs reacting against this P30. The results are given as optical density read at 492 nm and are presented in the table below (see REC P30 column). 1 Recombinant P30: microplate evaluation VIDAS REC. P30 VIDAS REC. P30 VIDAS REC. P30 TITERS OD TITERS OD TITERS OD AG 1 >300 * AG 51 67 1140 AG 21 0 180 AG 54 >300 1638 AG 67 58 1432 AG 28 0 116 AG 73 >300 * AG 44 49 1309 AG 38 0 90 AG 37 289 * AG 31 46 1088 AG 39 0 207 AG 70 268 1385 AG 29 41 966 AG 40 0 105 AG 59 243 2049 AG 20 35 1096 AG 46 0 340 AG 10 240 * AG 22 34 706 AG 47 0 317 AG 64 222 * AG 25 22 598 AG 56 0 391 AG 72 208 * AG 4 21 1129 AG 58 0 210 AG 34 191 1271 AG 42 21 609 AG 60 0 494 AG 35 191 1986 AG 45 20 708 AG 61 0 304 AG 23 189 1409 AG 43 15 677 AC 65 0 312 AG 32 182 1760 AG 50 14 1209 AC 66 0 214 AG 27 172 * AG 85 14 526 AC 67 0 251 AG 33 156 * AG 41 10 319 AC 68 0 170 AG 36 153 1090 AG 75 10 440 AC 74 0 119 AG 2 153 1578 AG 86 9 270 AC 76 0 335 AG 24 153 1435 AG 57 7 234 AC 78 0 433 AG 48 128 1718 AG 12 3 166 AC 79 0 250 AG 15 114 1770 AG 5 0 284 AC 81 0 189 AG 16 107 * AG 6 0 204 AC 82 0 202 AG 71 105 986 AG 11 0 376 AC 83 0 73 AG 9 104 1458 AG 14 0 160 AC 89 0 481 AG 77 70 1927 AG 17 0 550 AG 90 61 769 AG 18 0 98 AG 30 60 754 AG 19 0 433 VIDAS REC. P30 VIDAS REC. P30 VIDAS REC. P30 TITERS OD TITERS OD TITERS OD AG 1 >300 * AG 51 67 1140 AG 21 0 180 G 10 >300 * G 99 >300 * G 8 >300 * G 13 >300 * G 160 >300 * G 155 >300 2281 G 152 >300 * G 161 >300 * G 165 >300 * G 153 257 2435 G 162 >300 * G 164 >300 * G 169 246 * G 163 >300 * G 182 94 * G 154 228 2456 G 168 >300 * G 189 210 2365 G 170 222 * G 159 283 * G 200 168 * G 180 119 1981 G 197 187 * G 174 74 1542 G 195 99 2076 G 183 163 2280 G 119 72 1407 G 48 82 1477 G 178 145 * G 96 71 1336 G 158 82 1544 G 187 142 2169 G 127 26 944 G 115 27 1127 G 202 68 1702 G 121 24 722 G 81 17 692 G 157 69 1568 G 87 21 796 G 177 13 427 G 198 53 1532 G 88 20 765 G 172 12 871 G 104 39 2464 G 81 16 649 G 185 12 801 G 100 35 1212 G 176 16 455 G 143 5 476 G 126 28 1269 G 142 5 182 G 148 2 139 G 115 27 942 G 139 2 417 G 150 2 318 G 111 24 896 G 133 1 281 G 151 2 324 G 110 12 319 G 118 0 2787 G 136 1 141 G 173 10 517 G 97 0 304 G 146 1 155 G 132 4 264 G 204 0 279 G 93 0 175 G 145 2 409 G 112 0 173 G 141 2 281 G 138 0 121 G 144 1 390 G 147 0 131 G 131 1 172 G 134 0 82 G 105 0 125 *: means an optical density greater than 2400 (OD expressed as a thousandth) &equals; limit of the reader As can be seen on reading the preceding table, there is a very good correlation between the results obtained with the tests carried out on the VIDAS apparatus in the presence of Toxoplasma membrane and cytoplasmic antigens and those obtained in a conventional microplate assay in the presence of the recombinant P30 protein of the invention.