Fig 1.
The principle of the nanoflower based-competitive ELISA immunoassay.
(A) Preparation of functional nanoflowers. (B) The procedure for detecting using the nano-cELISA method.
Fig 2.
Optimization of the generating conditions for McAb-HRP-HNFs.
(A) The morphology of McAb-HRP@Cu-HNFs observed under SEM at 12-48h. (B) Verification of catalytic ability of nanoflowers. a, blank control of reaction substrate (TMB + H2O2). b, McAb and reaction substrates. c, HRP and reaction substrates. d, functional nanoflowers and reaction substrates. (C) The detection effect of the nanoflowers at different growth stages. (D) Screening the optimal Cu2+ concentration. (E) Screening of the proportion of McAb and HRP (*: P < 0.05, ns: no significant difference).
Fig 3.
Optimization of the nano-cELISA reaction system.
(A) Selection of optimal dilution ratio for McAb-HRP@Cu-HNFs. (B) Determination of the usage ratio between test serum and nanoflowers. (C, D) Optimization of coating buffer and coating time. (E, F) Optimization of blocking buffer and blocking time. (G) Optimization of the competition time. (H) Optimization of the substrate reaction conditions.
Fig 4.
Preliminary validation of the nano-ELISA in humans.
(A) Determination of the cut-off value for human sera. (B) Specificity tests. (C) Sensitivity test. (D) Detection of human clinical samples. (E) ROC curve analysis.
Fig 5.
Analytical performance of the nano-cELISA in dog sera.
(A) Determination of the cut-off value for dog sera. (B) Evaluation of seroconversion responses in dog after experimental infection with C. sinensis. (C) Specificity tests. (D) Detection of dog clinical samples. (E) ROC curve analysis.
Fig 6.
Evaluation of the nano-cELISA using artificially infected mice sera.
(A) Determination of the cut-off value for mice sera. (B) Assessment of the seroconversion in mice after artificially infected with C. sinensis.