Meritev adsorpcije in agregacije protiteles na fazni meji voda-zrak

magistrsko delo

Timotej Žuntar (Author), Natan Osterman (Mentor)

Abstract

Protitelesa so ena izmed glavnih komponent imunskega sistema in čedalje širše uporabljana v terapevtske namene. Zaradi močne odvisnosti oblike teh velikih proteinskih molekul in s tem medmolekulskih sil od lokalnega okolja so protitelesa nagnjena tako k adsorpciji na faznih mejah kot tudi samoagregaciji. V tem magistrskem delu sta bila procesa spremljana na dva načina. S pomočjo mikroskopije pod Brewstrovim kotom (BAM) smo pomerili adsorpcijsko dinamiko proteinskih filmov na fazni meji voda-zrak. Filme dveh protiteles smo kontrolirano prebodli in spremljali odbojnost površine med njihovo rekonstitucijo. Iz meritev smo določili odvisnost hitrosti rekonstitucije filma od koncentracije protiteles; pri koncentraciji $\mathrm{0.1\ mg/mL}$ se npr. film popolnoma rekonstituira v času reda $\mathrm{10\ s}$. Pri natančnosti izvedenih meritev ni bilo opaženih sistematskih razlik med opazovanima tipoma proteina. Proces smo modelirali s preprostim 2D modelom po metodi končnih elementov in izboljšanim 3D modelom prekurzorsko mediirane adsorpcije. Dodatno smo proteinske filme kontrolirano motili in agregirane proteinske delce analizirali z optično mikroskopijo. V mehansko motenih vzorcih smo periodično zajemali slike dna posod z raztopinami in razvili skripto za njihovo avtomatsko analizo. Za obe opazovalni metodi smo preverili tudi odvisnost rekonstitucije oz. agregacije od koncentracije surfaktantskih molekul. Mejni koncentraciji za tvorbo proteinskega filma in agregacijo sta bili znatno nižji od kritične micelarne koncentracije surfaktantov.

Keywords

protitelesa;proteini;elipsometrija;Brewstrov kot;adsorpcija;agregati;

Data

Language:	Slovenian
Year of publishing:	2021
Typology:	2.09 - Master's Thesis
Organization:	UL FMF - Faculty of Mathematics and Physics
Publisher:	[T. Žuntar]
UDC:	577.3
COBISS:	90424067
Views:	182
Downloads:	36
Average score:	0 (0 votes)
Metadata:

Other data

Secondary language:	English
Secondary title:	Measurement of adsorption and aggregation of antibodies at the water-air phase boundary
Secondary abstract:	Antibodies are a crucial part of the immune system and increasingly used for therapeutic purposes. Due to the strong dependance of conformations of these large protein molecules and ensuing intermolecular forces on local conditions, antibodies are prone to both adsorption at interfaces and formation of aggregates. In this master's thesis, the processes were monitored in two ways. We measured the adsorption dynamics of protein films at the water-air phase boundary using Brewster angle microscopy (BAM) by puncturing films of two antibodies in a controlled manner and monitoring changes in reflectivity during their reconstitution. From the data, we determined the dependance of reconstitution speed on antibody concentrations; at a concentration of $\mathrm{0.1\ mg/mL}$, for example, the film fully reforms in a timespan of $\mathrm{10\ s}$. At the precision of the measurements carried out, a systematic difference between the observed protein types could not be detected. We modelled the process with a simple 2D model based on the finite element method and an improved 3D model of precursor-mediated adsorption. Additionally, we subjected the protein films to controlled agitation and analysed the aggregated protein particles using optical microscopy. Images of container bottoms of agitated samples were periodically taken and a script was developed for their automatic analysis. For both observation methods, we also examined the effect of surfactant molecules on reconstitution/aggregation. Concentration limits for the formation of a viscoelastic film and aggregation were significantly below the critical micelle concentration of surfactants.
Secondary keywords:	antibodies;proteins;ellipsometry;Brewster angle;adsorption;aggregates;
Type (COBISS):	Master's thesis/paper
Study programme:	0
Thesis comment:	Univ. v Ljubljani, Fak. za matematiko in fiziko, Oddelek za fiziko
Pages:	65 str.
ID:	14444870