T.gondii RH strain was used for the extraction of genomic DNA. Tachyzoites were injected into the peritoneal cavity of Swiss mice. Three days later, they were harvested from peritoneal fluid, washed with phosphate-buffered saline (PBS) and stored at -70 °C.

The E.coli DH5α strain (Invitrogen) was used for cloning and E.coli BL21 (DE3), BL21 (DE3) plysS and Rosetta (DE3) strains (Promega) were used for expression of the recombinant antigen. The pET-28b(+) (Novagen) plasmid was utilized for the construction of the expression system.

A total number of 68 serum samples from pregnant women, each sample corresponding to a woman (provided from three medical diagnostic laboratories in Tehran, Iran), were divided into 2 groups according to the results of the standard toxoplasma-specific IgG, IgM, and IgG avidity ELISA (Euroimmun, Lubeck, Germany). Group I (acute-phase sera) consisted of 33 serum samples collected from pregnant women with clinical signs of suspected acute toxoplasmosis. They were IgG and IgM positive and have low avidity of toxoplasma-specific IgG antibodies. Group II (chronic sera) were from 35 women with the positive IgG and negative IgM response.

Genomic DNA was extracted from the tachyzoites of T.gondii RH strain using the DNA extraction kit (Bioneer) and used as the template for the PCR amplification. A truncated fragment of gra8, encoding amino acid 23 to 169, was PCR amplified using a pair of specific primers (Forward primer: 5′-GCG GAT CCC GCC ATG AAC GGT CCT TTG AGT TAT-3′ and Reverse primer: 5′-GCC TCG AGA GCT GTC GTG GTT GTC GTA ACG TAG G-3′) designed based on the published sequence of gra8 on GenBank database (AF150729). Forward and reverse primers were designed to contain the BamHI and XhoI restriction sequences, respectively. PCR was performed as follows: 94 °C for 5 min, followed by 35 cycles at 94 °C for 1 min, 57°C (+ 0.1 °C increment per cycle) for 1 min, and 72 °C for 50 sec. The final extension was 7 min at 72 °C.

The PCR product was digested with BamHI and XhoI and inserted into the pET-28b(+) expression vector digested with the same enzymes. The resulting recombinant plasmid, containing the truncated sequence of gra8, was named pET-28b(+)-GRA8. The presence of the gra8 fragment in the recombinant plasmid was investigated by the EcoRV restriction enzyme digestion and DNA sequencing determination.

To obtain the recombinant GRA8, E.coli BL21(DE3), BL21(DE3)plysS and Rosetta (DE3) strains harboring the pET-28b(+)-GRA8 plasmid was cultivated in Luria Bertani (LB), 2XTY and Terrific broth (TB) media supplemented with 25 μg/ml kanamycin and 35 μg/ml chloramphenicol, where appropriate, at 37°C with shaking at 200 rpm. Expression of GRA8 was induced at OD₆₀₀ of 0.6 by addition of various concentration of Isopropyl-β-D-Thiogalactopyranoside (IPTG) ranged from 0.1-1 mM. Following incubation at 30°C for an additional 5 hr, the induced bacteria were harvested by centrifugation at 6000 × g for 15 min. The expression of GRA8 was analyzed by 12% SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE).

To assess the solubility of GRA8, the pellet of 1 ml of induced bacteria was incubated in 100 μl of the lysis buffer (500 mM NaCl, 50 mM Tris-Cl, pH = 9.25, 1 mg/ml lysozyme) at 4°C for 30 min. The cells were lyzed by gentle vortexing and centrifuged at 16000 × g for 15 min at 4°C to separate the insoluble proteins and cell debris from the soluble ones. The pellet and supernatant were analyzed by SDS-PAGE.

The purification of GRA8 was performed by IMAC using HisTrap FF columns (5 ml) (GE Healthcare) and the Bio-Rad FPLC instrument. The induced cells from 200 ml culture were collected by centrifugation and the resulting pellet was resuspended in 15 ml of binding buffer (500 mM NaCl, 50 mM Tris-Cl, 5 mM imidazole). The purification process performed at four pH values of binding buffer: 7.5, 8.1, 8.75 and 9.25. The cells suspension was sonicated for 6 min bursts at 200-300 W using an MSE ultrasonic disintegrator (Fisons). The cell lysate was subsequently centrifuged at 12000 × g at 4°C for 20 min. The supernatant was passed through a 0.2 μm (pore size) filter. The cleared lysate was applied to the column previously equilibrated with the binding buffer at a flow rate of 1 ml/min. The column was then washed step-wised with 10 column volumes of binding buffer until the absorbance at 280 nm of the effluent was minimal. Afterwards, the column was washed step-wised with 25 ml, 20 ml and 15 ml of the binding buffer containing 35, 60 and 90 mM of imidazole at 4 ml/min. Absorbed materiel was eluted at 1 ml/min with binding buffer containing 500 mM imidazole. The purified samples were analyzed on SDS-PAGE stained with Coomassie Blue G-250 as well as silver nitrate.

