Microcystin-LR heterologous genetically engineered antibody recombinant and its binding activity improvement and application in immunoassay.
J Hazard Mater
; 406: 124596, 2021 03 15.
Article
em En
| MEDLINE
| ID: mdl-33307449
ABSTRACT
Microcystin-LR (MC-LR) is a high-toxic biohazard that pollutes ecological environment and agroproducts. In this study, a newly recombined genetically engineered antibody (AVHH-MVH) with higher thermal stability and binding activity was designed by chain shuffling and based on our previously obtained anti-MC-LR scFv and nanobody. Based on AVHH-MVH template, a capacity of 8.99 × 105 CFU/mL of phage display AVHH-MVH mutagenesis library was constructed by site-directed mutagenesis in MVH-CDR3 region, and then used for ultrasensitive mutants screening. Afterwards, a total of five positive AVHH-MVH mutants were isolated from the mutagenesis library, and their binding activity was higher than AVHH-MVH for MC-LR. The AVHH-MVH mutant 3 was cloned into pET-25b vector for soluble expression, and the concentration of target protein expressed in culture system was 43.5 mg/L. An indirect competitive enzyme-linked immunosorbent assay (IC-ELISA) was established based on purified AVHH-MVH mutant 3 protein, and it showed ultrasensitive binding activity for MC-LR with the detection limit of 0.0075 µg/L, which was far below the maximum residue limit standard of 1.0 µg/L in drinking water proposed by World Health Organization. The established IC-ELISA shows good accuracy, repeatability, stability and applicability for MC-LR spiked samples, and it is promising for MC-LR ultrasensitive monitoring.
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Base de dados:
MEDLINE
Assunto principal:
Microcistinas
/
Toxinas Marinhas
Idioma:
En
Ano de publicação:
2021
Tipo de documento:
Article