The development of a lateral flow immunochromatographic test strip for measurement of specific IgA and IgG antibodies level against porcine epidemic diarrhea virus in pig milk

Figures

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  Figure 1.. Schematic diagram of the LFICS. The LFICS consists of three pad components: sample pad, conjugate pad, and absorbent pad, along with a nitrocellulose membrane and a backing pad. Gold-labeled mouse IgG mAb and gold-labeled S protein are applied to the conjugate pad. The nitrocellulose membrane features two lines: test line and control line. The test line included goat anti-pig IgA antibody (A) and goat anti-pig IgG antibody (B), while the control line contained goat anti-mouse IgG antibody.

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  Figure 2.. Expression and purification of recombination S protein. (A) Purification profile of S protein. Lane M: NativeMARK™ Unstained Protein Standard molecular weight marker (20-720 kDa); lane 1: purified SP protein. (B) Analysis of purified S protein by immunoblotting. Lane M: NativeMARK™ Unstained Protein Standard; lane 1: anti-6xHis antibody. (C) Antigenic reactivity of S protein to positive and negative sera against PEDV was evaluated using an indirect ELISA, measured by S/P ratio.

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  Figure 3.. Limited of detection (LOD) of the immunochromatographic strip test. Different dilution of anti-PEDV specific IgA and IgG positive milk (1:5, 1:10, 1:20, 1:40, 1:80 and 1:160). The strip detection limits were observed to be 1:40 for IgA (A) and 1:80 for IgG (B), as indicated by the blue and green boxes, respectively.

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  Figure 4.. Limited of detection (LOD) of neutralizing antibody (nAb) levels against PEDV on immunochromatographic strip test. Positive nAb sample was diluted ten-fold at 4log2, 3log2, 2log2, 1log2 and 0log2, respectively. The sensitivity of the specific IgA (A) and IgG (B) strips was 1log2 (purple box).

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  Figure 5.. Cross-reactivity of the immunochromatographic strip test. The strip detection of anti-PEDV specific IgA (A) and IgG (C) positive milk, as well as anti-PDCoV specific IgA (B) and IgG (D) positive milk. The samples were diluted at 1:5 in TWS buffer, and then each strip was immersed with the dilution sample. Positive signals were observed on T-line of the anti-PEDV specific IgA (A) and IgG (C) positive milk samples. C and T represent control line and test line, respectively.

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  Figure 6.. Correlation of antibody levels between VN titer and ELISA S/P ratio. A scatter plot of antibody levels in colostrum (A,B) and milk (C,D) is shown. Linear regression, r and P values were analyzed by Pearson (two-tails) with a fitted regression line and 95% confidence intervals.

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  Figure 7.. Correlation of antibody levels between VN titer and strip test T/C ratio. A scatter plot of antibody levels in colostrum (A,B) and milk (C,D) is shown. Linear regression, r and P values were analyzed by Pearson (two-tails) with a fitted regression line and 95% confidence intervals.

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  Figure 8.. Correlation between ELISA S/P ratio and strip test T/C ratio. Levels of IgA (A,C) and IgG (B,D) antibodies against PEDV in pig colostrum and milk were evaluated using ELISA and strip test. All samples were analyzed with Pearson r and P values (two-tails) with a fitted regression line and 95% confidence intervals.

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  Figure 9.. Relationship between semiquantitative (visual scores) and quantitative measurements by ELISA and test strip. Relationship between visual scores and T/C ratio by the test strip for IgA and IgG ELISA in colostrum (a and B) and milk (C and D), respectively. Relationship between visual scores and S/P ratio by ELISA for IgA and IgG ELISA in colostrum (E and F) and milk (G and H), respectively. Horizontal lines represent as mean ± SD using Cuzick’s trend test with 95% confidence interval.

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  Figure 10.. Stability and repeatability tests of the LFICS. The LFICS demonstrated stability for 12 months at RT (A). Repeatability was determined using CV percentage at different storage times (B).