Development of Monoclonal Antibodies Recognizing Linear Epitope: Illustration by Three Bacillus thuringiensis Crystal Proteins of Genetically Modified Cotton, Maize, and Tobacco

Abstract

Bacillus thuringiensis Cry1Ac, Cry1Ia1, and Cry1Ie are δ-endotoxin insecticidal proteins widely implemented in genetically modified organisms (GMO), such as cotton, maize, and potato. Western blot assay integrates electrophoresis separation power and antibody high specificity for monitoring specific exogenous proteins expressed in GMO. Procedures for evoking monoclonal antibody (mAb) for Western blot were poorly documented. In the present study, Cry1Ac partially denatured at 100 °C for 5 min was used as an immunogen to develop mAbs selectively recognizing a linear epitope of Cry1Ac for Western blot. mAb 5E9C6 and 3E6E2 selected with sandwich ELISA strongly recognized the heat semidenatured Cry1Ac. Particularly, 3E6E2 recognized both E. coli and cotton seed expressed Cry1Ac in Western blot. Such strategy of using partially denatured proteins as immunogens and using sandwich ELISA for mAb screening was also successfully demonstrated with production of mAbs against Cry1Ie for Western blot assay in maize.

Immunoglobulin E and immunoglobulin G cross-reactive allergens and epitopes between cow milk αS1-casein and soybean proteins

“…Anti-α S1 -casein monoclonal antibodies were prepared according to the methods of Cao et al (2017) and Restani et al (1999) with some modifications. Briefly, Balb/c mice were injected intraperitoneally with 50 mg of total α S1 -casein (purity ≥ 85%; purification of α S1casein from raw CM in our laboratory according to the method of Hodgkinson et al, 2012) solution mixed with Freund's adjuvant.…”

“…First, the gene sequences of α S1 -casein and Gly m Bd 60K protein were retrieved by Gen Bank (https: / / www .ncbi .nlm .nih .gov/ genbank/ ), then the AA sequences of α S1 -casein and Gly m Bd 60K protein were derived by DNAStar software (version 8.0; https: / / www .dnastar .com/ ). Amino acid sequence alignment was carried out by Blastp software at NCBI web (Li et al, 2017;Foroutan et al, 2018).…”

Urea-doped carbon dots as fluorescent switches for the selective detection of iodide ions and their mechanistic study

“…31 Cotton is one of the most abundantly available and important material resources, which can be comprehensively used. [32][33][34] The main component of white cotton is cellulose, which contains plenty of hydroxyl groups. Herein, considering the environmental concerns, we report a green strategy for the synthesis of highly uorescent CDs with white cotton as the raw material and urea as the nitrogen source.…”