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Generation and characterization of anti-α-enolase single-chain antibodies in chicken
It was previously reported that up-regulation of α-enolase protein was detected in 65% of patients with non-small cell lung cancers (NSCLC). Moreover, a high titer of anti-α-enolase antibodies was developed in a smaller proportion (7.4%) of these patients than in non-tumor-associated patients and he...
Autores principales: | , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Elsevier B.V.
2010
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7112641/ https://www.ncbi.nlm.nih.gov/pubmed/20655599 http://dx.doi.org/10.1016/j.vetimm.2010.06.001 |
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author | Leu, Sy-Jye Lee, Yu-Ching Shih, Neng-Yao Huang, I-Jen Liu, Ko-Jiunn Lu, Hsu-Feng Huang, Shih-Yi Yang, Yi-Yuan |
author_facet | Leu, Sy-Jye Lee, Yu-Ching Shih, Neng-Yao Huang, I-Jen Liu, Ko-Jiunn Lu, Hsu-Feng Huang, Shih-Yi Yang, Yi-Yuan |
author_sort | Leu, Sy-Jye |
collection | PubMed |
description | It was previously reported that up-regulation of α-enolase protein was detected in 65% of patients with non-small cell lung cancers (NSCLC). Moreover, a high titer of anti-α-enolase antibodies was developed in a smaller proportion (7.4%) of these patients than in non-tumor-associated patients and healthy subjects. In the present study, we characterized polyclonal and single-chain variable fragment (scFv) anti-α-enolase antibodies from immunized chickens. The E. coli-derived recombinant α-enolase protein was purified to its high homogenicity as verified by SDS-PAGE. After the 4th immunization, a high titer of specific polyclonal anti-α-enolase antibodies was elicited in immunized chickens and specifically recognized the purified human α-enolase antigen as determined by Western blot and ELISA. The expressed heavy and light chain variable genes (VH and VL) were isolated from spleen B cells and amplified to construct phage antibody libraries containing scFv molecules. After four rounds of panning selection, the scFv antibodies of randomly chosen clones were expressed and their binding specificity to α-enolase protein was verified using competitive ELISA, flow cytometry and immunofluorescence staining. Nucleotide sequence analysis from 10 α-enolase binding clones showed that 3 (30%) clones used identical heavy and light genes for scFv antibody expression, as represented by EnL5. Notably, amino acid changes in complementarity-determining regions (CDRs) were more frequently observed than those in framework regions (FRs) in all clones, indicating a strong affinity selection through mutations. All together, it is believed that these polyclonal and scFv IgY antibodies may be helpful in the development of molecular diagnostic and therapeutic agents for lung cancers in the future. |
format | Online Article Text |
id | pubmed-7112641 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2010 |
publisher | Elsevier B.V. |
record_format | MEDLINE/PubMed |
spelling | pubmed-71126412020-04-02 Generation and characterization of anti-α-enolase single-chain antibodies in chicken Leu, Sy-Jye Lee, Yu-Ching Shih, Neng-Yao Huang, I-Jen Liu, Ko-Jiunn Lu, Hsu-Feng Huang, Shih-Yi Yang, Yi-Yuan Vet Immunol Immunopathol Article It was previously reported that up-regulation of α-enolase protein was detected in 65% of patients with non-small cell lung cancers (NSCLC). Moreover, a high titer of anti-α-enolase antibodies was developed in a smaller proportion (7.4%) of these patients than in non-tumor-associated patients and healthy subjects. In the present study, we characterized polyclonal and single-chain variable fragment (scFv) anti-α-enolase antibodies from immunized chickens. The E. coli-derived recombinant α-enolase protein was purified to its high homogenicity as verified by SDS-PAGE. After the 4th immunization, a high titer of specific polyclonal anti-α-enolase antibodies was elicited in immunized chickens and specifically recognized the purified human α-enolase antigen as determined by Western blot and ELISA. The expressed heavy and light chain variable genes (VH and VL) were isolated from spleen B cells and amplified to construct phage antibody libraries containing scFv molecules. After four rounds of panning selection, the scFv antibodies of randomly chosen clones were expressed and their binding specificity to α-enolase protein was verified using competitive ELISA, flow cytometry and immunofluorescence staining. Nucleotide sequence analysis from 10 α-enolase binding clones showed that 3 (30%) clones used identical heavy and light genes for scFv antibody expression, as represented by EnL5. Notably, amino acid changes in complementarity-determining regions (CDRs) were more frequently observed than those in framework regions (FRs) in all clones, indicating a strong affinity selection through mutations. All together, it is believed that these polyclonal and scFv IgY antibodies may be helpful in the development of molecular diagnostic and therapeutic agents for lung cancers in the future. Elsevier B.V. 2010-10-15 2010-06-22 /pmc/articles/PMC7112641/ /pubmed/20655599 http://dx.doi.org/10.1016/j.vetimm.2010.06.001 Text en Copyright © 2010 Elsevier B.V. All rights reserved. Since January 2020 Elsevier has created a COVID-19 resource centre with free information in English and Mandarin on the novel coronavirus COVID-19. The COVID-19 resource centre is hosted on Elsevier Connect, the company's public news and information website. Elsevier hereby grants permission to make all its COVID-19-related research that is available on the COVID-19 resource centre - including this research content - immediately available in PubMed Central and other publicly funded repositories, such as the WHO COVID database with rights for unrestricted research re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for free by Elsevier for as long as the COVID-19 resource centre remains active. |
spellingShingle | Article Leu, Sy-Jye Lee, Yu-Ching Shih, Neng-Yao Huang, I-Jen Liu, Ko-Jiunn Lu, Hsu-Feng Huang, Shih-Yi Yang, Yi-Yuan Generation and characterization of anti-α-enolase single-chain antibodies in chicken |
title | Generation and characterization of anti-α-enolase single-chain antibodies in chicken |
title_full | Generation and characterization of anti-α-enolase single-chain antibodies in chicken |
title_fullStr | Generation and characterization of anti-α-enolase single-chain antibodies in chicken |
title_full_unstemmed | Generation and characterization of anti-α-enolase single-chain antibodies in chicken |
title_short | Generation and characterization of anti-α-enolase single-chain antibodies in chicken |
title_sort | generation and characterization of anti-α-enolase single-chain antibodies in chicken |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7112641/ https://www.ncbi.nlm.nih.gov/pubmed/20655599 http://dx.doi.org/10.1016/j.vetimm.2010.06.001 |
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