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

Gorawit Yusakul, Seiichi Sakamoto, Hiroyuki Tanaka, Satoshi Morimoto

研究成果: ジャーナルへの寄稿記事

抄録

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.

元の言語英語
記事番号e2822
ジャーナルBiotechnology Progress
35
発行部数4
DOI
出版物ステータス出版済み - 1 1 2019

Fingerprint

Colforsin
Immunoglobulin G
Antigens
Enzyme-Linked Immunosorbent Assay
Bombyx
Nucleopolyhedrovirus
Immunoglobulin Fragments
Hemolymph
Inclusion Bodies
Hybridomas
Adenylyl Cyclases
Antibody Formation
Larva
Dialysis
Monoclonal Antibodies
Escherichia coli
Light
Costs and Cost Analysis
Polymerase Chain Reaction
Antibodies

All Science Journal Classification (ASJC) codes

  • Biotechnology

これを引用

@article{550583e8ef0a4e8680a3bfe0e99e3a9a,
title = "Modification of the first constant domain of heavy chain enabled effective folding of functional anti-forskolin antigen-binding fragment for sensitive quantitative analysis",
abstract = "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.",
author = "Gorawit Yusakul and Seiichi Sakamoto and Hiroyuki Tanaka and Satoshi Morimoto",
year = "2019",
month = "1",
day = "1",
doi = "10.1002/btpr.2822",
language = "English",
volume = "35",
journal = "Biotechnology Progress",
issn = "8756-7938",
publisher = "John Wiley and Sons Ltd",
number = "4",

}

TY - JOUR

T1 - Modification of the first constant domain of heavy chain enabled effective folding of functional anti-forskolin antigen-binding fragment for sensitive quantitative analysis

AU - Yusakul, Gorawit

AU - Sakamoto, Seiichi

AU - Tanaka, Hiroyuki

AU - Morimoto, Satoshi

PY - 2019/1/1

Y1 - 2019/1/1

N2 - 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.

AB - 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.

UR - http://www.scopus.com/inward/record.url?scp=85065170295&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=85065170295&partnerID=8YFLogxK

U2 - 10.1002/btpr.2822

DO - 10.1002/btpr.2822

M3 - Article

AN - SCOPUS:85065170295

VL - 35

JO - Biotechnology Progress

JF - Biotechnology Progress

SN - 8756-7938

IS - 4

M1 - e2822

ER -