Development of Antibodies for Cyanobacterial Neurotoxin: Anatoxin-a
Callus, Irene (2024-08-01)
Development of Antibodies for Cyanobacterial Neurotoxin: Anatoxin-a
(01.08.2024)
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-fe2024090468913
https://urn.fi/URN:NBN:fi-fe2024090468913
Tiivistelmä
Anatoxin-a is a neurotoxin produced by certain cyanobacteria in water bodies worldwide. It may cause neurological symptoms in animals and humans when ingested and several deaths in animals and livestock have been reported due to anatoxin-a intoxication. As immunoassay-based tests for anatoxin-a are limited, there is a need for more straightforward tests for the detection and monitoring of anatoxin-a levels in water. Thus, this thesis aimed to find antibodies for anatoxin-a that could be used in an immunoassay.
In this master thesis work, three antigen-binding fragment (Fab) libraries, previously converted from a single-chain variable fragment library, were first enriched against biotinylated anatoxin-a by three panning rounds of phage display. As this stage identified no Fab binders, the panning was continued with two rounds against HRP-anatoxin conjugate. The three original Fab libraries and the Fab libraries that had already been panned once against biotinylated-anatoxin were used. Fab genes were cloned into Escherichia coli to produce soluble Fab in microtiter plate-based cultures. The Fab clones were then screened to measure their binding ability to both biotinylated anatoxin-a and HRP-anatoxin conjugate.
In the end, 30 clones were identified to recognize both biotinylated- and HRP-anatoxin conjugates. We hypothesize that if Fab binders recognize both of these anatoxin-a conjugates, then they are most likely binding to the anatoxin-a target in both cases. This would then mean that they can also recognize free anatoxin-a.
From the results obtained, it is highly recommended to continue with this research to see if any of the 30 isolated clones can bind to free anatoxin-a. This could then help pave the way to develop a user-friendly non-competitive immunoassay for anatoxin-a.
In this master thesis work, three antigen-binding fragment (Fab) libraries, previously converted from a single-chain variable fragment library, were first enriched against biotinylated anatoxin-a by three panning rounds of phage display. As this stage identified no Fab binders, the panning was continued with two rounds against HRP-anatoxin conjugate. The three original Fab libraries and the Fab libraries that had already been panned once against biotinylated-anatoxin were used. Fab genes were cloned into Escherichia coli to produce soluble Fab in microtiter plate-based cultures. The Fab clones were then screened to measure their binding ability to both biotinylated anatoxin-a and HRP-anatoxin conjugate.
In the end, 30 clones were identified to recognize both biotinylated- and HRP-anatoxin conjugates. We hypothesize that if Fab binders recognize both of these anatoxin-a conjugates, then they are most likely binding to the anatoxin-a target in both cases. This would then mean that they can also recognize free anatoxin-a.
From the results obtained, it is highly recommended to continue with this research to see if any of the 30 isolated clones can bind to free anatoxin-a. This could then help pave the way to develop a user-friendly non-competitive immunoassay for anatoxin-a.