Isolation of rhinovirus-specific Fab antibodies from universal phage display antibody libraries
Herath Mudiyanselage, Adeesha (2023-06-15)
Isolation of rhinovirus-specific Fab antibodies from universal phage display antibody libraries
(15.06.2023)
Julkaisu on tekijänoikeussäännösten alainen. Teosta voi lukea ja tulostaa henkilökohtaista käyttöä varten. Käyttö kaupallisiin tarkoituksiin on kielletty.
suljettu
Julkaisun pysyvä osoite on:
https://urn.fi/URN:NBN:fi-fe2023073192083
https://urn.fi/URN:NBN:fi-fe2023073192083
Tiivistelmä
Human rhino viral (HRV) diseases continue to be under-diagnosed, despite their negative impact on society. Besides nucleic acid-based methods, no antibodies are available for immunodiagnostics due to high antigenic variation of over 150 different HRV serotypes.
The study aimed to discover recombinant antibodies that recognize a broad range of different HRV serotypes for the development of a generic HRV immunoassay. The objective was to generate antibodies specific for a conserved region of HRV 3C protease by selecting them from universal fragment-antigen-binding phage display libraries.
Two different selection strategies have tested in the current study. First, bio-panning was performed by switching between the antigens of HRV 3C protease and a 3C-peptide derived from the selected conserved region of the protein. In the second approach, the gene coding for the HRV 3C protein was mutated and the mutated protein was used as a soluble blocker in the bio-panning against the HRV 3C protein. Enriched phages have analyzed in the phage immunoassay and cloned into a screening vector to express soluble Fabs in E. coli. In total, 976 Fab clones were analyzed in sandwich immunoassay. Eighty-six clones that gave signal for the target protein and/or the peptide were further studied in secondary screening and direct sequencing.
The phage immunoassay data confirmed a good enrichment of libraries, and screening and sequencing data revealed several different specific binders. Ten binders that gave the highest signals for both the protein and the peptide have selected and Fabs were expressed in bigger volumes, purified, and used in kinetic measurements. The best 9 Fabs will be tested on viral infected cell lines by fluorescence microscopy and promising Fabs recognize the desired target epitope are interesting candidates for further development into a diagnostic assay for HRV.
The study aimed to discover recombinant antibodies that recognize a broad range of different HRV serotypes for the development of a generic HRV immunoassay. The objective was to generate antibodies specific for a conserved region of HRV 3C protease by selecting them from universal fragment-antigen-binding phage display libraries.
Two different selection strategies have tested in the current study. First, bio-panning was performed by switching between the antigens of HRV 3C protease and a 3C-peptide derived from the selected conserved region of the protein. In the second approach, the gene coding for the HRV 3C protein was mutated and the mutated protein was used as a soluble blocker in the bio-panning against the HRV 3C protein. Enriched phages have analyzed in the phage immunoassay and cloned into a screening vector to express soluble Fabs in E. coli. In total, 976 Fab clones were analyzed in sandwich immunoassay. Eighty-six clones that gave signal for the target protein and/or the peptide were further studied in secondary screening and direct sequencing.
The phage immunoassay data confirmed a good enrichment of libraries, and screening and sequencing data revealed several different specific binders. Ten binders that gave the highest signals for both the protein and the peptide have selected and Fabs were expressed in bigger volumes, purified, and used in kinetic measurements. The best 9 Fabs will be tested on viral infected cell lines by fluorescence microscopy and promising Fabs recognize the desired target epitope are interesting candidates for further development into a diagnostic assay for HRV.