Reagents and materials
Sodium chloride (NaCl), Triton X-100, Tween 20, 2-amino-2-hydroxymethyl-propane-1,3-diol (Tris), sodium hydroxide (NaOH), glycine, bovine serum albumin (BSA), Coomassie blue dye solution, and 4-(2-hydroxyethyl) piperazine-1-ethanesulfonic acid (HEPES) were obtained from Beijing Solarbio Science & Technology Co., Ltd. (Beijing, China). Enhanced chemiluminescent (ECL) reagent was purchased from Thermo Fisher Scientific Inc. (Waltham, MA, USA). N-hydroxysulfosuccinimide sodium salt (sulfo-NHS), and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) were purchased from Aladdin Company (Shanghai, China). Sodium azide (NaN3) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Methanol and NaH2PO4·2H2O were purchased from Beijing Chemical Reagent Company (Beijing, China). Millipore Milli-Q water (> 18 MΩ cm) was used for all solutions. Horseradish peroxidase (HRP)-conjugated goat anti-swine IgG, goat anti-swine IgG, rabbit anti-goat IgG, and goat anti-rabbit IgG were purchased from Beijing Baiolaibo Technology Co., Ltd. (Beijing, China).
NH2-modified UCNPs (NaYF4: Yb3+, Er3+) were obtained from Shanghai Shunna Biotech Co. Ltd. (Shanghai, China). The excitation peak was 980 nm and that of emission was 545 nm. 0.45 μm polyvinylidene fluoride (PVDF) membrane was purchased from GE Healthcare (Chicago, IL, USA). Nitrocellulose membranes (Hi-Flow Plus HF135) and sample pads (SureWick glass fiber) were purchased from Millipore Corporation, and the absorbent pads and plastic backing were purchased from Shanghai Jieyi Biotechnology Co., Ltd. (Shanghai, China).
Instruments
A multifunctional imaging system was purchased from Analytik Jena GmbH (Upland, CA, USA). A XYZ3060 Biostrip Dispenser and a CM 4000 Guillotine Cutter were purchased from BioDot (Irvine, CA, USA). A vortex mixer was obtained from Tiangen Biochemical Technology (Beijing) Co., Ltd. (Beijing, China). An Allegra™ X-22 centrifuge was obtained from Beckman Coulter Inc. (Carlsbad, CA, USA). An ultrasonic water bath (power 20 kHz, max power 320 W) was obtained from Dongguan Kangshijie Ultrasonic Technology Co., Ltd. (Dongguan, China). A transmission electron microscope (TEM, H-7650) was purchased from Hitachi (Tokyo, Japan). A fluorescence spectrometer system was purchased from Beijing ZOLIX Instruments Co. (Beijing, China). A 980 nm optical laser (Max 4 W) was obtained from Ningbo Yuanming Laser Technology Co., Ltd. (Ningbo, China). An upconverting phosphor technology-based biosensor (UPT-based biosensor) was obtained from the Shanghai Institute of Optics and Fine Mechanics, Chinese Academy of Science (Shanghai, China).
Animals and parasites
Female Wistar rats weighing approximately 120 g were purchased from Norman Bethune University of Medical Science (NBUMS), China. Trichinella spiralis (ISS534) preserved in the Food-Borne Parasitology Laboratory of Key Laboratory for Zoonoses, Jilin University, was confirmed by the OIE Collaborating Center on Foodborne Parasites in the Asian Pacific Region and maintained by continuous passage infection in our laboratory.
