Monoclonal Antibody Generation and Characterization for Vip3Aa20 Protein Quantification in Transgenic Corn Plants

by Daily Hernández, Hasel Aragón, Marcos González, Gabriela González, Amarilis González, Andrés Tamayo, Ivis Morán, Pilar Téllez, Eduardo Sánchez, Miguel Castillo, Williams Ferro, Camilo Ayra, and Rodolfo Valdés
Volume 15, Issue 3 (Fall 2016)

Numerous standardized techniques for detection and quantification of proteins are based on polyclonal antibody (pAb) use. However, because pAbs are a heterogeneous mixture of antibodies, there is the possibility of non-specific interactions or cross-reactions with non-related proteins, which is a disadvantage in the detection and quantification of target proteins. Therefore, the main objective of this study was to generate and characterize monoclonal antibodies (mAbs) for quantifying the Vip3Aa20 protein of Bacillus thuringiensis (Bt) expressed in event MIR162 transgenic corn plants.

The Vip3Aa20 bioinsecticidal protein of Bt shares 99.7% of homology with the Vip3Aa1 protein, which has already been successfully isolated by affinity chromatography on metal chelate. For this reason, Vip3Aa1 was chosen as an antigen for obtaining mouse mAbs directed against these Bt δ-endotoxins. The mAbs were characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)/Western blot, high-pressure liquid chromatography gel filtration (HPLC-GF), enzyme-linked immunosorbent assay (ELISA), and association constant. In general, results showed that the use of the highly purified mAbs not only allowed the specific and accurate quantification of Vip3Aa20 in extracts of macerated leaves of event MIR162 transgenic corn plants, but also statistically discriminated (p=0.000) between homozygous (range: 180–360 ng/mL, mean ± standard deviation [SD]: 241.7 ± 43.2 ng/mL) and hemizygous (range: 0–140 ng/mL, mean ± SD: 51.7 ± 40.8 ng/mL) transgenic corn plants grown under the same experimental conditions...

Hernández D, Aragón H, González M, González G, González A, Tamayo A, Morán I, Téllez P, Sánchez E, Castillo M, Ferro W, Ayra C, Valdés R. Monoclonal antibody generation and characterization for Vip3Aa20 protein quantification in transgenic corn plants. BioProcess J, 2016; 15(3): 30–43.

Posted online November 15, 2016.