SAG1, SAG2, and SAG3 genes were cloned in pQE30, pGMEX1, and pGMEX1 vectors, respectively, and sequenced by Kazemi and Arabpour method [21,22,23]. pGEM43, pGEM22, and pQE30 containing P43, P22, and P30 genes were confirmed by PCR using Tox43, Tox22, and Tox30 primers [21,22,23]. PCR products were electrophoresed on 1.5% agarose gel, stained with ethidium bromide, and visualized under ultraviolet. PCR products with 1,158, 957, and 560 bp bands confirmed the insertion genes, respectively.

The recombinant plasmids pGEM43, pGEM22, and pQE30 were transformed into Escherichia coli JM109 competent cells using Hanahan method [24]. The reaction was spread on LB agar plate containing 50 µg/ml ampicillin.

A single colony was cultured in LB medium (Merck, Darmstadt, Germany) containing 100 µg/ml ampicillin and was incubated overnight at 37℃. It was then sub-cultured using 10-fold fresh LB medium containing ampicillin at 37℃. The cells were grown to OD₆₀₀=0.6 at 37℃ with 200 rpm shaking. The plasmid promoter was induced by 1 mM iso-propyl-D-thiogalactopyranoside (IPTG) for 5 hr and mass cultivated in LB medium. The bacterial cells were harvested by 20,000 g centrifugation for 15 min, and expressed recombinant P43 and P22 proteins were purified using T7-affinity chromatography (Novagen, Foster City, California, USA) according to the manufacturers' instruction with some modifications. The cell pellet was extracted from 25 ml of liquid culture and resuspended in 5 ml of equilibrating buffer with 10 mM of PMSF (Sigma, St. Louis, Missouri, USA) on ice for 2 hr. The suspension was then sonicated and centrifuged (700 g for 15 min at 4℃). The supernatant was transferred to a column containing 2 ml of equilibrated resin. The column was incubated overnight at 4℃ and then washed with 10 ml washing buffer (KH₂PO₄, Na₂HPO₄, KCl, NaCl, and Tween 20). Finally, elution buffer was added (citric acid, 1 mol/L) and incubated for 2 hr, the recombinant P43 and P22 proteins were eluted and neutralized with Tris-HCl 1 mol/L), and the protein concentration was measured by a biophotometer (Eppendorf, Hamburg, Germany). The P30 recombinant protein was purified using Ni-affinity chromatography (Novagen, Madison, Wisconsin, USA) according to the manufacturer's instruction with some modifications [25]. The recombinant proteins were analyzed by SDS-PAGE and western blotting as in previous studies [22,25,26].

Serum samples were collected from different laboratories around Shiraz and Tehran using commercial IgG and IgM ELISA kits (Euroimmun, Lubeck, Germany). For evaluation of recombinant IgG ELISA, 27 cases with infections other than toxoplasmosis, 30 sera from healthy individuals, and 110 anti-T. gondii IgG positive cases were used. The cut-off point was set at mean±2SD of 20 anti-Toxoplasma IgG negative serum samples. For determination of anti-T. gondii IgM, 30 negative and 28 positive samples were applied. The cut-off point was determined by calculating mean and SDs for 30 IgM and IgG negative serum samples.

Flat-bottomed 96-well microtiter plates (Greiner Bio One, Frickenhausen, Germany) were coated with 3 µg/ml of each of P22, P30, and P43 antigens in 50 mM carbonate bicarbonate buffer (Na₂Co₃: 15 mM, NaHCo₃: 35 mM) (pH=9.6) and incubated overnight at 4℃. The plate was washed 3 times with PBS-T. After blocking with 300 µl of 1% bovine serum albumin in PBS-T for 2 hr, the plates were washed as before and 100 µl of sera (1:200 dilution in PBS-T with 1% bovine serum albumin) was added to each well. The plates were incubated for 1.5 hr and washed as before.

