Modification of the first constant domain of heavy chain enabled effective folding of functional anti‐forskolin antigen‐binding fragment for sensitive quantitative analysis

• Published in: Biotechnology progress 2019-07, Vol.35 (4), p.e2822-n/a
• Main Authors: Yusakul, Gorawit, Sakamoto, Seiichi, Tanaka, Hiroyuki, Morimoto, Satoshi
• Format: Article
• Language: English
• Subjects: Adenylate cyclase; Animals; Antibodies, Monoclonal - genetics; Antigen-Antibody Reactions; Antigens; antigen‐binding fragment antibody; Binding; Bombyx - genetics; Bombyx mori; Coleus - chemistry; Colforsin - analysis; Colforsin - immunology; Dialysis; E coli; Enzyme-Linked Immunosorbent Assay - methods; enzyme‐linked immunosorbent assay; Escherichia coli - genetics; Fab; Folding; Forskolin; Functional analysis; Gene expression; heavy chain constant domain 1; Hemolymph; Hemolymph - physiology; Immunoglobulin Fab Fragments - genetics; Immunoglobulin Fab Fragments - immunology; Immunoglobulin Fab Fragments - metabolism; Immunoglobulin G; Immunoglobulin G - genetics; Immunoglobulin G - immunology; Immunoglobulin G - metabolism; Immunoglobulin Heavy Chains - genetics; Immunoglobulin Heavy Chains - immunology; Immunoglobulin Heavy Chains - metabolism; Immunoglobulins; Inclusion bodies; Larvae; Light chains; Monoclonal antibodies; Nucleopolyhedroviruses - genetics; Polymerase chain reaction; Protein Engineering - methods; Protein Folding; Quantitative analysis; Recombinant Proteins - genetics; Recombinant Proteins - immunology; Recombinant Proteins - metabolism; refolding; Reproducibility of Results; Silkworms; Viruses
• ISSN: 8756-7938; 1520-6033
• DOI: 10.1002/btpr.2822

The assembly between heavy and light chains is a critical step of immunoglobulin (Ig) and fragment antigen‐binding (Fab) antibody expression and of their binding activity. The genes encoding Fab were obtained from hybridoma cells secreting monoclonal antibody (MAb, IgG2b) against adenylate cyclase activator forskolin (FOR). The subclass of the first constant domain of heavy chain (CH1) of IgG2b was modified to IgG1 via overlap extension polymerase chain reaction and expressed via Escherichia coli bacterial system. Since both Fabs (IgG2b and IgG1) were expressed as inclusion bodies, functional analysis was performed after in vitro refolding via stepwise dialysis. The result indicated that the folding efficiency between VH‐CH1 and VL‐CL was improved by the CH1 modification from IgG2b to IgG1 subclass, although their specificity for FOR was not altered. Effective folding of IgG1 was also observed when they were expressed in the hemolymph of silkworm larvae using the Bombyx mori nuclear polyhedrosis virus bacmid system. An indirect competitive enzyme‐linked immunosorbent assay (icELISA) was then developed for the determination of FOR using effectively prepared Fab IgG1. The sensitivity of FOR determination was in the range of 3.91–62.5 ng/mL with less than 9% relative standard deviation, implying the sensitive and reliable analysis of developed icELISA. In addition, high accuracy of the icELISA was supported by the results of spiked‐and‐recovery tests, ranging from 100.2 to 102.3%. Therefore, Fab could be utilized reliably for icELISA instead of the more expensive MAb. Collectively, this approach improved productivity of Fab and reduced the cost of antibody production.