Figure 3

Validation of developed S-ELISA assay for detection of two Brucella spp. in different matrices spiked with 10 fold serial diluted WC Ag of Brucella. (a,b) S-ELISA graph plot showing detection in spiked matrices (WC Ag at 10⁸ CFU mL⁻¹ to 10¹ CFU mL⁻¹) for Brucella abortus S99 and Brucella melitensis 16 M along x-axis and absorbance at 495 nm along y-axis respectively. Linear regression co-efficient value for initial validation in human sera, cow urine and cow milk is R² = 0.7409 with linear model equation, y = - 0.0593×+ 0.7424 and, R² = 0.9116 with linear model equation, y = - 0.0737×+ 0.765 for validation against Brucella abortus S99 and Brucella melitensis 16 M WC detection respectively. (c,d) S-ELISA graph plot showing detection in spiked matrices for Brucella with three different human sera and one foetal bovine sera for validation along x-axis and absorbance at 495 nm along y-axis for Brucella abortus S99 and Brucella melitensis 16 M WC detection respectively. (e) S-ELISA graph plot showing detection of Brucella spp. in spiked human whole blood along x-axis and absorbance at 495 nm along y-axis. Linear regression co-efficient value, R² = 1 at no bias showing linearity with model equation, y = - 0.073×+ 2.588 for validation against Brucella abortus S99 and Brucella melitensis 16 M WC detection. OD value obtained is <1 at lower limits for both the spp. (f) S-ELISA graph plot showing detection in spiked matrices with 10 fold serial diluted WC Ag (10⁸ CFU mL⁻¹ to 10² CFU mL⁻¹) of Brucella melitensis 16 M along with WC Ag of Brucella abortus S99 and closely Brucella related bacterial spp. for validation along x-axis and absorbance at 495 nm along y-axis for evaluation of cross-reactivity. Both Brucella spp. are cross-reactive on validation.