Preparation and characterization of ES antigens from ML (ML-ES)
Some previously reported methods were used to prepare ES antigens [17, 28]. Briefly, ten Wistar rats were inoculated with 3000 T. spiralis ML per rat by the oral route. The infected rats were euthanized 35 days post-infection (dpi), and the ML were recovered from the muscle tissues of rats with artificial digestion fluid (1% pepsin/HCl) [29]. After washing three times with 0.01 M phosphate-buffered saline (PBS, pH 7.2), the ML were resuspended in RPMI-1640 medium (Gibco BRL, Grand Island, NY, USA) containing antibiotics (100 U/ml penicillin, 100 μg/ml streptomycin) at approximately 5000 worms/ml and then incubated at 37 °C in an atmosphere containing 5% CO2 for 18 h. The ML were removed by filtration (0.22 μm filter, Millipore, USA) to obtain the filtrate containing ES antigens. The filtrate was concentrated using a 3 kDa ultrafilter (Millipore, USA), and then PBS was used to exchange the medium. The concentration of ES antigens from the ML was measured by the bicinchoninic acid method (BCA Kits, Beyotime Biotechnology, Shanghai, China). Moreover, the ES antigens were characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE) and western blotting (WB). Briefly, 10 μg and 15 μg ES antigens were separately incubated in loading buffer at 100 °C for 10 min, and the mixed samples were subjected to SDS-PAGE on a 4% concentration gel and 12% separation gel. The separation gel was stained with Coomassie blue dye solution at room temperature (RT) for 2 h, and the gel was subsequently recorded using a camera. Meanwhile, the ES antigens in the paralleled separation gels were transferred to PVDF membranes. After blocking in TBST-B (25 mM Tris, pH 8.0, 125 mM NaCl, 0.05% Tween 20 (V/V), 3.7% BSA) at RT for 2 h, the membranes were incubated with the primary antibodies (10,000 T. spiralis-infected pig serum, 60 dpi or normal pig serum) at a dilution of 1:200 in TBST-B for 12 h at 4 °C. Secondary antibody (HRP-conjugated goat anti-swine IgG) at a dilution of 1:1000 in TBST-B was incubated with the membranes for 2 h at RT. The membranes were reacted with ECL reagent and exposed to a multifunctional imaging system. The ES antigens were stored at − 80 °C until use.
Preparation and characterization of UCNPs-ES/goat anti-rabbit IgG
ES-COOH/goat anti-rabbit IgG-COOH was pre-activated to its succinimide by using EDC and sulfo-NHS and then reacted with an NH2-UCNP fragment [30]. Briefly, 1 ml of ES/goat anti-rabbit IgG solution (300 μg/ml in 10 mM NaH2PO4, pH 6.0) was incubated with 10 μl of 50 mg/ml EDC and 10 μl of 50 mg/ml sulfo-NHS overnight at RT with gentle shaking. Then, HEPES buffer (100 mM pH 7.4) was used to exchange the NaH2PO4 buffer of the reaction system with a 3 kDa ultrafilter. The resulting sulfo-NHS-activated ES/antibody was covalently linked to 500 μl of NH2-UCNPs (10 mg/ml in HEPES, pH 7.4) overnight at RT. After the conjugation reaction, the UCNPs-ES/goat anti-rabbit IgG was separated from free ES/goat anti-rabbit IgG by centrifugation at 28,500×g for 10 min. Next, the UCNPs-ES/goat anti-rabbit IgG was resuspended in UCP storage buffer (50 mM glycine, 0.03% Triton and 0.1% NaN3, pH 8.0) at 1 μg/μl and stored at 4 °C for up to 6 months. Morphological examination of UCNPs-ES/goat anti-rabbit IgG was performed by transmission electron microscopy (TEM), which was compared with unconjugated UCNPs, and from the sizes of the conjugated and unconjugated UCNPs were measured by software (Image-Pro Plus). Upconversion (UC) emission spectra of the conjugated and unconjugated UCNPs were recorded under 980 nm excitation light, and physical images were obtained in dark and light environments, respectively.
Development and optimization of the UPT-LF-ES assay
The strip was composed of a sample pad, an NC membrane, an absorbing pad, and a plastic backing. A schematic illustration of the strip is shown in Fig. 1a. After the strip was assembled, the assay was optimized in the goat anti-swine IgG volume (T line) (400, 600, 800, 1000 ng/4 mm) and the sample dilution (1:100, 1:150, 1:200, 1:250, 1:300). According to the optimization results, the strip was prepared as follows: goat anti-swine IgG (800 ng/4 mm) and rabbit anti-goat IgG (100 ng/4 mm) in 10 mM Tris–HCl (pH 8.0) were dispensed as the T-line and C-line on the NC membrane, respectively. The membrane was dried at 37 °C for 2 h and then assembled on the plastic backing, which was positioned for a 2 mm overlap between the NC pad and the sample pad or absorbing pad. The assembled cards were cut into a strip 4 mm wide using a CM 4000 Guillotine Cutter. The finished strips were stored in a container with a dry pack at RT (expiration date: 6 months) until testing.
