PJB-2019-388
Preparation and variable region sequencing of an anti-diclazuril monoclonal antibody and its application in food safety
HONG SHEN
Abstract
The present study worked toward generating an anti-diclazuril antibody for use in the establishment of an enzyme-linked immunosorbent assay (ELISA)-based method of diclazuril detection with high sensitivity and specificity that could be applied widely for measuring anti-coccidial drug residues. The artificial immunogens were investigated by the molecular modification, synthesis. Monoclonal antibodies were obtained by immunizing mice and preparation of hybridoma cell. Using the RNA of total RNA as the template, the corresponding antibody gene was re-transcribed and the corresponding antibody heavy chain variable region and the light chain variable region were obtained by PCR amplification, and cloned and sequenced. Our prepared anti-diclazuril monoclonal antibody has a half-maximal inhibitory concentration (IC50) ranging from 0.13 to 0.2 ppb, and the sensitivity of an ELISA using this antibody was 0.05 ppb. Its linear correlation coefficient, R2, is ≥0.99, and its cross-reaction with toltrazuril was <0.04%. Our work provides a method for preparation of an anti-diclazuril antibody and a protocol to determine the nucleotide sequence of the variable region of the prepared antibody. The gene sequence of the monoclonal antibody can be recombined and expressed to ensure a more stable target antibody is produced, and this represents a future direction.
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