## 3 Results and Discussion  
### Optimizing Conditions  
ADHP is non-fluorescent; however, after the addition of H2O2 and HRP, strong excitation and emission signals are displayed, as shown in Figure 3a, proving that resorufin generated by ADHP produces an effective fluorescent signal in the HRP enzymatic reaction. Therefore, developing an ELISA method on the basis of ADHP fluorogenic substrate is feasible. Before developing the fluorescent sandwich ELISA method, the effects of ADHP concentration, the total volume of ADHP and H2O2, temperature, and pH on the fluorescent sandwich ELISA working solution were studied to examine the fluorescent conditions. As shown in Figure 3b, with an increasing concentration of ADHP, the fluorescence intensity was significantly enhanced and was then kept stable, indicating that the fluorescence intensity finally reached saturation in the case of excessive ADHP. The fluorescence intensity reached the highest point with 250 uM ADHP, so this concentration of ADHP was selected to prepare the fluorescent working solution. When the total volume of ADHP and H2O2 increased, a similar trend was shown and the fluorescence intensity values were slightly different at a total volume between 40 and 80 uL (Figure 3c). To save fluorescent substrate and ensure high signal intensity, the fluorescent working solution with a total volume of 40 uL was finally selected for fluorescent sandwich ELISA determination.  
The results of the enzymatic reaction of ADHP were also greatly influenced by environmental factors. Incubation at 37 \({}^{\circ}\)C gave a stronger fluorescence signal than incubation at room temperature because appropriate heating had a positive effect on the enzymatic reaction (Figure 3d). However, the fluorescence intensity decreased after the temperature continued rising to 50 \({}^{\circ}\)C, which was because high temperature reduced the activity of HRP, leading to a decrease in the enzymatic reaction rate and thus affecting the intensity  
Figure 2: Pretreatment steps for bovine colostrum powders using different methods: bovine colostrum powders (A) were dissolved and dispersed in water (B), then the solution was divided into three layers after centrifugation (C), after removing the upper and lower layers, skimmed milk was obtained (D), then the acid treatment and centrifugation followed to obtain the supernatant and precipitation (E), and finally a transparent and light-yellow solution was obtained after throwing the precipitation (F).  
of the fluorescence signal. Therefore, 37 \({}^{\circ}\)C was chosen as the reaction temperature of ADHP. Moreover, the effect of pH given in Figure 3e displays that the fluorescence intensity was higher at a pH of 7 than at a pH of 5 or 9. This is probably because alkaline buffer accelerates the decomposition of H\({}_{2}\)O\({}_{2}\), thereby reducing the content of substrates in enzymatic reactions. Meanwhile, ADHP exists mainly as non-deprotonated neutral molecules under acidic conditions, making it difficult for oxidation reactions to occur [33]. In addition, HRP had the best enzymatic activity at a pH of 7. Therefore, the optimal working temperature and pH for the fluorescent sandwich ELISA working solution were 37 \({}^{\circ}\)C and a pH of 7, respectively.