Studies On Anti-idiotypic Nanobody Based Green Immunoassay For Citrinin

Citrinin(CIT) is a secondary metabolite produced by several fungal species of the genera Penicillium, Aspergillus, and Monascus, which is harmful to human and animals. This mycotoxin occurs on a wide range of cereals and Monascus fermentation products. Owing to the level of CIT contamination in food commodities and dietary exposure, many countries have set the maximum level of CIT in food supplements based on Monascus fermented rices. At present, there are a diversity of analysis methods for CIT, including thin layer chromatography, high-performance liquid chromatography(HPLC), immunoassays and electrochemical sensor, etc. By contrast, immunoassay methods are specific to analytes based on antibody-antigen interactions, which also have advantages such as high sensitivity, rapid sample preparation, fast detection and cost effective, etc. Thus, immunoassays are suitable for rapid screening of a large number of samples. However, huge amount of CIT standard and organic reagents are needed for synthesis of chemically-derived detection antigen, as well as in the development of an immunoassay, which causes potential hazardous to analysts and the environment. In this present study, the anti-idiotypic nanobody technology was applied for CIT molecular mimicry research. CIT substitute antigens based on nanobody were obtained, and CIT green immunoassays based on surrogate antigen or standard were developed in this research, which improved operator safety by reducing the risk of exposure to toxic reagents. The study provides a sensitive, fast, simple, cost effective and environmental friendly method to monitor CIT contamination. The main contents and innovations are as follow:1 This is the first straightforward acquisition of AIds from an alpaca na?ve nanobody library and application of nanobody technique in mimicry of CIT antigen. The green immunoassays based on nanobody are developed, which provide a new tool for other toxic small molecules detection.1.1 Development of phage-ELISA for CIT analysis. After four rounds bio-panning, one anti-idiotypic nanobody(named X27) was obtained, which showed specificbinding to paratope of CIT-Mc Ab 4G6. The selected phage-displayed VHH was used as surrogate for CIT hapten to develop an indirect competitive phage enzyme-linked immunosorbent assay(P-ELSA). After a series of optimization of coating antibody concentration, phage input, p H value, ionic strength and methanol concentration, the established assay exihibited a good sensitivity of IC50 value of 10.9 ng/m L, with a dynamic linear range of 2.5 ~ 100.0 μg/kg. Moreover, the assay was highly speci?c to CIT with negligible cross reactivity with other common mycotoxins(AFB1, OTA, ZEN, DON). The average recovery from rice samples and wheat powder ranged from 82.0% to 100.3% and 79.2% to 110.1%, respectively. Results obtained by P-ELISA of the CIT-positive samples were in good agreement with those obtained by ic-ELISA.1.2 The development of ELISA based on the nanodoby as substitute coating antigen. After expression and purification, the nanobody X27 was able to specifically bind to CIT-Mc Ab 4G6 with no cross-activity of other anti-mycotoxins Mc Abs. After optimization of coating concentration, antibody concentration, and methanol concentration, V-ELISA based on nanobody X27 as surrogate of CIT antigen was developed for CIT analysis. The method exhibited an IC50 value of 44.6 ng/m L, with a dynamic linear range of 5.0 ~ 300.0 μg/kg. There was no cross-reactivity toward four common mycotoxins(AFB1, OTA, ZEN, DON). The average recovery from rice samples and wheat powder ranged from 82.7% to 101.5% and 83.7% to 115.6%, respectively. Results obtained by V-ELISA of eight Monascus-fermented rice samples were in good agreement with those obtained by ic-ELISA. These results indicated that X27 nanobody can functionally mimic the epitope of CIT hapten and act as a surrogate antigen for monitoring of CIT contamination in Monascus-fermented samples.1.3 Determination of binding kinetics of antibody to antigen. The binding kinetics of VHH X27 or CIT-OVA to Mc Ab was estimated by bio-layer interferometry using BLItz. KDs of VHH X27 to anti-CIT Mc Ab and CIT-OVA to anti-CIT Mc Ab were 160 n M and 68 n M, respectively. Owing to the lower binding affinity between X27 and anti-CIT Mc Ab, the sensitivity of V-ELISA using VHH as a coating antigen was better than that of the conventional ic-ELISA.2 Construction of a two-humped camel na?ve nanobody library and applicationof nanobodies as CIT surrogate standard and coating antigen, which provides a way to develop a non-toxic immunoassay.2.1 Construction of a two-humped camel na?ve nanobody library. Total RNA was extracted from peripheral blood lymphocytes of ten two-humped non-immunized camels. VHH genes(~ 500 bp) were cloned by PCR using three pairs of hinge specified primers. The VHH genes library was constructed by ligating amplified VHH genes with plasmid p HEN1 and electrotransformated to E.coli TG1. The camel na?ve nanobody library contains 5.0 ×108 independent transformants. After rescued with helper phage M13K07, the titer of phage displayed VHH library is 1.2 × 1013 c.f.u/m L.2.2 The solid-phase affinity biopanning of CIT-McAb 4G6. The Gly-HCl elution and competition elution strategy was used to obtain specific anti-idiotypic VHHs from the camel na?ve VHH library. Five selected clones were found to be anti-idiotypic, as shown in its inhibitory binding to antibody by free CIT. These clones exhibited different affininty. Among them, the nanobody C1 showed a highest affininty to CIT-Mc Ab(KD = 38 n M). Moreover, the nanobody C1 kept its ability to bind to CIT-Mc Ab under treatment at 70 ℃ for 20 min, which showed a better thermal stability.2.3 The development of non-toxic ELISA based on anti-idiotypic nanobodies. The nanobody A9 was used as CIT coating antigen, two standard curves of competitive ELISA was developed based on CIT standard and surrogate standard of nanobody C1, respectively. The concentration of nanobody C1 showed excellent linear relationship with CIT concentration at the same inhibition value at the same inhibition ratio. The linear equation is y = 49.404x- 10.395(R2 = 0.9977), in which y is CIT concentration and x is the concentration of nanobody C1. The concentration of CIT in samples was calculated by a two-step calculation: the concentration of C1 was first achieved by a logistic regression from the inhibition ratio of samples, and then converted to CIT concentration by a linear equation. The assay was compared to a high-performance liquid chromatographic(HPLC) method for determination of 8 Monascus-fermented rice samples, displaying a good correlation(R2 = 0.957). In conclusion, the established non-toxic ELISA based on anti-idiotypic nanobodies could be applied inthe contamination monitoring of CIT in real samples.