Boosted Electrochemical Immunosensing of Genetically Modified Crop Markers Using Nanobody and Mesoporous Carbon.

The problems of environmental security and the potential risks of human health caused by transgenic crops have attracted much attention. Recent studies reveal 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) from Agrobacterium sp. strain CP4 protein (CP4-EPSPS), which shows very high resistance to herbicide glyphosate, is a typical biomarker of genetically modified (GM) crops. For this reason, it is highly anticipated to devise a sensitive and convenient strategy to detect CP4-EPSPS protein in crops. Herein, we report a simple electrochemical immunosensor by coupling nanobody, ordered mesoporous carbon (OMC), and thionine (Th). As a capture agent, the nanobody was screened out from an immunized Bactrian camel, and exhibited superior properties with respect to conventional antibody, such as higher stability and stronger heat resistance. Moreover, OMC offered an effective platform with high surface area, electrical conductivity, and biocompatibility, which greatly facilitated the assembly of redox probe Th, and further coupling of large amount of capture nanobodies. As a result, the CP4-EPSPS protein could be determined with high sensitivity and efficiency by differential pulse voltammetry (DPV) in a wide linear range from 0.001 to 100 ng·mL-1 with a low detection limit of 0.72 pg·mL-1, which was more than 3 orders of magnitude lower than those of previously reported works. As an example, the proposed electrochemical immunosensor was successfully applied to spiked samples, demonstrating its great potential in CP4-EPSPS screening and detection.

In silico peptide prediction for antibody generation to recognize 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) in genetically modified organisms.

For the prospective immunorecognition of 5-enolpyruvylshikimate-3-phosphate synthase (CP4-EPSPS) as a biomarker protein expressed by transgenic soybean, an extensive in silico evaluation of the referred protein was performed. The main objective of this study was the selection of a set of peptides that could function as potential immunogens for the production of novel antibodies against CP4-EPSPS protein. For this purpose, the protein was in silico cleaved with trypsin/chymotrypsin and the resultant peptides were extensively analyzed for further selection of the best candidates for antibody production. The analysis enabled the successful proposal of four peptides with potential immunogenicity for their future use as screening biomarkers of genetically modified organisms. To our knowledge, this is the first attempt to select and define potential linear epitopes for the immunization of animals and, subsequently, to generate adequate antibodies for CP4-EPSPS recognition. The present work will be followed by the synthesis of the candidate peptides to be incubated in animals for antibody generation and potential applicability for the development of an immunosensor for CP4-EPSPS detection.

Serum testing of genetically modified soybeans with special emphasis on potential allergenicity of the heterologous protein CP4 EPSPS.

Roundup Ready soy contains the CP4-enolpyruvylshikimate-3-phosphate synthase (CP4 EPSPS) protein. Serum IgE from two distinct populations of soy-allergic patients were recruited to determine their IgE-binding specificity. One population consisted of 10 adult patients from Europe, whose primary diagnosis was soy food allergy with some also having mite allergy. In addition, 6 primarily mite-allergic, 6 food-allergic (celery, carrot, milk, shrimp, walnut, and apple), and 5 non-allergic patients were tested. Another population consisted of 13 children from Korea, whose primary diagnosis was atopic dermatitis and secondarily soy and egg sensitization. In addition, 11 non-allergic patients were tested. Each patient population was extensively characterized with respect to clinical symptoms, specific IgE (CAP) scores, and total IgE. Immunoblots and ELISA assays were developed using serum IgE from these patients and soy extracts, CP4 EPSPS, rice extract, ovalbumin, rubisco, purified major peanut allergen Ara h 2, the putative soy allergen Gly m Bd 30k and mite allergen Der f 2 proteins as the intended targets. Immunoblot results indicated that soy-allergic patients bound soy extracts but did not specifically bind rubisco or CP4 EPSPS. ELISA results were in general agreement with the immunoblot results except that rubisco bound significant quantities of serum IgE from some patients. These results indicate that the CP4 EPSPS protein does not bind significant quantities of IgE from two geographically distinct sensitive populations and there is no evidence for an increased allergenic potential of this biotech protein.

[Preparation and characterization of monoclonal antibody against CP4-EPSPS].

AIM: To prepare monoclonal antibody (mAb) against CP4-EPSPS, which could be applied to the development of gold colloidal rapid diagnostic kit for the specific detection of GMO. METHODS: BALB/c mice were immunized with CP4-EPSPS. Splenocytes of the immunized mice were collected and fused with the mouse myeloma cell line Sp2/0 cells. The hybridoma cells that secreted CP4-EPSPS mAb antibodies were cloned with limited dilution method. The immunoglobulin (Ig) subtype, the ascites titers, the binding site, and the affinity of the obtained mAbs were determined by indirect ELISA. The specificity of mAbs was tested by ELISA and Western blot analysis. RESULTS: From over 80 positive hybridomas which secreted anti-cp4-EPSPS mAbs, a pair of hybridomas were screened out, designated III5A3 and III13A2. Chromosome analysis revealed that the obtained hybridomas were with the universal characteristics of the monoclonal hybridoma cells which secreted mAb, and the Ig subtype of III5A3 and III13A2 mAb was both IgG1. The ascites titers of III5A3 and III13A2 mAb were 1:10(6) and 1:10(8), respectively. Our study also demonstrated that these two mAbs, which recognized the same epitope, could both specifically bind to the CP4-EPSPS protein. The relative affinity constant of III5A3 and III13A2 mAb was determined as 10(5) and 10(6) respectively. CONCLUSION: A pair of high titer, specific mAbs against CP4-EPSPS have been successfully prepared and primarily identified, which may be useful in the development of a rapid and convenient diagnostic kit for detection of GMO.

Improved ELISA method for screening human antigen-specific IgE and its application for monitoring specific IgE for novel proteins in genetically modified foods.

For monitoring the occurrence of IgE antibody specific for novel proteins in genetically modified (GM) foods, ELISA is the most convenient method. The levels of IgE specific for recombinant proteins, phosphinothricin-N-acetyltransferase (PAT), CP4-EPSPS, and Cry9C were determined by ELISA using the sera from patients allergic to known allergens. Ovalbumin (OVA) and OVA-positive patient sera were used as positive control. In the ELISA, 20-fold-diluted sera tested were mostly negative for the specific IgE. However, the PAT-specific, but not CP4-EPSPS- or Cry9C-specific IgE in some patients was apparently higher than that of the healthy volunteers. To clarify the binding specificity of the antibody, we pre-incubated the sera with soluble PAT, but the inhibition was marginal, suggesting that the binding was non-specific. Therefore, we used 1M NaCl as a washing buffer to remove IgE non-specifically bound to the coated PAT. This washing step efficiently decreased non-specific binding. In contrast, OVA-specific IgE binding to OVA-coated plate was not affected by the washing. Finally, in this pilot study significant levels of IgE antibodies specific for the three proteins were not detected in the sera of Japanese food-allergy patients.