UPT-LF-ES assay procedure
A schematic illustration of the assay is depicted in Fig. 1b. In detail, the assay included four steps. UCP storage buffer containing UCNPs-ES/goat anti-rabbit IgG was suspended by vortexing for 10 s. The desired amounts of UCNPs-ES (200 ng) and UCNPs-goat anti-rabbit IgG (50 ng) were diluted in UPT-LF-ES assay buffer (100 mM HEPES pH 7.5, 270 mM NaCl, 0.5% v/v Tween 20, 1% v/v BSA) to a final volume of 100 μl and sonicated to homogenize potential aggregates [26]. A 0.7 μl serum sample was added to the UPT-LF-ES assay buffer containing UCNPs-ES and UCNPs-goat anti-rabbit IgG and mixed. The mixed solution was incubated at RT for 15–20 min. The mixture was added to the sample pad of the UPT-LF-ES strip, which was placed for 15 min at RT and then for 5–10 min at 37 °C. The strip was scanned with a UPT-based biosensor. The results are expressed as a ratio signal (UPT value T/C).
Cut-off threshold
To assess clinical specificity, the UPT-LF-ES assay was performed with 169 known negative pig sera samples obtained from the Food-Borne Parasitology Laboratory of Key Laboratory for Zoonoses, Jilin University.
Serum samples
Serum samples from T. spiralis experimental infection
Nine Large White pigs (females, 2 months old, 20 kg) were obtained from the Experimental Animal Center of Norman Bethune University of Medical Science (NBUMS), China. Before the experiment, all pigs were tested using routine blood examination and fecal samples, and the results indicated that these pigs were healthy. Then the pigs were randomly divided into three groups and experimentally inoculated with 100, 1000, or 10,000 T. spiralis ML. Serum samples were collected and prepared on 0, 7, 9, 11, 13, 15, 17, 19, 21, 25, 30, 45, 60, 90, and 120 dpi, according to the collection method from this article [31]. The average larvae per gram (LPG) value was calculated and presented in a previously published article from our lab [31]. These serum samples were also tested in the UPT-LF-ES assay.
Serum samples with heterologous infections
A set of 8 samples from pigs infected with Toxoplasma gondii (n = 2), cysticerci of Taenia solium (n = 2), and cysticerci of Taenia asiatica (n = 2) was tested to evaluate potential cross-reactivity with other parasitic infections. These sera were provided by the Food-Borne Parasitology Laboratory of Key Laboratory for Zoonoses, Jilin University.
Single-blinded experimental validation
Validation of the assay was performed using the sera from 35 positive samples that included different infections at 120 dpi, and these samples were randomly arranged between a set of 20 negative samples. These 35 positive serum samples were randomly selected in the serum sample bank of our laboratory [31]. These 55 serum samples were confirmed using the magnetic stirrer method and WB by the Food-Borne Parasitology Laboratory of Key Laboratory for Zoonoses, Jilin University, and were tested with the assay in a single-blinded experiment that was performed by a common tester who did not participate in this assay’s early development. The results from this experiment were evaluated by Student’s t-test and receiver operating characteristic curve (ROC curve) analysis.
Data analysis
Peak areas for the T- and C-lines were calculated as relative intensity units by software from the Shanghai Institute of Optics and Fine Mechanics, Chinese Academy of Science, and the ratio values at which the T-line signals were divided by the C-line signals of each strip. Data were entered into Origin 2020b and are presented as histograms. The kappa (K) coefficient was employed to measure agreement between two methods, which allowed us to measure agreement beyond what was expected by chance alone [32, 33]. The generic formula for K is
$$K = \, (P_{o} - P_{e} )/\left( {1 - P_{e} } \right),$$
where Po and Pe are the observed and expected proportions of agreement [34]. K > 0.8 and ≤ 1.0 show an almost perfect agreement between two methods, and K > 0.61 and ≤ 0.8 show a substantial agreement between two methods, and K > 0.41 and ≤ 0.61 show a moderate agreement between two methods [34].
