3.1.4. The Ab Amount

The Ab amount plays a decisive role in the signal intensity and inhibitory effect of the immunoassay method [10,33]. The detection performance of four different Ab amounts (0.8, 1.0, 1.2, and 1.4 μg for milk and 0.6, 0.8, 1.0, and 1.2 μg for pork) were investigated. As shown in Figure S3, with the increased of Ab amount, the fluorescent signal of T-line increased gradually, but the inhibition effect became worse. Combined with the results of quantitative detection, it was not difficult to find that, when the amount of Ab was 0.8 μg, which was equivalent to adding 80 μL of Ab solution, the inhibition rate of the test strip was the best. Therefore, the optimal amount of Ab was 0.8 μg for both milk and pork detection.

#### 3.1.5. Key Reagents of Sample Pad Treatment Solution

The main function of the sample pad is the carrier of the sample, which can promote the release of Ab probe and eliminate the matrix interference of the sample [24,28]. Therefore, the handling of the sample pad is very important.

Surfactant. Surfactant can promote the release of immunoprobe, affect the behavior of immunoprobe on chromatographic pads, and reduce non-specific reaction [28,34]. In this study, we employed Tween-20 as a surfactant and studied the effect of its concentration changes on the performance of test strips. As shown in Figure S4, we prepared a series of different concentrations of Tween-20 in the sample pad treatment solution and found that, as the concentration of Tween-20 increased, the release rate of immunoprobe accelerated. The results of quantitative analysis show that the inhibition rate was the best when the concentration of Tween-20 was 0.5% for milk samples and 0.2% for pork samples. The reason for the inconsistency of the optimal concentration should be closely related to the viscosity and fluidity of the samples.

Sample pad treatment buffer. It is particularly important for the sample pad treatment buffer to protect Ab activity and eliminate the interference of the sample matrix [24,29]. In this study, the effects of three kinds of buffers with different ionic strength and pH values on the detection performance of test strips were compared. The results (Figure 3) show that pH value > 9.0 was not conducive to the performance of Ab activity because the fluorescence intensity of test strip was not ideal. However, high ionic strength was conducive to inhibition. Considering the fluorescence signal intensity, inhibition effect, and inhibition rate, 0.05 M PB was finally determined as the buffer of the sample pad treatment solution.

**Figure 3.** The fluorescence intensity, inhibition effect, and inhibition rate results of the sample pad treatment buffer: ( **A**) ultraviolet lamp results for milk detection; (**B**) ultraviolet lamp results for pork detection; ( **C**) fluorescence quantitative results for milk detection; and ( **D**) fluorescence quantitative results for pork detection.

To sum up, the formula of the sample pad treatment solution for milk detection was 0.05 M PB (pH 7.4, 0.5% Tween-20, 0.3% PVP) and for pork detection was 0.05 M PB (pH 7.4, 0.2% Tween-20, 0.3% PVP).