The plates were incubated with 100 µl of peroxidase-conjugated goat anti-human IgG and IgM (1:20,000 and 1:10,000 dilutions, respectively) and were incubated for 1 hr, washed, and then 100 µl of TMB (Sigma) was added. After 15 min of incubation at room temperature, the reaction was stopped using 50 µl of 2N H₂SO₄ as a stopping solution. Finally, the optical density was measured by an ELISA reader (Sunrise-can, Mannedorf, Switzerland).

The sensitivity and specificity were calculated from TP/(TP+FN)×100, and TN/(TN+FP)×100, respectively. Positive predictive and negative predictive values were achieved from TP/(TP+FP) and TN/(TN+FN), respectively. Agreement was calculated as follows: (TP+TN)/(TP+TN+FP+FN)×100 (TP, TN, FP, and FN represent the number of true positive, true negative, false positive, false negative, respectively). All results were assessed by software program of the website URL (http://vassarstats.net/clin1.html).

The first set of samples consisting of 110 IgG positive, 30 IgG negative, and 27 non-relative serum samples were evaluated by SAG1 antigen. The cut-off point was 0.31. Out of 110 positive serum samples, 103 were found to be positive by recombinant SAG1. Four out of 30 healthy controls were found positive, and no cross reactivity was found in non-relative cases. The results of commercial and recombinant ELISA are summarized in Table 1. The assay showed sensitivity of 93.6% (95% CI, 86.8-97.1%) and specificity of 92.9% (95% CI, 82.1-97.7%) for recombinant SAG1 in comparison with commercial ELISA (Table 2).

The set of IgG positive and negative sera were tested by recombinant SAG2 in the ELISA system with the same protocol used for evaluating SAG1. The cut-off point was 0.13. As shown in Table 2, all the 110 positive sera were positive by this antigen, although 5 out of 30 healthy controls were found positive and 1 cross reactivity was found with diseases other than toxoplasmosis. As shown in Table 3, the sensitivity and specificity of this antigen were 100.0% and 89.4%, respectively and a high degree of agreement (96.9%) was observed. There was marked difference in the absorbance values between sera from IgG positive and IgG negative groups with rSAG2 ELISA in comparison with 2 other recombinant antigens (Fig. 1).

Using SAG3 in the ELISA system and the cut-off point of 0.28, 105 out of 110 positive sera were detected as positive, and 5 out of 30 health individuals were determined as positive. No cross reactivity was found with other diseases. According to the results, the sensitivity of 95.4% and the specificity of 91.2% were calculated for the assay. A fair agreement (94%) was found between this antigen and commercial ELISA.

Twenty-eight IgM positive and 30 IgM negative sera were tested in the ELISA system for detecting Toxoplasma-specific IgM. The cut-off (0.6) was determined by calculating mean and SD for 30 IgM and IgG negative serum samples. The assay showed a positive reaction in 11 out of 28 IgM positive and 6 out of 30 IgM negative samples (Fig. 2). Accordingly, the sensitivity and specificity of 39.3% (95% CI, 22.1-59.2%) and 80.0% (95% CI, 60.8-91.5%) were calculated for the assay. The sensitivity and specificity of IgM rec-ELISA and commercial ELISA are shown in Table 3.

The cut-off point of 0.31 was calculated for SAG2-ELISA. Positive reaction was noticed in 18 out of 28 IgM positive and 5 out of 30 IgM negative sera samples (Fig. 2). All 18 IgM positive and 5 IgM negative sera that were detected by SAG1 and SAG3 recombinant antigens were also detected by SAG2 recombinant antigen. Accordingly, the sensitivity of 64.3% (95% CI, 44.1-80.6%) and the specificity of 83.3% (95% CI, 64.5-93.6%) were measured for the test. The best agreement (74.1%) was observed between recombinant SAG2 and commercial ELISA (Table 3).

When the serum samples were tested by SAG3-ELISA, positive reactions were noticed in 5 out of 28 IgM positive and 7 out of 30 IgM negative sera samples (considering the cut-off point of 0.44). The sensitivity of 17.9% (95% CI, 6.7-37.5%) and the specificity of 76.7% (95% CI, 57.2-89.3%) were found for the assay (Table 3).