Different Strategies used in the Production of human monoclonal scfv Antibodies Specific to Dimers of Membrane Receptors

• Main Authors: Sylwia Łukasiewicz (Author), Agata Stachowicz (Author), Ewelina Fic (Author), Ewa Błasiak (Author), Agata Kowalik (Author), Marta Dziedzicka-Wasylewska (Author)
• Format: Book
• Published: Journal of Biology and Medicine - Peertechz Publications, 2019-04-19.
• Subjects: Research Article
• Online Access: Connect to this object online.

<p>Phage display technology is one of the methods widely adopted for the production of human monoclonal antibodies. The subject of the present research was the scFv - single chain variable fragment antibody - which was able to recognize the D2-5-HT1A heteromer formed by dopamine D2 and serotonin 5-HT1A receptors. The antibody was selected using the phage display technique and the human antibody scFv phagemid library Tomlinson J. We investigated two expression systems: prokaryotic and eukaryotic. The results indicate that both of these systems (E.coli HB2151 and High Five cells (BTI-TN-5B1-4) of Trichopulsia ni) are suitable for the production of scFv antibodies, but the scFvs obtained from insect cells could attain a higher protein concentration. Next, we examined a few purification methods: immobilized-metal affinity chromatography (IMAC), affinity chromatography with the c-myc tag and the methods using protein L or A agarose, which are applicable to scFvs lacking tags. In the bacterial as well as insect cell expression systems, protein L agarose chromatography gave satisfactory results, and in our opinion, it is the best choice as a purification resin. Additionally, circular dichroism measurements confirmed the predominant content of secondary structures in the tested antibodies, which is consistent with the literature data.</p>