Preparation of Anti-Aristolochic Acid I Monoclonal Antibody and Development of Chemiluminescent Immunoassay and Carbon Dot-Based Fluoroimmunoassay for Sensitive Detection of Aristolochic Acid I.

Ai-Fen Ou,Qi-Yi He,Zi-Jian Chen,Su-Qing Zhao,Zhen-Lin Xu,Yi-Feng Zhang

doi:10.3390/foods10112647

Abstract

Aristolochic acid (AA) toxicity has been shown in humans regarding carcinogenesis, nephrotoxicity, and mutagenicity. Monitoring the AA content in drug homologous and healthy foods is necessary for the health of humans. In this study, a monoclonal antibody (mAb) with high sensitivity for aristolochic acid I (AA-I) was prepared. Based on the obtained mAb, a chemiluminescent immunoassay (CLEIA) against AA-I was developed, which showed the 50% decrease in the RLUmax (IC50) value of 1.8 ng/mL and the limit of detection (LOD) of 0.4 ng/mL. Carbon dots with red emission at 620 nm, namely rCDs, were synthesized and employed in conventional indirect competitive enzyme-linked immunosorbent assay (icELISA) to improve the assay sensitivity of a fluoroimmunoassay (FIA). Oxidized 3,3′′,5,5′′-tetramethylbenzidine dihydrochloride (oxTMB) can quench the emission of the rCDs through the inner-filter effect; therefore, the fluorescence intensity of rCDs can be regulated by the concentration of mAb. As a result, the assay sensitivity of FIA was improved by five-fold compared to CLEIA. A good relationship between the results of the proposed assays and the standard ultra-high performance liquid chromatography-triple quadrupole mass spectrometer (UPLC-QQQ-MS/MS) of real samples indicated good accuracy and practicability of CLEIA and FIA.

Highlights

Introduction published maps and institutional affilAristolochic acids (AAs) are a mixture of structurally related nitrophenanthrene carboxylic acids, mainly consisting of aristolochic acid I (AA-I) and aristolochic acid II (AA-II), which exist in Aristolochia spp. [1,2], a kind of Chinese herb
Based on the above principle, we developed horseradish peroxidase (HRP) and red CDs (rCDs) based on FIA for AAs analysis
The AA-I was conjugated to a carrier protein to prepare artificial antigen, and a sensitive anti-AA-I monoclonal antibody (mAb) was generated after animal immunization and hybridoma screening

Summary

Introduction

Introduction published maps and institutional affilAristolochic acids (AAs) are a mixture of structurally related nitrophenanthrene carboxylic acids, mainly consisting of aristolochic acid I (AA-I) and aristolochic acid II (AA-II), which exist in Aristolochia spp. [1,2], a kind of Chinese herb. [1,2], a kind of Chinese herb. Aristolochic acids (AAs) are a mixture of structurally related nitrophenanthrene carboxylic acids, mainly consisting of aristolochic acid I (AA-I) and aristolochic acid II (AA-II), which exist in Aristolochia spp. These herbs can be used as raw materials of some drug homologous and healthy foods, and even dietary supplements [3,4,5]. Some cases reported that the intake of slimming products containing AA resulted in nephropathy [13,14]. Many countries have prohibited products containing AAs. it is necessary to develop effective methods for detecting and monitoring AA in related food products

Methods

Results

Conclusion