The Development and Validation of a Novel Nanobody-Based Competitive ELISA for the Detection of Foot and Mouth Disease 3ABC Antibodies in Cattle

Figures

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  Figure 1. Selection of Nanobodies (Nbs) from an immune phage-displayed Nb library against FMDV 3ABC protein. Periplasmic Extract (PE) ELISA results demonstrating 19 Nb colonies analyzed for the immunorecognition to FMDV 3ABC protein and peptides (3A, 3B, 3C1, and 3C2). Luria-Bertani (LB) broth medium was used as negative control. Assay cutoff is indicated by dash line.

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  Figure 2. Binding affinity analysis of selected Nanobodies (Nbs) against FMDV 3ABC protein. (A) The binding affinity results of six anti-FMDV 3ABC Nbs, evaluated by indirect ELISA. During experiments Nb9 was used as a negative control. The results are presented in Relative Light Units (RLU). (B). The immunorecognition performance of six selected anti-FMDV 3ABC Nbs in a competitive ELISA format using a set of 8 infected and noninfected control samples. Mean percentage of inhibition (PI) for each Nb was calculated using the formula: 100–(X Aver infected/X Aver noninfected) × 100.

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  Figure 3. Diagnostics performance of the Nanobody (Nb94) based 3ABC competitive ELISA. (A) Receiver-Operating Characteristic (ROC) analysis results from a total of 222 cattle sera. Dotted lines represent 95 % confidence interval. (B). Lower limit of detection (LLD) using positive (infected sera) and negative (noninfected sera) control serum. Cutoff assay is indicated by dash line. (C) Intra assay repeatability performance was assessed using a set of positive and negative internal control sera tested in the assays in different days and by different operators. Lower and upper limits (2 × STDV) are indicated by dot lines. Cutoff assay is indicated by dash line. The total average and STDV of positive and negative internal control sera across the different experiments is also presented. Statistical analysis: *P < 0.05, **P < 0.01, ***P < 0.001.

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  Figure 4. Sera screening analysis of infected and noninfected cattle samples using the Nanobody (Nb94) based 3ABC competitive ELISA. Box plot representing 100 noninfected (negative) and 150 infected (confirmed by reverse transcription PCR or virus isolation) (positive) sera collected in Uganda. FMDV serotype of infected cattle, included serotype O, SAT1 and SAT2 (50 samples for each). Cutoff assay is indicated by dash line. Numbers in brackets indicate the total number of tested sera.

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  Figure 5. Sera screening analysis of naïve and vaccinated calves' samples using the Nanobody (Nb94) based 3ABC competitive ELISA and PrioCHECK NSP test. A total of 72 calves were serially sampled three times; before first vaccination (naïve state), after first vaccination, and after second vaccination. The prevalence of FMDV NSP antibodies were determined by the Nb (Nb94) based 3ABC competitive ELISA (A) and PrioCHECK NSP test (B). Cutoff for each assay is indicated by dash line.

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  Figure 6. Comparison analysis of serum samples tested by the Nanobody (Nb94) based 3ABC competitive ELISA. Serum collected from cattle with different FMD status were analyzed for NSP antibodies presence by the Nb (Nb94) based 3ABC competitive ELISA. These included free (naïve and noninfected), infected (FMDV serotype O, SAT1 and SAT2) and vaccinated (first and second vaccination). The mean results of percentage of inhibition (PI) is presented. Cutoff assay is indicated by dash line. Statistical analysis: *P < 0.05, **P < 0.01, ***P < 0.001.

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  Figure 7. Sera screening of randomly collected field samples tested by the Nanobody (Nb94) based 3ABC competitive ELISA. A total of 165 cattle serums previously collected from four different districts (Mbale, Nakasake, Insingiro, and Gomba) in Uganda, by the Uganda Virus Research Institute (UVRI), were obtained and analyzed using the Nb (Nb94) based 3ABC competitive ELISA. Samples were collected as part of an FMD surveillance program for assessing the levels of NSP antibodies prevalence in the country. Cutoff assay is indicated by dash line. Numbers in brackets indicate the total number of sera tested in each district.