Expression of scFv-anti-CHIKV-E2 in Escherichia coli with chaperones Co-expression, and its functional assay by electrochemical immunosensor.
J Immunoassay Immunochem
; 45(4): 307-324, 2024 Jul 03.
Article
en En
| MEDLINE
| ID: mdl-38776466
ABSTRACT
Single Chain Variable Fragment (scFv), a small fragment of antibody can be used to substitute the monoclonal antibody for diagnostic purposes. Production of scFv in Escherichia coli host has been a challenge due to the potential miss-folding and formation of inclusion bodies. This study aimed to express anti-CHIKV E2 scFv which previously designed specifically for Asian strains by co-expression of three chaperones that play a role in increasing protein solubility; GroEL, GroES, and Trigger Factor. The scFv and chaperones were expressed in Origami B E. coli host under the control of the T7 promoter, and purified using a Ni-NTA column. Functional assay of anti-CHIKV-E2 scFv was examined by electrochemical immunosensor using gold modified Screen Printed Carbon Electrode (SPCE), and characterized by differential pulses voltammetry (DPV) using K3[Fe(CN)6] redox system and scanning microscope electron (SEM). The experimental condition was optimized using the Box-Behnken design. The results showed that co-expression of chaperone increased the soluble scFv yield from 54.405 µg/mL to 220.097 µg/mL (~5×). Furthermore, scFv can be used to detect CHIKV-E2 in immunosensor electrochemistry with a detection limit of 0.74048 ng/mL and a quantification limit of 2,24388 ng/mL. Thus, the scFv-anti-CHIKV-E2 can be applied as a bioreceptor in another immunoassay method.
Palabras clave
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Técnicas Biosensibles
/
Chaperonas Moleculares
/
Escherichia coli
/
Técnicas Electroquímicas
/
Anticuerpos de Cadena Única
Idioma:
En
Revista:
J Immunoassay Immunochem
Asunto de la revista:
ALERGIA E IMUNOLOGIA
Año:
2024
Tipo del documento:
Article
País de afiliación:
Indonesia