Combination Screening of a Naïve Antibody Library Using <i>E. coli</i> Display and Single-Step Colony Assay
The use of single-domain camelid antibodies, termed VHHs or nanobodies, has found increasing application in diagnosis, pharmaceutical development, and research because of their superior properties, such as small size, elevated stability, high water solubility, and excellent affinity for the antigen....
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| Format: | Article |
| Language: | English |
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MDPI AG
2024-01-01
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| Series: | Applied Microbiology |
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| Online Access: | https://www.mdpi.com/2673-8007/4/1/16 |
| _version_ | 1797242237336158208 |
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| author | Mieko Kato Yoshiro Hanyu |
| author_facet | Mieko Kato Yoshiro Hanyu |
| author_sort | Mieko Kato |
| collection | DOAJ |
| description | The use of single-domain camelid antibodies, termed VHHs or nanobodies, has found increasing application in diagnosis, pharmaceutical development, and research because of their superior properties, such as small size, elevated stability, high water solubility, and excellent affinity for the antigen. Antigen-specific VHHs are generated by screening VHH display libraries via bio-panning. However, the bio-panning step needs to be repeated multiple times, which is time-consuming and laborious. Here, we developed a simple and rapid screening method that combined <i>Escherichia coli</i> display and a single-step colony assay to successfully identify positive clones from a naïve VHH library. The library was constructed from peripheral blood mononuclear cells of alpaca, and VHHs were displayed on the surface of <i>E. coli</i> using the inverse autotransporter intimin. Libraries enriched by magnetic cell sorting were screened directly using a single-step colony assay. Colonies formed on the hydrophilic filter and antigen-coated membrane. The expression of VHHs was induced, and those bound to the antigen on the membrane were detected as positive clones. Screening and identification of positive clones required only two days, which saves considerable time and resources compared to existing protocols. |
| first_indexed | 2024-04-24T18:36:02Z |
| format | Article |
| id | doaj.art-cd7d46eecf19420abf305219af9bc2f5 |
| institution | Directory Open Access Journal |
| issn | 2673-8007 |
| language | English |
| last_indexed | 2024-04-24T18:36:02Z |
| publishDate | 2024-01-01 |
| publisher | MDPI AG |
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| series | Applied Microbiology |
| spelling | doaj.art-cd7d46eecf19420abf305219af9bc2f52024-03-27T13:18:57ZengMDPI AGApplied Microbiology2673-80072024-01-014123724910.3390/applmicrobiol4010016Combination Screening of a Naïve Antibody Library Using <i>E. coli</i> Display and Single-Step Colony AssayMieko Kato0Yoshiro Hanyu1Department of Biochemistry, Bio-Peak Co., Ltd., 584-70 Shimonojo, Takasaki 370-0854, JapanBiomaterials Research Group, Health and Medical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1 Higashi, Tsukuba 305-8566, JapanThe use of single-domain camelid antibodies, termed VHHs or nanobodies, has found increasing application in diagnosis, pharmaceutical development, and research because of their superior properties, such as small size, elevated stability, high water solubility, and excellent affinity for the antigen. Antigen-specific VHHs are generated by screening VHH display libraries via bio-panning. However, the bio-panning step needs to be repeated multiple times, which is time-consuming and laborious. Here, we developed a simple and rapid screening method that combined <i>Escherichia coli</i> display and a single-step colony assay to successfully identify positive clones from a naïve VHH library. The library was constructed from peripheral blood mononuclear cells of alpaca, and VHHs were displayed on the surface of <i>E. coli</i> using the inverse autotransporter intimin. Libraries enriched by magnetic cell sorting were screened directly using a single-step colony assay. Colonies formed on the hydrophilic filter and antigen-coated membrane. The expression of VHHs was induced, and those bound to the antigen on the membrane were detected as positive clones. Screening and identification of positive clones required only two days, which saves considerable time and resources compared to existing protocols.https://www.mdpi.com/2673-8007/4/1/16<i>E. coli</i>displayVHHcolony assayscreeningantibody |
| spellingShingle | Mieko Kato Yoshiro Hanyu Combination Screening of a Naïve Antibody Library Using <i>E. coli</i> Display and Single-Step Colony Assay Applied Microbiology <i>E. coli</i> display VHH colony assay screening antibody |
| title | Combination Screening of a Naïve Antibody Library Using <i>E. coli</i> Display and Single-Step Colony Assay |
| title_full | Combination Screening of a Naïve Antibody Library Using <i>E. coli</i> Display and Single-Step Colony Assay |
| title_fullStr | Combination Screening of a Naïve Antibody Library Using <i>E. coli</i> Display and Single-Step Colony Assay |
| title_full_unstemmed | Combination Screening of a Naïve Antibody Library Using <i>E. coli</i> Display and Single-Step Colony Assay |
| title_short | Combination Screening of a Naïve Antibody Library Using <i>E. coli</i> Display and Single-Step Colony Assay |
| title_sort | combination screening of a naive antibody library using i e coli i display and single step colony assay |
| topic | <i>E. coli</i> display VHH colony assay screening antibody |
| url | https://www.mdpi.com/2673-8007/4/1/16 |
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