Characterisation of Anti-glucagon-like Peptide-1 Antibodies Utilising Surface Plasmon Resonance
Translated title
Karakterisering af anti-glucagon-like peptide-1 antistoffer ved brug af overflade plasmon resonans
Author
Thomsen, Lasse
Term
4. term
Education
Publication year
2015
Submitted on
2015-09-15
Pages
104
Abstract
Specialet undersøgte, hvor alsidig overfladeplasmonresonans (SPR) er til at karakterisere anti‑GLP‑1‑antistoffer. Metoden blev brugt til både kinetisk analyse (hvor hurtigt antistoffer binder og slipper) og termodynamisk analyse (hvordan temperatur påvirker bindingsstyrken) samt til såkaldt sandwich‑analyse, hvor to antistoffer binder målstoffet på forskellige steder. Antistofferne havde affiniteter på 2.93e-8–3.56e-7 M. Bindingerne var entalpistyrede, hvilket peger på dannelse og stabilisering af hydrogenbindinger ved lavere temperaturer og dermed højere affiniteter. Når antistofferne fungerede som sandwich‑antistoffer, steg affiniteten yderligere til 1.21e-9–4.87e-9 M. Parvis epitopkortlægning blev udført for at vurdere, hvilke antistofpar der kan kombineres i en sandwich, og hvor specifikke de er. Resultaterne indikerede en bestemt rækkefølge af epitopgenkendelse fra N‑terminalen: ABS 033‑04, HYB 147‑13, HYB 147‑12, HYB 147‑08 og ABS 047‑03. Sammenlignet med enzymkoblet immunosorbentassay (ELISA) tilbyder SPR flere fordele, men kræver betydelig optimering for at give valide og pålidelige resultater.
This thesis examined how versatile surface plasmon resonance (SPR) is for characterizing anti‑GLP‑1 antibodies. The work combined kinetic analysis (how quickly antibodies bind and dissociate) and thermodynamic analysis (how temperature affects binding strength) with “sandwich” analysis, in which two antibodies bind the target at different sites. The antibodies showed affinities of 2.93e-8–3.56e-7 M. The interactions were enthalpy‑driven, consistent with the formation and stabilization of hydrogen bonds at lower temperatures, which led to higher affinities. When the antibodies were used in sandwich configurations, affinities improved to 1.21e-9–4.87e-9 M. Pair‑wise epitope mapping was performed to assess which antibody pairs can form sandwich combinations and to evaluate specificity. The results suggested a specific order of epitope recognition from the N‑terminus: ABS 033‑04, HYB 147‑13, HYB 147‑12, HYB 147‑08, and ABS 047‑03. Compared with enzyme‑linked immunosorbent assay (ELISA), SPR offers several advantages but requires considerable optimization to produce valid, reliable results.
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