Sensitive detection of Bacillus thuringiensis Cry1B toxin based on camel single-domain antibodies

Abstract

Bt Cry1B toxin, a residue in insect-resistant transgenic plants, has been identified to be harmful to human health. Therefore, it is urgent to detect the Cry1B toxin level in each kind of transgenic plant. Nbs, with prominently unique physiochemical properties, are becoming more and more promising tools in the detection of target antigens. In this study, an immune phage display library that was of high quality was successfully constructed for the screening of Cry1B-specific Nbs with excellent specificity, affinity, and thermostable. Subsequently, a novel sandwich ELISA for Cry1B detection was established, which was based on the biotin-streptavidin system using these aforementioned Nbs. This established detection system presented a linear working range from 5 to 1000 ng ml^-1 and a low detection limit of 3.46 ng ml^-1 . The recoveries from spiked samples were in the range of 82.51%-113.56% with a relative standard deviation (RSD) lower than 5.00%. Taken together, the proposed sandwich ELISA would be a potential method for the detection of Cry1B toxin in transgenic Bt plants specifically and sensitively.

Keywords: Cry1B; biotin-streptavidin system; nanobody; phage display; sandwich ELISA.

Figure 1

Identification of Cry1B‐specific Nbs. (a) 68 positive clones (ratio ≥3) were selected from PE‐ELISA. (b) Three kinds of different amino acid sequences of anti‐Cry1B Nbs were identified. (c) Three Nbs having different sequences were purified

Figure 2

Feature analysis of purified Cry1B Nbs. (a) The specificity of purified Cry1B Nbs was tested by indirect ELISA with different types of Cry toxins. (b) The equilibrium dissociation constants between Cry1B and the paired Nbs (Nb2 and Nb3) was measured with the kinetic analysis by SPRi. Cry1B dilutions were injected at concentrations of 1, 3, 9, 27, 81, 243, and 729 nmol L^‐1 (C7–C1). (c) Relevant parameters of kinetic analysis. (d) The thermal stability of Nb2 and Nb3 was measured by a fluorescence‐based assay with Sypro Orange dye. The temperature was set from 25 to 98°C in increments of 1°C and three repeats were performed

Figure 3

Cry1B detection by the improved sandwich ELISA and recoveries study. (a) Outline of strategies to detect Cry1B by sandwich ELISA based on biotin–streptavidin system. (b) Calibration curve for Cry1B detection. The linear relationship between the absorbance at 450 nm and the Cry1B concentration was in the range from 5 to 1,000 ng ml^‐1. (c) Recoveries of Cry1B toxin from spiked samples. (d) Determination of real Bt rice sample. The leaves from a Bt rice that could express Cry1B toxin and the corresponding nontransformed rice leaves used as control were detected by the proposed sandwich ELISA