Nanobody-based electrochemical immunoassay for Bacillus thuringiensis Cry1Ab toxin by detecting the enzymatic formation of polyaniline

Abstract

We describe an electrochemical immunoassay for the Cry1Ab toxin that is produced by Bacillus thuringiensis. It is making use of a nanobody (a heavy-chain only antibody) that was selected from an immune phage displayed library. A biotinylated primary nanobody and a HRP-conjugated secondary nanobody were applied in a sandwich immunoassay where horseradish peroxidase (HRP) is used to produce polyaniline (PANI) from aniline. PANI can be easily detected by differential pulse voltammetry at a working voltage as low as 40 mV (vs. Ag/AgCl) which makes the assay fairly selective. This immunoassay for Cry1Ab has an analytical range from 0.1 to 1000 ng mL -1 and a 0.07 ng mL -1 lower limit of detection. The average recoveries of the toxin from spiked samples are in the range from 102 to 114 %, with a relative standard deviation of <7.5 %. The results demonstrated that the assay represented an attractive alternative to existing immunoassays in enabling affordable, sensitive, robust and specific determination of this toxin.

Impedimetric immunosensors for the detection of Cry1Ab protein from genetically modified maize seeds

“…An immunosensor to detect the Bacillus thuringiensis Cry1Ab protein based on surface plasmon resonance was reported [19] and exhibited satisfactory linear responses in the detection of Cry1Ab protein concentration ranges from 10 to 500 ng mL -1 and from 8 to 1000 ng mL -1 , with detection limits of 5.0 and 4.8 ng mL -1 . Another interesting study was published by Zhu et al [17] who developed an electrochemical immunoassay that indirectly detect Cry1Ab toxin by cyclic voltammetry. In this case, the authors used a HRP-conjugated secondary nanobody applied in a sandwich immunoassay where horseradish peroxidase (HRP) is used to produce polyaniline (PANI) from aniline.…”

“…However, the sensor platform was laborious due to the several immuno-recognition steps that require large number of pure antibodies to make the recognition of Cry proteins. In the work reported by Zhu et al, [17] an electrochemical immunoassay for Cry1Ab detection was also developed however it was based on nanobody with HRP-catalyzed deposition of PANI, by DPV technique, using glassy carbon electrodes. A primary nanobody and a HRP-conjugated secondary nanobody were applied in the construction of a sandwich assay.…”

“…In the field of protein-based sensors, few sensing methods to detect Cry1Ab protein using different transducers have been reported, mainly electrochemical [16][17][18] and optical methods [19]. Nevertheless, electrochemical biosensors are an attractive way to detect GMOs due to their simplicity, low-cost instrumentation and sensitive limit of detection.…”

Phage-displayed nanobody based double antibody sandwich chemiluminescent immunoassay for the detection of Cry2A toxin in cereals

“…Nanobodies provide an alternative reagent for the development of ELISA for Cry toxins, the smallest functional antigen-binding unit, are derived from heavy-chain antibodies of camelid (De Genst et al, 2006;Hamers-Casterman et al, 1993). Compared with conventional antibodies, nanobodies have high stability, high affinity, the advantages of easy to produce, and can recognize inaccessible epitopes (Shu et al, 2019;Zhu et al, 2015). Moreover, phage-displayed nanobodies can provide an advantage over the soluble nanobodies being used as reporter elements for immunoassays development.…”

Phage-Mediated Competitive Chemiluminescent Immunoassay for Detecting Cry1Ab Toxin by Using an Anti-Idiotypic Camel Nanobody

“…At present, various methodologies have been applied for GMO analysis. Among these approaches, polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA) are the two most frequently used formats. − PCR methods are highly sensitive and accurate but cannot identify the level of expression of transgenic proteins; and the requirements of skilled operator and well-equipped laboratory are non-negligible factors that limit their application. , By contrast, ELISA methods are suitable for detecting transgenic proteins expressed in GMOs with the advantages of simplicity, cost-effectiveness, and high throughput . To date, the most commonly used ELISA method for Cry proteins is double antibody sandwich ELISA (DAS-ELISA). − However, the main drawback of DAS-ELISA is the difficulty of matching between two antibodies and complicated procedures of the assay.…”