Selection, affinity maturation, and characterization of a human scFv antibody against CEA protein

Abstract Background Pseudomonas aeruginosa is the leading cause of nosocomial infections, especially in people with a compromised immune system. Targeting virulence factors by neutralizing antibodies is a novel paradigm for the treatment of antibiotic-resistant pseudomonas infections. In this respect, exotoxin A is one of the most potent virulence factors in P. aeruginosa. The present study was carried out to identify a novel human scFv antibody against the P. aeruginosa exotoxin A domain I (ExoA-DI) from a human scFv phage library. Methods The recombinant ExoA-DI of P. aeruginosa was expressed in E. coli, purified by Ni-NTA column, and used for screening of human antibody phage library. A novel screening procedure was conducted to prevent the elimination of rare specific clones. The phage clone with high reactivity was evaluated by ELISA and western blot. Results Based on the results of polyclonal phage ELISA, the fifth round of biopanning leads to the isolation of several ExoA-DI reactive clones. One positive clone with high affinity was selected by monoclonal phage ELISA and used for antibody expression. The purified scFv showed high reactivity with the recombinant domain I and full-length native exotoxin A. Conclusions The purified anti-exotoxin A scFv displayed high specificity against exotoxin A. The human scFv identified in this study could be the groundwork for developing a novel therapeutic agent to control P. aeruginosa infections.

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A Biosensor-Based Characterization of the Affinity Maturation of the Immune Response Against Interferon-β and Correlations with Neutralizing Antibodies in Treated Multiple Sclerosis Patients

Journal of Interferon & Cytokine Research ◽

10.1089/jir.2008.0144 ◽

2008 ◽

Vol 28 (12) ◽

pp. 713-724 ◽

Cited By ~ 13

Author(s):

Ebrima Gibbs ◽

Joel Oger

Keyword(s):

Multiple Sclerosis ◽

Immune Response ◽

Neutralizing Antibodies ◽

Affinity Maturation ◽

Interferon Β ◽

Multiple Sclerosis Patients

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Development of a human scFv antibody targeting the lethal Iranian cobra (Naja oxiana) snake venom

Toxicon ◽

10.1016/j.toxicon.2019.10.006 ◽

2019 ◽

Vol 171 ◽

pp. 78-85

Author(s):

Fatemeh Kazemi-Lomedasht ◽

Montarop Yamabhai ◽

Jean-Marc Sabatier ◽

Mahdi Behdani ◽

Mohammad Reza Zareinejad ◽

...

Keyword(s):

Snake Venom ◽

Scfv Antibody ◽

Human Scfv ◽

Antibody Targeting

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A Highly Sensitive Detection System based on Proximity-dependent Hybridization with Computer-aided Affinity Maturation of a scFv Antibody

Scientific Reports ◽

10.1038/s41598-018-22111-4 ◽

2018 ◽

Vol 8 (1) ◽

Cited By ~ 5

Author(s):

Zhiheng Wang ◽

Yan Li ◽

Wenbin Liang ◽

Junsong Zheng ◽

Shuhui Li ◽

...

Keyword(s):

Detection System ◽

Affinity Maturation ◽

Scfv Antibody ◽

Sensitive Detection ◽

Highly Sensitive ◽

Computer Aided ◽

Highly Sensitive Detection

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Construction and characterization of a highly reactive chicken-derived single-chain variable fragment (scFv) antibody against Staphylococcus aureus developed with the T7 phage display system

International Immunopharmacology ◽

10.1016/j.intimp.2016.02.024 ◽

2016 ◽

Vol 35 ◽

pp. 149-154 ◽

Cited By ~ 7

Author(s):

Jingquan Li ◽

Yongping Xu ◽

Xitao Wang ◽

Yuan Li ◽

Lili Wang ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Phage Display ◽

Scfv Antibody ◽

Display System ◽

Single Chain Variable Fragment ◽

Single Chain ◽

T7 Phage Display ◽

T7 Phage

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BP 7 Characterization of affinity maturation in trout

Developmental & Comparative Immunology ◽

10.1016/s0145-305x(97)88536-9 ◽

1997 ◽

Vol 21 (2) ◽

pp. 100

Author(s):

H.L. Zhang ◽

I.W. Khor ◽

D.A. Evans ◽

S.L. Kaattari

Keyword(s):

