The animal model was established according to the Guidelines for the Laboratory Animal Use and Care Committee of the Ministry of Health, China, and the Ethics Committee on Animal Research of Xuzhou Medical University (No. SCXK < SU > 2014–0003). All serum samples from pregnant women were provided by Xuzhou Maternity and Child Health Care Hospital. Written informed consent was provided by each participant.
Preparation of rTgPrx
The recombinant plasmid pGEX-6P-1/TgPrx was constructed. The positive plasmid was transformed into E. coli BL21 and induced by isopropyl-β-D-thiogalactoside (IPTG) . Soluble rTgPrx was purified via glutathione S-transferase (GST) affinity chromatography and identified by Western blotting. PreScission Protease (GE Healthcare, U.S.A.) was used to cleave the GST tag from the rTgPrx fusion protein. The concentration of rTgPrx was measured using a BCA protein assay kit (Thermo Scientific, U.S.A.).
Parasite and animals
T. gondii tachyzoites (RH strain) were provided by Peking University Health Science Center (Beijing, China). Snails confirmed to be infected with Schistosoma japonicum cercariae were purchased from the Jiangsu Institute of Parasitic Diseases. Plasmodium berghei was passaged in the laboratory. Fish confirmed to be infected with Clonorchis sinensis metacercariae were donated by the Department of Parasitology, Sun Yat-Sen University. Six-week-old female BALB/c mice were purchased from Beijing Vital River Laboratory Animal Technology, and feeding in specific pathogens free environment. After collecting blood from the canthus, mice were anesthetized with isoflurane and then sacrificed by cervical dislocation.
Preparation of test serum
T. gondii tachyzoites were cultured in human foreskin fibroblasts (HFFs) . HFFs infected with tachyzoites were collected, centrifuged at 985 × g for 10 min and washed twice with phosphate-buffered saline (PBS). Each pellet was resuspended in an appropriate amount of PBS, sonicated, and centrifuged at 12 000 × g for 15 min at 4°C . The supernatant, which contained STAg from T. gondii, was collected, aliquoted, and stored at -80°C. The concentration of STAg was measured using a BCA protein assay kit.
Forty mice were subcutaneously injected with a mixture of STAg (20 μg per mouse) and an equivalent volume of Freund’s complete adjuvant (Sigma, U.S.A.). Two weeks later, a second immunization was performed with an emulsion of STAg in an equal amount of Freund’s incomplete adjuvant (Sigma, U.S.A.). One week later, a third immunization was performed with the same dose and method as the second immunization. One week after the final immunization, serum was collected and analyzed by ELISA. Negative control serum was prepared from twenty mice immunized with PBS.
Thirty mice randomly divided into 10 mice per group were used to prepare serum samples of C. sinensis, S. japonicum and P. berghei infection. Each mouse was gavaged with 45 metacercariae, and C. sinensis-positive serum was collected one month later . S. japonicum-positive serum was prepared by infecting mice (40 cercariae per mouse) percutaneously in a shaved region of the abdomen . Mice were intraperitoneally inoculated with 1×106 P. berghei parasites . Tail vein blood smears were prepared and stained with Giemsa. Serum was collected when the percentage of infected red blood cells exceeded 50%. All serum samples were maintained at -20°C for later use.
Detection of serum by Dot-IGSS assay
Preparation of a colloidal gold-labeled secondary antibody
Colloidal gold particles (5 nm) were prepared by the tannic acid-trisodium citrate mixed reduction method . Solution A (2.5 mL of 1% HAuCl and 197.5 mL of ddH2O) and solution B (10 mL of 1% sodium citrate, 1.75 mL of 1% citric acid, 0.5 mL of 0.1 M K2CO3, and 37.5 mL of ddH2O) were prepared separately and preheated to 60°C with magnetic stirring. Solution B was quickly poured into solution A, and the mixture was boiled for 5 min after it turned dark red. The pH of the colloidal gold solution was adjusted to 9.0 using 0.1 mol/L K2CO3. Then, 0.2 mL of goat anti-mouse or goat anti-human IgG (2 mg/mL)（Zhongshan Gold Bridge, China） was added to 40 mL of the above solution, and the mixture was stirred continuously for 20 min. Then, 4 mL of 10% bovine serum albumin (BSA) was added, and the mixture was stirred for 20 min. The supernatant was collected after centrifugation at 1500 × g for 30 min. The precipitate was collected after centrifugation at 12 000 × g for 60 min and dissolved in 4 mL of TBS buffer (10 mL of 1 M Tris-HCl [pH 7.5], 8.8 g of NaCl, and 1 L of ddH2O]. The colloidal gold- labeled secondary antibody was stored at -20°C.
