Upconverting phosphor technology-based lateral flow assay for the rapid and sensitive detection of anti-Trichinella spiralis IgG antibodies in pig serum

Abstract

Background: Trichinella spiralis is a zoonotic food-borne parasite. A disease caused by infection with T. spiralis is called trichinellosis in humans. It is important to investigate the epidemic situation and the surveillance of herds and then prevent infection in humans. Therefore, this study is to develop a rapid and sensitive diagnostic method for on-site test in domestic and wild animals.

Methods: Upconverting phosphor nanoparticles (UCNPs), an excellent optical label, were conjugated with the excretory-secretory (ES) antigens from T. spiralis muscle larvae (ML) or goat anti-rabbit IgG, and a lateral flow (LF) assay based on these probes (UCNPs-ES/goat anti-rabbit IgG) was developed for the rapid and sensitive detection of anti-T. spiralis IgG antibodies in pig serum. The assay is named the UPT-LF-ES assay. In addition, the probes were characterized, and the assay was optimized. A cut-off threshold of the assay was also identified by using 169 known negative pig samples. Performance of the assay to T. spiralis with different infective numbers, cross-reactivity with other parasitic infections, the single-blinded experiment, and coincidence were evaluated with the assay.

Results: The UPT-LF-ES assay was successfully constructed and optimized based on the probes of UCNPs-ES/goat anti-rabbit IgG. In the pigs infected with 100, 1000, and 10,000 ML, positive results were first presented at 35 days post-infection (dpi), 30 dpi, and 25 dpi, respectively. The assay had no cross-reaction with other parasitic infections. A single-blinded experiment indicated that the sensitivity and specificity of the UPT-LF-ES assay were 100% and 100%, respectively, the area under the receiver operating characteristic (ROC) curve was 1.000. In addition, the value detected by the UPT-LF-ES assay was significantly different between positive and negative samples. Moreover, compared with the "gold standard" magnetic stirrer method, the coincidence rate of the UPT-LF-ES assay was 87.27%, and the kappa (K) coefficient was 0.7454, showing a substantial agreement.

Conclusions: The UPT-LF-ES assay is a useful point-of-care test (POCT) with T. spiralis in the detection of pig, which contributes to preventing human trichinellosis.

Keywords: Excretory-secretory antigens; Lateral flow assay; Serodiagnosis; Trichinella spiralis; Upconverting phosphor technology.

Fig. 1

The UPT-LF-ES assay. a Schematic illustration of the UPT-LF-ES strip: test line 100 ng of goat anti-swine IgG and control line 100 ng of rabbit anti-goat IgG. b The LF protocol for antibody detection included four steps in series

Fig. 2

SDS-PAGE and WB identification of ML-ES. a ML-ES were characterized by SDS-PAGE. b ML-ES antigens were detected by T. spiralis-infected pig serum. c ML-ES antigens were detected by uninfected pig serum as negative control. These experiments included two repeats (1, 2). M, marker proteins

Fig. 3

Characterization of UCNPs-ES/goat anti-rabbit IgG. TEM images of unconjugated UCNPs (a), UCNPs-ES (b), and UCNPs-goat anti-rabbit IgG (c) are shown. The spectra stimulated in 980 nm laser light are shown (d), and the physical pictures were also obtained in dark and light environments (e), including unconjugated UCNPs solution (1), UCNPs-ES solution (2), and UCNPs-goat anti-rabbit IgG solution (3)

Fig. 4

Optimization of the UPT-LF-ES assay and the optimized results. a The dosage of goat anti-swine IgG (400, 600, 800, 1000 ng/4 mm) in the T-line and the dilution of serum (1:100, 1:150, 1:200, 1:250, 1:300) were optimized to obtain better conditions (T-line: 800 ng of goat anti-swine IgG; dilution of serum: 1:150). Positive serum (b) and negative serum samples (c) were tested under the optimized conditions, and the inserted photographs showed the results under 980 nm laser light

Fig. 5

Cut-off threshold of the UPT-LF-ES assay. The cut-off thresholds were obtained by 169 known negative serum samples, including a low specificity cut-off (ratio: 0.1906) and a high specificity cut-off (ratio: 0.3233). AV, Average value; HA, Highest value

Fig. 6

Performance of the T. spiralis assay with different infective numbers. Serum samples from pigs experimentally infected with 100 (a), 1000 (b), or 10,000 (c) T. spiralis were tested using this assay. The green line is the low specificity cut-off of 0.1906, and the red line is the high specificity cut-off of 0.3233. Data are presented as the mean ± SD from three independent experiments

Fig. 7

Cross-reactivity of the assay with sera infected with other parasites

Fig. 8

The results of single-blinded experimental validation. a The results of the 55 pig serum samples tested by the assay. b All of the results are expressed as the box plot. The ratio values of these two groups were significantly different (t₍₅₃₎ = 9.827, P < 0.0001). Student’s t-test was used to determine significant differences. c ROC analysis of the 55 pig serum samples based on the ratio values of the assay. The integrated area under the ROC curve was 1.000