Reagents and instruments
Bovine serum albumin (BSA) and Tween-20 were obtained from Solarbio (Beijing, China). EDC was obtained from TCI (Shanghai, China). COOH-modified europium nanoparticles (EuNPs) were obtained from Thermo (USA). Mouse anti-pig monoclonal IgG antibodies and rabbit anti-goat IgG antibodies were obtained from Beijing Biolab Technology (Beijing, China). Goat anti-rabbit IgG was obtained from Cell Signal Technology (USA). NC membranes (Millipore 135) and Ultra-15 3 kDa centrifugal filters were obtained from Millipore (USA). The sample pads, absorbent paper and plastic backing were obtained from Jinbiao Biotech (Shanghai, China). BCA kits were obtained from Beyotime Biotechnology (Shanghai, China). The ES commercial ELISA kit was obtained from Qiagen (Germany).
XYZ3060 dispenser was obtained from Biodot (USA). A TRF fluorescence quantitative analyzer was obtained from Weice Biotech (Nanjing, China). A UV lamp was obtained from Shenzhen Feike Technology (Shenzhen, China).
Preparation of ES antigens
The collection of ML-ES antigens followed the previous methods and procedures [27, 28]. A total of 10 specific pathogen-free (SPF) SD rats were orally inoculated with 3500 T. spiralis ML (T1, ISS534) and were euthanized at 35 dpi, and tissues were used for the recovery of ML by the artificial digestion method. After washing three times with 0.9% saline solution, the ML were cultured in serum-free RPMI-1640 medium containing antibiotics (90 U/mL penicillin and 90 μg/mL streptomycin) at 37 °C for 18 h in 5% CO2. After separation by centrifugation at 1000 × g, the ML culture supernatant was concentrated by Ultra-15 3 kDa centrifugal filters and the protein concentration was measured by BCA kits.
The collection of PAW-ES antigens followed the previous methods and procedures [14]. After infection with 10 000 T. spiralis ML, the SD rats were euthanized at 6 h after infection. The entire small intestine was removed from the abdominal cavity and soaked in 0.9% saline solution at 37 °C for 2 h (with 180 U/mL penicillin and 180 μg/mL streptomycin). The culture and collection of PAW-ES antigens were performed as ML-ES antigens.
Finally, ML-ES antigens and PAW-ES antigens were analyzed by SDS-PAGE (Additional file 1).
Pigs and serum samples
The experiment of this part has been done by our laboratory before [27]. Fifteen Large White pigs were divided into three groups (five pigs per group) and inoculated with 100, 1000 and 10 000 T. spiralis ML (T1, ISS534). Serum samples were collected from pigs at 0, 7, 9, 11, 13, 15, 17, 19, 21, 25, 30, 35, 45, 60, 90 and 120 dpi. Finally, all pigs were sacrificed to calculate larvae per gram of muscle (lpg) at 120 dpi. These results were showed in supplement material, which indicated all pigs were successfully infected with T. spiralis ML (Additional file 4).
One hundred serum samples were collected from 25 Large White pigs infected with 400 T. spiralis ML at 25, 30, 35, 45 dpi. Another 170 serum samples were collected from 170 parasite-free Large White pigs as negative controls to calculate the cut-off value. A total of 38 serum samples were collected from pigs infected with Clonorchis sinensis (2), Cryptosporidium parvum (2), Taenia sodium (2), Toxoplasma gondii (2), Ascaris suum (15), Trichuris suis (5) and Metastrongylus elongatus (10). All pigs were healthy according to the Chinese Laboratory General Requirements for Animal Experiments. Before the experiment, all pigs underwent a week-long healthy observation and were examined for parasite eggs in feces and blood by the flotation and sedimentation method. Furthermore, all pigs were fed basic diet without adding antibiotics and were kept under standard pig houses in our laboratory under the care of a professional breeder.
Preparation of fluorescent probe
EuNPs were conjugated with ES antigens as follows [29]: firstly, 10 μL of EuNPs were centrifuged at 14 000×g for 15 min to remove glycerol and phosphate, and then 10 µL of 1 mg/mL EDC, 10 µL of 1 mg/mL NHS, 100 µL of MES and EuNPs were mixed and stirred for 45 min to activate the EuNPs completely at room temperature. After removing the unbound EDC and NHS by centrifugation at 14 000 × g for 15 min, 20 µg the ML-ES and 20 µg PAW-ES antigens were mixed into the solution, and incubated for 3 h. Then, 200 µL of 5% BSA was added to the mixtures to block the unreacted active sites overnight at 4 °C. Finally, the EuNPs-ML-ES and EuNPs-PAW-ES fluorescent probes were resuspended in 200 µL of preservation buffer (50 mm/L PBS containing 1% BSA and 1% ProClin). The EuNPs-goat anti-rabbit IgG fluorescent probe was also prepared by the same procedure described above.
