Ločevanje izooblik monoklonskih protiteles z lektini

magistrsko delo

Gašper Kosmač (Avtor), Aleš Podgornik (Mentor), Aleš Podgornik (Član komisije za zagovor), Mojca Narat (Član komisije za zagovor), Blaž Cigić (Član komisije za zagovor)

Povzetek

Preučevali smo interakcijo monoklonskih protiteles (mAb) z lektinom konkanavalinom A (ConA) v raztopini, ter pogoje, pri katerih vezava med proteinoma optimalno poteče. Z merjenjem intrinzične fluorescence triptofana, smo preučevali razvijanje strukturne konformacije mAb, ob spreminjanju parametrov kot so pH pufra, temperatura inkubiranja, ter dolžina časa inkubacije. Na podlagi teh meritev smo jim določili talilno temperaturo (Tm), ki je znašala 61 °C, ter denaturacijski čas (tm), ki je znašal približno 60 minut. Z metodo izključitvene kromatografije smo preučevali nastanek agregatov, ter tvorbo morebitne povezave med mAb in ConA. Povezave med proteinoma nismo zaznali. Z namenom popolne izpostavitve glikozidnih ostankov na mAb, smo le tem dodali reagent DTT, vendar do redukcije disulfidnih vezi, ter razcepitve verig mAb verjetno ni prišlo. Sklepali smo, da bi lahko bil vzrok tvorbe agregatov, ob sočasni izgubi nativne oblike, ter odsotnosti razcepitve mAb ob dodatku DTT, oksidativni učinek le tega na mAb. Učinek bi lahko sprožila povišana temperatura pri inkubaciji mAb. Verjetno predhodno oksidiranost, ter posledično neustreznost reagenta DTT smo pokazali tudi z detekcijo le tega pri 280 nm. Poskusili smo tudi z vezavo dekstrana, ter glikoziliranega BSA na ConA. V nobenem primeru vezave nismo zaznali. Z izključitveno kromatografijo smo ugotovili, da se uporabljani ConA neodvisno od okolja nahaja v monomerni obliki, ki je biološko neaktivna.

Ključne besede

monoklonska protitelesa;izključitvena kromatografija;lektini;glikozilacija;

Podatki

Jezik:	Slovenski jezik
Leto izida:	2019
Tipologija:	2.09 - Magistrsko delo
Organizacija:	UL FKKT - Fakulteta za kemijo in kemijsko tehnologijo
Založnik:	[G. Kosmač]
UDK:	602.44(043.2)
COBISS:	9361529
Št. ogledov:	917
Št. prenosov:	199
Ocena:	0 (0 glasov)
Metapodatki:

Ostali podatki

Sekundarni jezik:	Angleški jezik
Sekundarni naslov:	Separating of different isoforms of monoclonal antibodies with lectins.[!]
Sekundarni povzetek:	The interaction of monoclonal antibodies (mAbs) with lectin concanavalin A (ConA) in solution and the conditions under which protein binding occurs optimally were studied. By measuring the intrinsic fluorescence of tryptophan, we investigated the unfolding of the structural conformation of the mAb while changing parameters such as pH of the buffer, incubation temperature, and length of incubation time. Based on these measurements, a melting temperature (Tm) of 61 ° C and a denaturation time (tm) of approximately 60 minutes were determined. The formation of aggregates and the formation of a possible bond between mAb and ConA were investigated using the size exclusion chromatography method. No protein binding was detected. In order to completely expose the glycosidic residues on the mAb structure, we added DTT. However the reduction of disulfide bonds and consequently cleavage of the mAb on light and heavy chains most likely did not take part. We concluded that, the cause of aggregate formation, with the concomitant loss of the native form, and the absence of cleavage of the mAb by the addition of DTT, could possibly be the result of oxidative effect of the latter on the mAb. The effects could be triggered by the elevated temperature of mAb incubation. The likely pre-oxidation and consequent inadequacy of the DTT reagent was also demonstrated by detecting it at 280 nm. We also tried binding dextran and glycosylated BSA with ConA. In no case did the binding take place. Exclusion chromatography revealed that ConA, independently of the environmental factors, exists in the monomeric form which is biologically inactive.
Sekundarne ključne besede:	monoclonal antibodies;size exclusion chromatography;lectins;glycosylation;
Vrsta dela (COBISS):	Magistrsko delo/naloga
Študijski program:	0
Konec prepovedi (OpenAIRE):	1970-01-01
Komentar na gradivo:	Univ. v Ljubljani, Biotehniška fak., Študij biotehnologije
Strani:	IX, 67 f.
ID:	11230375