Production and characterization of antibody against Opisthorchis viverrini via phage display and molecular simulation

Abstract

In this study, a key issue to be addressed is the safe disposal of hybridoma instability. Hybridoma technology was used to produce anti-O. viverrini monoclonal antibody. Previous studies have shown that antibody production via antibody phage display can sustain the hybridoma technique. This paper presents the utility of antibody phage display technology for producing the phage displayed KKU505 Fab fragment and using experiments in concomitant with molecular simulation for characterization. The phage displayed KKU505 Fab fragment and characterization were successfully carried out. The KKU505 hybridoma cell line producing anti-O. viverrini antibody predicted to bind to myosin was used to synthesize cDNA so as to amplify the heavy chain and the light chain sequences. The KKU505 displayed phage was constructed and characterized by a molecular modeling in which the KKU505 Fab fragment and -O. viverrini myosin head were docked computationally and it is assumed that the Fab fragment was specific to -O. viverrini on the basis of mass spectrometry and Western blot. This complex interaction was confirmed by molecular simulation. Furthermore, the KKU505 displayed phage was validated using indirect enzyme-linked immunosorbent assays (ELISA) and immunohistochemistry. It is worthy to note that ELISA and immunohistochemistry results confirmed that the Fab fragment was specific to the -O. viverrini antigen. Results indicated that the approach presented herein can generate anti-O. viverrini antibody via the phage display technology. This study integrates the use of phage display technology together with molecular simulation for further development of monoclonal antibody production. Furthermore, the presented work has profound implications for antibody production, particularly by solving the problem of hybridoma stability issues.

Fig 1. Conceptual framework for this study.

This framework can be divided to 4 major cluster; Construction of phagemid vector carrying KKU505 Fab sequence (blue dashed box), characterization of KKU505 Fab fragment by experiment (green dashed box) and molecular simulation (yellow dashed box), and Thanan et al. study (red dashed box).

Fig 2. Validation of phage display KKU505 Fab fragment by indirect ELISA.

Crude OV antigens at concentration of 1 μg/well were captured on wells. 10¹² pfu/well of KKU505 Fab phage 1 and 8, 1H10 phage (phage display anti-MICA Fab fragment) and VSCM13 phage (helper phage) was performed using indirect ELISA, and traced by HRP-conjugated anti-M13 major coat protein antibody and 1H10 phage was used as a negative control. This phage ELISA signals ware normalized by reaction signal without phage (mean OD is 0.199).

Fig 3. Relation curve between crude OV antigens and bovine serum albumin (BSA) concentration and phage detection by indirect ELISA.

A standard curve was plotted with crude OV antigens bovine serum albumin (BSA) with concentrations ranging from 0.01 μg/well to 2 μg/well. 10¹² pfu/well of KKU505 Fab phage 8 was used as a detector using indirect ELISA technique. This phage ELISA signals ware normalized by reaction signal without antigens (mean OD is 0.224).

Fig 4. Immunohistochemistry staining on the biliary system of mouse infected with -O. viverrini.

Comparison of Immunohistochemistry staining on the biliary system of mouse infected with -O. viverrini which detected by PBS and KKU505 monoclonal antibody as a monoclonal system and KKU505 phage 1 and 8 and 1H10 phage (phage display anti-MICA Fab fragment) as a phage system. PBS and 1H10 phage as negative control of monoclonal system and phage system, respectively. KKU505 phage 1 and 8 for localization pattern comparison of phage display KKU505 Fab fragment specific to OV antigens and KKU505 mAb. “Conjugated” is conjugated control. “mAb” is KKU505 mAb. “1H10” is phage display anti-1H10 antibody. “Phage 1 and 8” is phage display antibody KKU508 clone 1 and 8, respectively.

Fig 5. Structure and sequence of KKU505 Fab modelling.

A. The 3D structure of KKU505 Fab was predicted using the I-TASSER server. This structure was colored following IMGT (cyan: Variable region and gray: Constant region). B. Electrostatic potential of the 3D structure of KKU505 Fab colored according to charge (blue: Positive charge and red: Negative charge). C. The sequence of KKU505 Fab was colored following IMGT (cyan: Variable region, gray: Constant region, red: HC-CDR1, orange: HC-CDR2, purple: HC-CDR3, blue: LC-CDR1, green: LC-CDR2 and forest: LC-CDR3). “HC” is heavy chain. “LC” is light chain. “CDR” is complementarity determining region.

Fig 6. Antibody–antigen docking output.

Molecular interactions between KKU505 Fab (green schematic ribbon) and OV myosin head (wheat surface) as antibody–antigen complex output by ClusPro 2.0. Zoomed regions show interactions of this antibody–antigen complex. Side-chains are ball and stick. Hydrogen and ionic bonds are dotted lines. Amino acids are abbreviated with a capital. The number is residue position.