To identify the change of fluorescent probe after coupling, the morphology of the fluorescent probe was observed by transmission electron microscopy (TEM) and the absorption peak of the fluorescent probe was detected by fluorescent microplate reader.
Preparation of the ICS
EuNPs-conjugated ML-ES or PAW-ES antigens were designed as probes to capture anti-T. spiralis antibodies, and conjugated goat anti-rabbit IgG antibodies were used as an indicator probe. Mouse anti-pig monoclonal IgG and rabbit anti-goat IgG were immobilized on the NC membrane as the test line (T-line) and control line (C-line), respectively. Using the XYZ3060 dispenser at a rate of 0.8 μL/cm, mouse anti-pig monoclonal IgG (1 mg/mL) and rabbit anti-goat IgG (1 mg/mL) were applied to the NC membrane as the T-line and C-line, respectively. Finally, NC membranes absorbed paper and sample pad were assembled into strips (3.8 mm-wide).
For detection, 100 µL the running buffer (0.9% NaCl, 1.5% BSA, 0.05% Tween-20), 1 µL serum sample, 1 µL EuNPs-ES and 0.5 µL EuNPs-goat anti-rabbit IgG were mixed in a bioclean tube. Then, after the sample was added into the ICS, the ICS was placed into a 37 °C incubator for 10 min. The fluorescent signal of the T-line was observed under a UV lamp at 365 nm wavelength and was analyzed by a TRF reader.
The cut-off value for the ICS and Qiagen ELISA
A total of 170 serum samples from 170 parasite-free pigs were detected by ICS, and the result of ICS were analyzed by a TRF reader. The cut-off value was calculated by \(\overline{{\text{x}}}\) ± 2 SD of the T-line fluorescence values of the 170 serum samples. A T-line values for a sample below the cut-off value was judged as negative, and a value above or equal to the cut-off value was judged as positive. The cut off value of Qiagen ELISA follow the manufacturer’s instructions: S/P values = (ODSample − ODNegative Control)/(ODPositive Control − ODNegative Control).
Cross-reactivity with other parasites
To evaluate the specificity of ICS, a total of 38 serum samples from pigs infected with Clonorchis sinensis, Cryptosporidium parvum, Taenia sodium, Toxoplasma gondii, Ascaris suum, Trichuris suis and Metastrongylus elongatus were detected by the EuNPs-ML-ES ICS and EuNPs-PAW-ES ICS.
Seroconversion of infected pigs detected by ICS and Qiagen ELISA
Serum samples from pigs infected with 100, 1000 and 10 000 ML (five animals per group) at 0, 7, 9, 11, 13, 15, 17, 19, 21, 25, 30, 35, 45, 60, 90 and 120 dpi were detected by EuNPs-ML-ES ICS, EuNPs-PAW-ES ICS and Qiagen ELISA. The test results for the middle- and high-dose ELISA groups have been published previously, and the results for the low-dose group are shown in Additional file 2 [30].
A standard indirect ELISA protocol was performed by Qiagen ELISA kit. Briefly, 90 µL of sample diluent and 10 µL pre-diluted serum were added into each sample well, then wells were incubated for 60 min at room temperature (18–25 °C). After removing solution by aspiration, wells were rinsed each well of Wash Buffer. 100 µL ready-to-use Conjugation was added into each well and incubated for 30 min at room temperature. After removing solution and wells were rinsed again. 100 µL TMB Substrate Solution was added into each well and incubated for 10 min at room temperature. Stop the reaction by adding 100 µL Stop Solution per well, and optical density (OD) values was measured by plate reader at 450 nm.
The sensitivity and specificity of EuNPs-ML-ES ICS and EuNPs-PAW-ES ICS
To investigate the sensitivity and specificity of ICS, 100 serum samples were collected from 25 Large White pigs infected with 400 T. spiralis ML at 25, 30, 35, 45 dpi, at the end of experiment, all pigs were confirmed to be infected with T. spiralis ML by artificial digestion method. 170 serum samples collected from 170 parasite-free pigs as negative, which also confirm by artificial digestion method. These serum samples were detected by EuNPs-ML-ES ICS and EuNPs-PAW-ES ICS.
Clinical application of ICS
To validate the method in the field in an endemic area where Trichinella circulates among the backyard pigs. We collected 1032 pork and 1032 serum samples from slaughterhouses and rural areas in Inner Mongolia, Sichuan and Guangxi provinces in China where T. spiralis infection were suspected. All pork samples were detected by artificial digestion method, and serum sample were detected by the two ICS.
Statistical analysis
After the results of ICS were analyzed by fluorescent reader, the T-line fluorescence values were imported into GraphPad Prism8 software. SPSS 19 was used for statistical analysis, Chi-square tests were performed on ICS with standard artificial digestion.