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.

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.

A SERS-based lateral flow immunochromatographic assay using Raman reporter mediated-gap AuNR@Au nanoparticles as the substrate for the detection of enrofloxacin in food samples.

A lateral flow immunochromatographic assay (LFIA) based on surface-enhanced Raman scattering (SERS) for sensitive and specific detection of antibiotic enrofloxacin (ENR) in food samples was developed. 1,4-benzenedithiol (BDT) was selected as the Raman reporter, and the BDT mediated-gap AuNR@Au nanoparticles (NPs) were synthesized, characterized and used as the substrate in SERS-LFIA due to the existence of the anisotropic gold nanorods (AuNRs) and the nano-gap with the high SERS enhancement. AuNRs were prepared, then covered by monolayer BDT. Under reduction condition and in presence of HAuCl4, the reduced gold was deposited and grown on AuNRs to form AuNRBDT@Au NPs. As the two thiol groups on para-positions in BDT were respectively linked to AuNR (core) and Au (shell), the gap size inside the NPs was uniform. The immunoprobe (e.g. AuNRBDT@Au-Ab) was obtained by immobilizing Ab against ENR on the surface of AuNRBDT@Au NPs. The performance of SERS-LFIA was similar to that in colloidal gold based-LFIA, and the entire assay time was within 15 min. After LFIA procedures, the specific SERS intensity of BDT at 1560 cm-1 on the test line was measured for the quantitative detection of ENR. The IC50 and limit of detection (LOD) of the LFIA for ENR were 59 pg mL-1 and 0.12 pg mL-1 (e.g. 71 pg g-1 and 0.14 pg g-1 in real sample), respectively. There was no cross-reactivity (CR) of the LFIA with other five antibiotics. The recoveries of ENR from spiked food samples were in range of 89.2%-102.4% with the relative standard deviation (RSD) of 1.70%-6.38%. It was proven that the proposed method was able to simply and rapidly detect ENR in food samples with high sensitivity, specificity, accuracy and precision. The platform can be also an alternative platform for the detection of other target analytes using corresponding Abs.