**5. Conclusions**

In this study, antigens with different coupling ratios were synthesized and used to prepare a sensitive mAb, 4D6. The mAb showed high affinity for PYR (100%) and BaP (38%). Based on the mAb, a sensitive, efficient and reproducible ic-ELISA method was found and used for the determination of PYR and BaP residues in fish, shrimp and crab for the first time. The LODs of PYR and BaP in fish, shrimp and crab range between 0.43 and 0.98 μg/kg. Confirmation and relevance with HPLC-FLD showed the ic-ELISA method is a feasible tool. Therefore, the ic-ELISA method established in this experiment provided a practical tool for usual screening of PYR and BaP in aquatic products and supplemented the lack of an ELISA method in aquatic products. It lays a foundation for the development and application of PAHs residue detection kit in aquatic products. It has important academic value and practical significance to guarantee the quality of aquatic products.

**Supplementary Materials:** The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/foods11203220/s1, Figure S1: Structure of PYR, BaP and PBA; Figure S2: Subtype determination of four monoclonal antibodies; Figure S3: The liquid chromatogram spectrum of PYR in fish sample (10 ng/mL); Table S1: Comparison of absorbance and inhibition rate of antigens with different coupling ratio on immunized mice; Table S2: Dilutions and IC50 values of four monoclonal antibodies; Table S3: Optimization of ELISA conditions; Table S4: Cross-reactivity of 4D6 mAb with 16 polycyclic aromatic hydrocarbon drugs.

**Author Contributions:** Conceptualization, S.W., H.L., Yu Si, L.Q., H.Y. and D.P.; software, L.Q.; validation, S.W., H.L. and H.Y.; formal analysis, S.W. and Y.S.; investigation, S.W. and X.Y.; data curation, S.W.; writing—original draft preparation S.W.; writing—review and editing, S.W. and H.L.; visualization, S.W. and J.X.; supervision, D.P.; project administration D.P.; funding acquisition, D.P. All authors have read and agreed to the published version of the manuscript.

**Funding:** The Key Research and Development Program of Hubei Province (CN) (2020BBB079) and the National Natural Science Foundation of China (32072920).

**Institutional Review Board Statement:** All animal experiments in this study adhered to the Huazhong Agricultural University animal experiment centre guidelines and were approved by the Animal Ethics Committee.

**Informed Consent Statement:** Informed consent was obtained from all subjects involved in the study.

**Data Availability Statement:** The data presented in this study are available on request from the corresponding author.

**Conflicts of Interest:** The authors declare that they have no known competing financial interest or personal relationships that could have appeared to influence the work reported in this paper.