Affinity Maturation

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Human scFv antibody fragments specific for the epithelial tumour marker MUC-1, selected by phage display on living cells

Cancer Immunology Immunotherapy ◽

10.1007/s002620100174 ◽

2001 ◽

Vol 50 (2) ◽

pp. 93-101 ◽

Cited By ~ 30

Author(s):

Cindy Wong ◽

Robert Waibel ◽

Michael Sheets ◽

Jean-Pierre Mach ◽

Ricarda Finnern

Keyword(s):

Phage Display ◽

Tumour Marker ◽

Living Cells ◽

Antibody Fragments ◽

Scfv Antibody ◽

Human Scfv ◽

Epithelial Tumour

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Characterization of a single-chain variable fragment (scFv) antibody directed against the human asialoglycoprotein receptor

Biotechnology and Applied Biochemistry ◽

10.1042/ba20050081 ◽

2006 ◽

Vol 44 (2) ◽

pp. 65 ◽

Cited By ~ 4

Author(s):

Guanxin Shen ◽

Limin Cao ◽

Yinchang Zhu ◽

Weiyu Wang ◽

Xiaorong Zhao ◽

...

Keyword(s):

Asialoglycoprotein Receptor ◽

Scfv Antibody ◽

Single Chain Variable Fragment ◽

Single Chain

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Developing and characterization of single chain variable fragment (scFv) antibody against frizzled 7 (Fzd7) receptor

Bioengineered ◽

10.1080/21655979.2016.1255383 ◽

2016 ◽

Vol 8 (5) ◽

pp. 501-510 ◽

Cited By ~ 12

Author(s):

Hamid Nickho ◽

Vahid Younesi ◽

Leili Aghebati-Maleki ◽

Morteza Motallebnezhad ◽

Jafar Majidi Zolbanin ◽

...

Keyword(s):

Scfv Antibody ◽

Single Chain Variable Fragment ◽

Single Chain

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Characterization of anti-interferon-γ antibodies in HIV-negative immunodeficient patients infected with unusual intracellular microorganisms

Experimental Biology and Medicine ◽

10.1177/1535370218764086 ◽

2018 ◽

Vol 243 (7) ◽

pp. 621-626 ◽

Cited By ~ 5

Author(s):

Jiraprapa Wipasa ◽

Romanee Chaiwarith ◽

Kriangkrai Chawansuntati ◽

Jutarat Praparattanapan ◽

Kritsadee Rattanathammethee ◽

...

Keyword(s):

Opportunistic Infections ◽

Interferon Γ ◽

Affinity Maturation ◽

Salmonella Spp ◽

Recombinant Interferon ◽

Intracellular Infection ◽

Downstream Signaling ◽

Fundamental Information ◽

Recall Antigen

A major characteristic of immunodeficiency associated with life-threatening intracellular infection in adults is the presence of anti-interferon-γ antibodies. Although little is known about the mechanism underlying this syndrome, it is believed that the antibodies inhibit the activity of downstream signaling pathway of interferon-γ. In this study, the characteristics of these antibodies in patients who presented, or have a history of, intracellular infection and were positive to anti-interferon-γ antibodies were investigated. The antibodies exhibited mainly the IgG1 and the IgG4 subtypes and recognized the C-terminal of the interferon-γ linear epitope containing the KRKR motif, which is required for the biological activity of interferon-γ. The antibodies bound to recombinant interferon-γ with significantly lower avidity than antibodies to a recall antigen, tetanus toxoid, suggesting that the antibodies might have not undergone affinity maturation. The data from this study may provide fundamental information to better understand the properties of anti-interferon-γ antibodies, which can be useful for future studies. Impact statement An increase in the number of immunodeficient patients related to autoantibodies to interferon (IFN)-γ has been observed particularly in East Asian adults. These patients are often presented with opportunistic infections caused by intracellular pathogens, including non-tuberculous mycobacteria (NTM), Cryptococcus neoformans, Penicillium marneffei (now called Talaromyces marneffei), and non-typhoidal Salmonella spp. The mortality rate for this syndrome is relatively high with 32% patients dying at the median time of 25 months after diagnosis. Characterization of these autoantibodies may promote better understanding of the syndrome, an emerging health problem affecting East Asia populations and impeding their welfare and economic development.

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