One rabbit was immunized i.m with 100 μg of the purified GRA8 emulsified in Freund's complete adjuvant. Injection was repeated twice with two weeks intervals using Freund's incomplete adjuvant. The serum sample was collected two weeks after the last immunization. The serum obtained from another rabbit injected with the adjuvant alone was taken as the control accordingly.

To assess the immunogenicity of GRA8, Western blotting was performed using sera of the immunized and non-immunized rabbits. About 5×10⁶ tachyzoites of the RH strain were loaded onto SDS-PAGE. The antigens then transferred onto the nitrocellulose membrane, blocked with 2% Bovine Serum Albumin (BSA) in PBS containing 0.1% Tween 20 (PBS-T) and probed with rabbit sera diluted 1:500 in blocking buffer. The membrane was washed three times with PBS-T and incubated with Horseradish Peroxidase (HRP)-conjugated goat anti-rabbit IgG antibody (Ray Bio-tech) diluted 1:500 in blocking buffer. Finally, the protein band was revealed by the peroxidase activity on 3, 3′-diaminobenzidine tetrahydrocholoride (DAB) substrate (Sigma) as the chromogenic substrate.

Furthermore, to assess the immunoreactivity of GRA8, Western blotting was performed using pooled sera from ten pregnant women with acute toxoplasma infection and ten women with chronic infection. The purified GRA8 antigen was analyzed by SDS-PAGE and transferred onto the nitrocellulose membrane. The membrane was blocked, as explained above, and probed with acute and chronic pooled sera diluted 1:100 in blocking buffer. Bound antibodies were detected using HRP-conjugated goat anti-human IgG and IgM antibodies (Sigma) diluted 1:2000 and 1:1000 in blocking buffer, respectively.

Maxisorp microtiter ELISA plates (Maxisorp, Nunc, Roskilde, Denmark) were coated overnight at 4°C with 4 µg/ml of GRA8 in 50 mM carbonate buffer (pH = 9.6). The plates were then washed three times with PBS-T. To avoid the nonspecific binding, the plates were blocked with PBS-T containing 1% bovine serum albumin (BSA) at 37°C for 1 hr. One hundred µl of the sample sera diluted 1:100 in blocking buffer were added to the plates. The plates were then incubated at 37°C for 1 hr and washed three times with PBS-T. One hundred µl of HRP conjugated goat anti-human IgM antibody (Sigma)-diluted 1:10000 in blocking buffer-was added to each well. The plates were incubated at 37°C for 30 min and washed 3 times. One hundred µl of tetra-methylene benzidine dihydrochloride (Kem-En-Tec) was then added as the substrate. After 15 min of incubation at room temperature, the reaction was stopped by addition of 100 µl of 1 M HCL. The optical density was measured at 450 nm by a BioTek ELISA reader (BioTek Instruments, Highland Park, VT) with a reference wave length of 630 nm. Each serum sample was screened in duplicate wells.

The result for each serum sample was determined by taking the mean value of the absorbency readings for the duplicate wells. A sample was considered positive if the final value was equal to or higher than the defined cutoff.

The gra8 gene fragment corresponding to amino acids 23 to 169 was PCR amplified yielding a single specific product of 458 bp. The PCR product was cloned into the pET-28b(+) vector. The presence of gra8 gene fragment in the recombinant plasmid was suggested by restriction digestion using EcoRV enzyme. Sequence analysis of the correct clone revealed 100% homology with the published sequence of gra8 (data not shown). In the pET-28b(+)-GRA8 construct, the sequences encoding the amino terminal signal peptide (amino acids 1 to 23) and the C-terminal transmembrane domain were excluded. Moreover, a short sequence encoding 35 aa including a T7 tag and a cluster of hexa-histidine residues were fused into the N-terminus of the gene. A second hexa-histidine tag was placed in C-terminal fusion of the target gene to further facilitate the subsequent purification of the recombinant protein. SDS-PAGE analysis of induced bacteria showed expression of a protein band of 27 kDa, which was absent in uninduced bacteria (Figure 1). The discrepancy observed between the calculated molecular weight of GRA8 (20.2 kDa with 189 amino acids) and the apparent band on SDS-PAGE is a common feature of GRA proteins (31). High proline content of GRA8 (53 of 189 residues) also likely contributes to the aberrant electrophoretic migration (21).

