2.7.3. Incubation Time

The sensitivity was also greatly affected by the incubation time of antigen and antibody. Under the above determined conditions, the competition time was set to 25, 35, 45 and 55 min; the HRP-IgG incubation time was set to 30, 40, 50 and 60 min to explore the optimal incubation time.

#### 2.7.4. Organic Solvents

To investigate the effect of different organic solvents on the binding ability of antigen and antibody, four solvents, DMF, acetone, acetonitrile and DMSO, were tested for assay optimization. Briefly, each solvent was diluted to 10%, 20%, 30% and 40% with PBS. Then, a series of PYR concentrations were prepared with the above dilution solvent, and standard curves were drawn according to the ic-ELISA steps.
