Fig 1.
Cartoon (not in scale) depicting the two 1G10 mAb-based ELISAs for detection of anti BoHV-1 antibodies: classic configuration using the antibody-HRP conjugate (left) and ANANAS integrated method (right).
For simplicity reasons, the mAb used for Bo-HV1 adsorption on the plate (see methods) is not included in the scheme.
Fig 2.
Optimization of detector mAb (1G10-biotin) concentration.
Net OD (grey columns), and ODAg- (black columns) registered in the analysis of negative diluting milk and an individual positive milk sample diluted 1:10 and 1:100 in negative diluting milk, as obtained with the ANANAS-integrated test using biotin-1G10 at different concentrations. TMB development was blocked after 10 min reaction. Each sample was analyzed in duplicate. These data are also reported in table format in (S1 Table).
Fig 3.
Assay sensitivity comparison in the pilot study.
Maximum, minimum and median net OD values registered in the analysis of individual positive milk samples (no. 8 from gE-negative and no. 10 from gE-positive cows) diluted 1:25, 1:50 in negative diluting milk, using 1G10 in the classic or ANANAS-integrated configuration.
Table 1.
ELISA test specificity comparison.
Assay specificity calculated for the classic method based on 1G10-HRP conjugate, the 1G10-ANANAS integrated, and the commercial IDEXX one. Results of the IDEXX test were interpreted adopting the S/P threshold indicated by the manufacturer (0.25) or the same one adopted for the 1G10 based methods (0.1). The results have been obtained using 252 negative tank milk samples from farms in the Bolzano province
Fig 4.
ELISA tests sensitivity comparison.
Sensitivity as a function of milk dilution using the (o) classic test, (▲) ANANAS integrated, (□) Commercial with 0.25 S/P threshold, (■) commercial with 0.1 S/P threshold methods. Data have been elaborated taking into consideration all animals (left panel) or separating the results of anti-gE positive (mid panel)/negative (right panel) animals. The dashed parts of the curves in mid panel were generated by linear extrapolations from the two closest data points. The vertical dotted lines highlight the interception of dilution curves with the 95/90% sensitivity. These data are also reported in table format in (S3 Table).