To optimize the bacterial production of GRA8, the expression was carried out in three E.coli expression hosts: BL21(DE3), BL21 (DE3)plysS and Rosetta(DE3) (Figure 1A) and three culture media: LB, 2XTY and TB (Figure 1B). The expression level of GRA8 seemed to be equal in the three bacterial hosts, however, the highest expression level of GRA8 was achieved in E.coli cells cultured in LB medium (Figure 1). To evaluate the solubility of GRA8, the recombinant Rosetta (DE3) cultivated in different medium were lyzed and fractionated into the soluble and insoluble proteins. Figure 1B showed that most of GRA8 in induced bacteria is in insoluble form, however, densitometry analysis using ImageJ software showed soluble expression of GRA8 was enhanced by cultivating in LB medium, as compared to the two other culture media (data not shown).

The soluble fraction of G RA8 was purified using IMAC. In the preliminary experiments, we observed marked variations in the purity and yield of GRA8 at different pH values of binding buffer. Therefore, IMAC was performed at four pH value of 7.5, 8.1, 8.75 and 9.25 as explained in the Materials and Methods. SDS-PAGE analysis of protein samples showed that the purity of GRA8 was improved when pH of binding buffer was increased from 7.5 to 9.25, and the highest purity of 97.97% was achieved at pH value of 9.25 (Figures 2A and 2B). Similarly, yield of GRA8 was increased from 10 mg/l to 13.6 mg/ ml when pH reached to 9.25 (Figure 2A).

The improved purity and yield of GRA8 at higher pH values was also evident from the corresponding chromatograms (Figure 2A). The peak of some impurities seen at pH = 7.5 and 8.1 was disappeared or weakened at highe r pH. In addition, the area under the peak of GRA8, representing the yield of protein, was apparently higher at pH = 8.75 and 9.25, compared to the lower pH values. Moreover, the bacterial impurities co-purified with GRA8 were also decreased from pH = 7.5 to pH = 9.25 (Figure 2B). The same results were obtained when Ni-NTA agarose resin was used for batch purification of GRA8 (data not shown).

The silver nitrate staining of the GRA8 purified at pH = 9.25 showed that protein was purified near to homogeneity (Figure 2C). The higher band revealed by silver nitrate staining, about 54 kDa, might be attributed to the dimmerization of GRA8. In fact, oligo-merisation of GRA proteins has been suggested in the previous studies (32–34).

Antigenicity of GRA8 was investigated in Western blotting using pooled human sera representing the acute and chronic infection. The purified antigen strongly reacted with IgG and IgM antibodies in acute sera. No specific band, however, was observed on the blot probed with chronic sera (Figure 3A). The results demonstrated the antigenicity of GRA8 and its superior reactivity towards acute sera in comparison to chronic ones.

Moreover, the immunoreactivity of GRA8 was confirmed by Western blotting using the serum sample from a GRA8-immunized rabbit. On the blot of RH tachyzoite probed with anti-GRA8 serum, a single protein band of 35 kDa, the same size of the native GRA8, was detected. In contrast, there was no specific band on the blot of tachyzoites probed with the serum from a rabbit injected with the adjuvant alone (Figure 3B).

A total number of 68 sera from pregnant women with acute and chronic infection were included in this study. The sera were examined individually by GRA8-IgM-ELISA. A cut-off value (0.31) was determined on the basis of the mean value plus two standard deviations of the OD values of chronic sera. One (2.8%) of 35 chronic serum samples scored above the cut-off (No. 19, OD: 0.36), resulting in a test specificity of 97.1% (Table 1). Twenty of 33 acute serum samples had OD values equal to or higher than the cut-off, leading to a sensitivity of 60.6% for the assay (Table 1). These results indicated that IgM ELISA using the purified GRA8 was useful in discriminating acute from chronic infection.