*3.4. The Standard Curve and Cross-Reactivity*

The PYR standard was diluted to 16.0, 8.0, 4.0, 2.0 and 1.0 μg/L. Under the above optimized conditions, the standard curve for PYR is shown in Figure 4. The equation was y = −44.558x + 76.59, ranging from 1 to 16 <sup>μ</sup>g/L, R2 = 0.9929, and the IC50 was 3.73 ± 0.43 <sup>μ</sup>g/L.

**Figure 4.** Standard curve for PYR in the ic-ELISA.

The specificity of developed ic-ELISA for PYR mAb was assessed by CR. The standards containing 16 PAHs were tested for CR using 4D6 mAb. The mAb exhibited high binding affinity with PYR (100%) and BaP (38%), low affinity with fluoranthene (8%) and negligible cross-reactivity (< 1%) with other PAHs (Table S4). This may be related to the structure of the hapten. The structures of PYR and BaP have the same four ring structures. Therefore, it is inevitable that antibodies prepared from PBA haptens cross with PYR and BaP. The CR results also confirmed our hypothesis. Previous studies on CR mainly focused on 2- to 5-ring PAHs [15,30,31]. However, this study mainly focused on 4- to 5-ring PAHs. Considering the CR for 16 PAHs, this mAb could specifically recognize PYR and BaP. Therefore, it can be used to set up an ic-ELISA kit to detect PYR and BaP in aquatic products.
