Development of Real-Time Immuno-PCR Based on Phage Displayed an Anti-Idiotypic Nanobody for Quantitative Determination of Citrinin in Monascus

Abstract

Citrinin (CIT) is a mycotoxin that has been detected in agricultural products, feedstuff, and Monascus products. At present, research has been performed to develop methods for CIT detection, mainly through TLC, HPLC, biosensor, and immunoassay. The immunoassay method is popular with researchers because of its speed, economy, simplicity, and ease of control. However, mycotoxins are inevitably introduced during the determination. Immunoassays require the use of toxins coupled to carrier proteins or enzymes to make competitive antigens. In this study, anti-idiotypic nanobody X27 as CIT mimetic antigen was used as non-toxic surrogate reagents in immunoassay. Therefore, the X27-based real-time immuno-PCR (rtIPCR) method had been established after optimal experiments of annealing temperature and amplification efficiency of real-time PCR, concentration of coating antibody, phage X27, and methyl alcohol. The IC50 value of the established method in the present study is 9.86 ± 2.52 ng/mL, which is nearly equivalent to the traditional phage ELISA method. However, the linear range is of 0.1–1000 ng/mL, which has been broadened 10-fold compared to the phage ELISA method. Besides, the X27-based rtIPCR method has no cross-reactivity to the common mycotoxins, like aflatoxin B1 (AFB1), deoxynivalenol (DON), ochratoxin A (OTA), and zearalenone (ZEN). The method has also been applied to the determination of CIT in rice flour and flour samples, and the recovery was found to be in the range of 90.0–104.6% and 75.8–110.0% respectively. There was no significant difference in the results between the rtIPCR and UPLC–MS. The anti-idiotypic nanobody as a non-toxic surrogate of CIT makes rtIPCR a promising method for actual CIT analysis in Monascus products.

Research on the Mechanism of Action of a Citrinin and Anti-Citrinin Antibody Based on Mimotope X27

“…We established a real-time immuno-PCR method for the quantitative determination of CIT based on mimotope X27. The IC 50 value of the established method in the present study is 9.86 ± 2.52 ng/mL, which is nearly equivalent to that obtained when using the traditional phage-ELISA method [ 30 ]. The sensitivity of this method in detecting citrinin has not been significantly improved.…”

“…The above mentioned concentrations of the X27 and X27 (28-32) phages were diluted 400 and 2 times, respectively, and indirect competitive ELISA was performed with different concentrations of CIT standards. The binding CIT MAb rate of the wild-type X27 gradually decreased linearly in the presence of CIT, but X27 (28)(29)(30)(31)(32) showed no obvious change (Figure 3B). Based on the above results, the wild-type X27 would gradually lose its binding ability to anti-CIT MAb and the competition ability of CIT when the amino acids 28-32 of the wild-type X27 were mutated to alanine at the same time.…”

“…In order to verify the correctness of the docking model, X27 (28)(29)(30)(31)(32) was constructed through the five amino acids 28-32 in the WT, mutated to T28AF29AN30AR31AY32A, and the binding activity of the mutant was identified by indirect competitive ELISA. As a result, the wild-type phage had a greater binding ability of the anti-CIT monoclonal antibody than X27 (28)(29)(30)(31)(32) at the same titer. Moreover, the X27 (28-32) phages basically lost the binding ability to CIT MAb (monoclonal antibody) when both the X27 and X27 (28-32) phages were diluted by more than 400 times (Figure 3A).…”

“…The reaction was performed at 95 • C for 3 min, followed by 30 cycles of 95 • C for 30 s, 55 • C for 30 s, 72 • C for 30 s, and then 72 • C for 10 min. The purified mutant X27 (28)(29)(30)(31)(32) and phagemid pHEN-1 were digested with restriction enzymes Nco I and Not I, respectively. Afterward, the validated X27 (28-32) and phagemid pHEN-1 were ligated and transformed into TG1 competent cells by heat shock at 42 • C for 90 s [35], and cultured in LB medium (containing 100 mg/mL ampicillin) at 37 • C. Next, the positive clones were selected and sequenced.…”

“…The sensitivity of this method in detecting citrinin has not been significantly improved. However, the linearity range of the established method is 0.1–1000 ng/mL, which is 10-fold broader than that of the phage-ELISA method [ 30 ]. Here, we adopted another strategy to improve the sensitivity of the citrinin detection in immunoassays.…”