Immunoassay Performance Of Nanobody Against Ochratoxin A And Its Molecular Interaction Mechanisms Study

Ochratoxin A(OTA) is a mycotoxin mainly produced by several Aspergillus and Penicillium fungal species, which can contaminate cereal and cereal products.Many researches have revealed the diverse toxicites of OTA, such as nephrotoxic and immunosuppressive effects. Effective detection and control of OTA contamination in food are the major ways to prevent the food safety issues.Currently, a number of analysis methods have been used for quantification of OTA,such as high performance liquid chromatography(HPLC), liquid chromatography with tandem mass detection and immunoassay. Immunoassay is fast, sensitive and suitable for testing large numbers of samples. Nanobody(Nb) is the smallest antigen-binding unit which is derived from the heavy chain variable of heavy chain antibody in camelids. Nb has characterics of easy production, good water-solubility and good stability, and it has been widely used for medical diagnosis, disease treatment and food safety analysis. Present work described the production of Nb against OTA and the study on its immunological detection performance and action mechanism.The results are shown as follows:1.The construction and biopanning of OTA nanobody phage display library and its application1.1 An OTA Nb primary library(2 × 107 cfu) was constructed successfully with good diversity.After rescue by helper phage, an OTA Nb phage display library was obtained. Combining the Gly-HCl method and competetion method,the phage display library was applied to four rounds of biopanning. In total, four unique Nb sequences were obtained after identification and DNA sequencing analysis.1.2 Phage VHH-28-based RT-IPCR for ultrasensitive detection of OTA in cereal was established with a LOD of 3.7 fg/m L, linear range of 0.01-1000 pg/m L, a cross-reactivity with OTB of 3.5%. The recovery of OTA spiked in cereal sample ranged from 80-126%, and a good correlation was obtained from the OTA content in cereal samples detected by RT-IPCR and commercial ELISA kit.2. Prokaryotic expression and activity analysis of OTA Nb and its application2.1 Nb15, 28, 32, 36 were produced successfuly and were subjected to activity analysis by competitive ELISA. The IC50 of four Nbs were 160.34、0.64、17.60、286.15 ng/m L, respectively. All of four Nbs have good temperature stability. At least50% and 25% of the antigen-binding capacity were remained when the Nbs were heated at 95 ℃ for 5 min and 90 ℃ for 60 min. The equilibrium dissociation constant of Nbs were determined by biosensor Bio-layer interferometry, meanwhile homology modeling and moecular docking were used for mechanisms analysis of interactions between Nb and OTA. The results indicated that the major interactions between Nb and OTA were hydrogen bond and hydrophobic effect, and a positive correlation was found between Nb affinity and sensitivity.2.2 Nb-ELISA based on Nb28 for the detection of OTA in cereal was developed with an IC50 value of 0.64 ng/m L, linear range of 0.27-1.47 ng/m L, LOD of 0.16 ng/m L and a cross-reactivity with OTB of 10.4%. The OTA recovery(%) of intra-assay ranged from 81% to 108% with coefficient variations of 1-9%. The OTA recovery(%)of inter-assay ranged from 85% to 99% with coefficient variations of 5-6%. A good correlation was obtained from the OTA content in cereal samples detected by Nb-ELISA and commmercial ELISA kit.3. Prokaryotic expression and activity analysis of OTA Nb-AP fusion protein and its applicationNb28-AP fusion protein was produced successfully with good anigen-binding capacity and alkaline phosphatase activity. A direct competitive fluorescence enzyme immunoassay(dc-FEIA) was developed using the fusion protein as detection probe, with an IC50 value of 0.13 ng/m L, linear range of 0.06-0.43 ng/m L and LOD of 0.04 ng/m L. Negligible cross-reactivity was observed with other common mycotoxins. The OTA recovery(%) of intra-assay ranged from 72% to121% with coefficient variations of 2-13%. The OTA recovery(%) of inter-assay ranged from 73% to 109% with coefficient variations of 5-9%. A good correlation was obtained from the OTA content in cereal samples detected by Nb-ELISA and LC-MS/MS.