Pieces of nitrocellulose (NC) membrane were placed in separate wells of a 96-well plate . rTgPrx (1 mg/mL, 1 μL) was added to the NC membranes, and the membranes were allowed to air dry. At the beginning of the experiment, the type of blocking solution, the blocking time, the dilution ratios of mouse serum and the colloidal gold-labeled secondary antibody were optimized(Additional files 1: Fig. S1, S2). Determined after exploration, the dried NC membranes were blocked in TBS containing 1% BSA and 10% goat serum at 37°C for 30 min. Diluted serum (mouse, 1:200; human, 1:100) was added to the sample wells and incubated at 37°C for 1.5 h. The serum was removed from each well, and the wells were washed three times with TBS for 5 min each. Diluted secondary antibody solution (1:20) was added to each well, and the plate was incubated at 37°C for 1.5 h. The secondary antibody solution was removed from each well, and the plate was washed sequentially with TBS, deionized water and distilled water. Silver nitrate solution was added to each well, and the plate was incubated in the dark for 7 min. The reaction was terminated by rinsing with ionized water, and the membranes were air dried. Brownish gray or brownish yellow spots on the NC membranes indicated positive serum. The negative control serum and blank control were also tested via the above method.
Assessment of the sensitivity, specificity, and reproducibility of the Dot-IGSS assay
Serum samples from mice and humans were simultaneously analyzed by Dot-IGSS with rTgPrx as the antigen (rTgPrx-Dot-IGSS), ELISA using rTgPrx as the antigen (rTgPrx-ELISA), Western blotting and a commercial ELISA kit for sensitivity analysis. Serum samples from mice infected with C. sinensis, S. japonicum and P. berghei were used for specificity analysis. Each serum sample was tested three times for repeatability analysis.
Diagnosis by ELISA with serum
Serum samples from mice and humans were analyzed with a commercial ELISA kit for the anti-Toxoplasma IgG antibody (Haitai Biotech, China) following the manufacturer’s protocol. In brief, diluted serum (1:100, 100 μL per well) was added to the wells and incubated at 37°C for 30 min. After washing, an enzyme-labeled antibody (50 μL per well) was added, and the plate was incubated at 37°C for 30 min. The colored substrate solution was added, and the plate was incubated at 37°C for 15 min in the dark. The reaction was then stopped, and the absorbance at 450 nm (A450 nm) was measured with a microplate reader(ASYS-Hitech GmbH, U.S.A.).
Serum samples from mice and humans were analyzed by rTgPrx-ELISA. A 96-well microtiter plate was coated with rTgPrx (1 μg/well) and incubated at 4°C for 12 h. After blocking at 37°C for 1 h, diluted serum (mouse, 1:200; human, 1:100) was added to the wells, and the plate was incubated at 37°C for 12 h. Unbound serum antibody was then washed away, and the samples were incubated with horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (diluted 1:5000) or goat anti-human IgG (diluted 1:2500) at 37°C for 2 h. O-phenylenediamine was the substrate solution; after it was added, the plate was protected from light for 15 min. The reaction was stopped, and the absorbance at 492 nm (A492 nm) was measured within 20 min. Each sample was independently analyzed three times, and the average value was calculated .
rTgPrx (20 μg) was separated via sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on 12% gels and transferred to polyvinylidene difluoride membranes. Membranes were incubated with mouse serum or human pregnancy serum (diluted 1:100) at 4°C overnight and were then probed with an HRP-conjugated goat anti-mouse or anti-human IgG antibody (diluted 1:5000, Zhongshan Gold Bridge, China ).
Data were analyzed using SPSS software. The positive rates were compared between groups with corrected chi-square tests or Fisher’s exact test. A significance level (α) of 0.05 was selected, and P<0.05 was considered to indicate statistical significance.