Back to Search
Start Over
Development of monoclonal antibody-based sensitive ELISA for the determination of Cry1Ie protein in transgenic plant
- Source :
- Analytical and Bioanalytical Chemistry. 408:8231-8239
- Publication Year :
- 2016
- Publisher :
- Springer Science and Business Media LLC, 2016.
-
Abstract
- Cry1Ie is a kind of Bacillus thuringiensis (Bt) toxin protein which has a different action model than the Cry1Ab and Cry1Ac protein. The transgenic maize expressing Cry1Ie might be commercially used in the near future and it is urgent to develop a method to detect Cry1Ie protein in transgenic plants and their products. To develop an ELISA method, Cry1Ie protein was expressed in Escherichia coli strain Transetta DE3, purified with the Ni-NTA spin columns, and then validated by sequencing. Bioassay results showed that the purified Cry1Ie protein was highly toxic to the Asian corn borer. The polyclonal antibody (pAb) and the specific monoclonal antibody (mAb) 1G42D6 were generated from rabbit and mice which were immunized with Cry1Ie protein, respectively. Western blotting of crude Cry1Ie protein extracts was established by employing mAb 1G42D6, whereas the mAb 1G42D6 negligibly recognized other Bt proteins. Sandwich ELISA against Cry1Ie protein was established by coating with pAb and detecting with mAb 1G42D6. The limit of detection (LOD), the limit of quantification (LOQ), and the quantification range of the assay in different matrices of maize plant were determined as 0.27–0.51, 0.29–0.78, and 0.45–15.71 ng/mL, respectively. Recoveries of Cry1Ie protein spiked in different maize tissues ranged from 75.1 to 99.5 %. The established sandwich ELISA was verified using transgenic maize overexpressing Cry1Ie. The results in this study suggested that the established ELISA method is effective for detecting Cry1Ie protein in transgenic plants.
- Subjects :
- 0301 basic medicine
medicine.drug_class
Bacillus thuringiensis
Enzyme-Linked Immunosorbent Assay
Genetically modified crops
medicine.disease_cause
Monoclonal antibody
Zea mays
01 natural sciences
Biochemistry
Analytical Chemistry
Hemolysin Proteins
03 medical and health sciences
Bacterial Proteins
Escherichia coli
medicine
Bioassay
Genetically modified maize
Bacillus thuringiensis Toxins
biology
Chemistry
010401 analytical chemistry
Antibodies, Monoclonal
Plants, Genetically Modified
biology.organism_classification
Molecular biology
0104 chemical sciences
Endotoxins
030104 developmental biology
Cry1Ac
Polyclonal antibodies
biology.protein
Subjects
Details
- ISSN :
- 16182650 and 16182642
- Volume :
- 408
- Database :
- OpenAIRE
- Journal :
- Analytical and Bioanalytical Chemistry
- Accession number :
- edsair.doi.dedup.....f90b0ce90c991b645672b777f6cec200