Production of soluble single chain variable fragment (scFv) against Chikungunya virus in Escherichia coli Origami B (DE3) and its application as bioreceptor in immunosensor
Shabarni Gaffar(1,2,3*), Siti Hesti Nurbayanti(1), Yeni Wahyuni Hartati(1), Mia Tria Novianti(2), Korry Novitriani(4), Safri Ishmayana(1), Muhammad Yusuf(1,3), Toto Subroto(1)

(1)Departement of Chemistry, Faculty of Mathematics and Natural Sciences, Universitas Padjadjaran, Bandung, Indonesia
(2)Research Center of Molecular Biology and Bioinformatic, Universitas Padjadjaran, Bandung, Indonesia
(3)Graduate School, Universitas Padjadjaran, Bandung, Indonesia
(4)Departement of Medical Laboratory Technology, Universitas Bakti Tunas Husada, Tasikmalaya, Indonesia.

Abstract

Chikungunya is an infectious disease caused by the chikungunya virus (CHIKV) with general symptoms of fever, skin rash, and joint pain that can last a long time. The available method for CHIKV detection is immunochromatography by using monoclonal antibodies to detect the E2 surface protein, since it was known to play a role in the attachment of CHIKV to the host cells. As a substitute for monoclonal antibodies, Single Chain Variable Fragment (scFv) can be used, which is part of the antibody that still has binding sites for E2. This study aimed to express anti-CHIKV E2 scFv in the Escherichia coli expression system and its application as bioreceptor in electrochemical immunosensor. The E. coli Origami B host cells carrying the pET21B-scFv plasmids were used to express scFv under control of the T7 promoter, and purified using a NiNTA column. Then, the scFv was immobilized on the surface of the 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 scFv was successfully expressed by obtaining a band of 30 kDa. Comparison of the expression results showed that co-expression of chaperone increased the soluble protein yield from 54.405 ug/mL to 220.097 ug/mL (5x). Furthermore, scFv can be used to detect CHIKV E2 in immunosensor electrochemistry with the optimum condition: scFv concentration (5.5 ug/ml), scFv immobilization (15 minutes) and CHIKV E2 incubation (45 minutes). The detection limit using the standard CHIKV E2 is 0.74048 ng/mL and the quantification limit of 2,24388 ng/mL. Thus, the scFv anti CHIKV E2 has the potential to be applied as a bioreceptor in another immunoassay method such as rapid test and ELISA.

Keywords: Chikungunya, E. coli Origami B, soluble scFv, immunosensor electrochemistry.

Topic: Biokimia Medis