An Extremely Highly Sensitive ELISA in pg mL-1 Level Based on a Newly Produced Monoclonal Antibody for the Detection of Ochratoxin A in Food Samples

Yexuan Ren; Ruwen Tian; Ting Wang; Junlin Cao; Jianguo Li; Anping Deng

doi:10.3390/molecules28155743

An Extremely Highly Sensitive ELISA in pg mL^-1 Level Based on a Newly Produced Monoclonal Antibody for the Detection of Ochratoxin A in Food Samples

Molecules. 2023 Jul 29;28(15):5743. doi: 10.3390/molecules28155743.

Authors

Yexuan Ren¹, Ruwen Tian¹, Ting Wang¹, Junlin Cao¹, Jianguo Li¹, Anping Deng¹

Affiliation

¹ The Key Lab of Health Chemistry and Molecular Diagnosis of Suzhou, College of Chemistry, Chemical Engineering and Materials Science, Soochow University, Renai Road 199, Suzhou 215123, China.

Abstract

In this study, an extremely highly sensitive enzyme-linked immunosorbent assay (ELISA) based on a newly produced monoclonal antibody (mAb) for the detection of ochratoxin A (OTA) in food samples was developed. OTA-Bovine serum albumin (BSA) conjugate was prepared and used as the immunogen for the production of the mAb. Among four hybridoma clones (8B10, 5C2, 9B7, and 5E11), the antibody from 8B10 displayed the highest affinity recognition for OTA. Based on the mAb (8B10), the IC₅₀ and LOD of the ELISA for OTA were 34.8 pg mL^-1 and 1.5 pg mL^-1, respectively, which was 1.53~147 times lower than those in published ELISAs, indicating the ultra-sensitivity of our assay. There was no cross-reactivity of the mAb with the other four mycotoxins (AFB₁, ZEN, DON, and T-2). Due to the high similarity in molecular structures among OTA, ochratoxin B (OTB), and ochratoxin C (OTC), the CR values of the mAb with OTB and OTC were 96.67% and 22.02%, respectively. Taking this advantage, the ELISA may be able to evaluate total ochratoxin levels in food samples. The recoveries of the ELISA for OTA in spiked samples (corn, wheat, and feed) were 96.5-110.8%, 89.5-94.4%, and 91.8-113.3%; and the RSDs were 5.2-13.6%, 8.2-13.0%, and 7.7-13.7% (n = 3), respectively. The spiked food samples (corn) were measured by ELISA and HPLC-FLD simultaneously. A good correlation between ELISA (x) and HPLC-FLD (y) with the linear regression equation of y = 0.918x - 0.034 (R² = 0.985, n = 5) was obtained. These results demonstrated that the newly produced mAb-based ELISA was a feasible and ultra-sensitive analytical method for the detection of OTA in food samples.

Keywords: enzyme-linked immunosorbent assay (ELISA); food samples; monoclonal antibody (mAb); mycotoxin; ochratoxin A (OTA); sensitive detection.

MeSH terms

Antibodies, Monoclonal
Enzyme-Linked Immunosorbent Assay / methods
Food Contamination / analysis
Mycotoxins* / analysis
Ochratoxins* / analysis

Substances

ochratoxin A
Ochratoxins
Antibodies, Monoclonal
Mycotoxins

Grants and funding

This research was funded by the National Natural Science Foundation of China (NSFC, contact nos. 21175097 and 31772053), the Supporting Funds for key research and development project of the Ministry of Science and Technology (5010900122), and a project funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (No. YX10900212).