Awọn ọja

Ayẹwo chromatography olomi tuntun ti o ga julọ fun awọn glycosyltransferases ti o da lori itọsẹ pẹlu acid anthranilic ati wiwa fluorescence.

A ṣe agbekalẹ awọn igbelewọn nipa lilo kemistri isamisi alailẹgbẹ ti 2-aminobenzoic acid (2AA; anthranilic acid, AA) fun wiwọn awọn iṣẹ ṣiṣe ti mejeeji β1-4 galactosyltransferase (GalT-1) ati α2-6 sialyltransferase (ST-6) nipasẹ omi iṣẹ ṣiṣe giga. chromatography (HPLC) pẹlu wiwa fluorescence (Anumula KR. 2006. Awọn ilọsiwaju ni awọn ọna itọsẹ fluorescence fun ṣiṣe-giga-iṣiro chromatographic olomi ti awọn carbohydrates glycoprotein. Anal Biochem. 350: 1-23).N-Acetylglucosamine (GlcNAc) ati N-acetyllactosamine ni a lo bi awọn olugba ati uridine diphosphate (UDP) -galactose ati cytidine monophosphate (CMP) -N-acetylneuraminic acid (NANA) gẹgẹbi awọn oluranlọwọ fun GalT-1 ati ST-6, lẹsẹsẹ.Awọn ọja enzymatic ti wa ni aami ni ipo pẹlu AA ati pe a yapa kuro ninu awọn sobusitireti lori iwe TSKgel Amide 80 nipa lilo awọn ipo deede-alakoso.Awọn ẹya enzymu ni a pinnu lati awọn agbegbe ti o ga julọ nipa ifiwera pẹlu awọn iṣedede itọsẹ ni igbakanna Gal-β1-4GlcNAc ati NANA-α2-6 Gal-β1-4GlcNAc.Linearity (akoko ati ifọkansi enzymu), konge (intra- ati interassay) ati atunṣe fun awọn igbelewọn ni a fi idi mulẹ.Awọn igbelewọn ni a rii pe o wulo ni mimojuto awọn iṣẹ-ṣiṣe henensiamu lakoko ipinya ati isọdọmọ.Awọn idanwo naa jẹ ifarabalẹ gaan ati ṣe deede tabi dara julọ ju awọn wiwọn orisun ipanilara ti ibile.Ọna kika ayẹwo tun le ṣee lo fun wiwọn iṣẹ ṣiṣe ti awọn gbigbe miiran, ti o ba jẹ pe awọn olugba carbohydrate ni opin idinku fun isamisi.Ayẹwo fun awọn olugba glycoprotein ni idagbasoke ni lilo IgG.Ọna profaili HPLC kukuru kan ni idagbasoke fun ipinya ti IgG glycans (biantennary G0, G1, G2, mono- and diialylated), eyiti o jẹ ki ipinnu awọn iṣẹ GalT-1 ati ST-6 ṣiṣẹ ni ọna iyara.Pẹlupẹlu, ọna profaili yii yẹ ki o jẹri iwulo fun mimojuto awọn ayipada ninu glycans IgG ni awọn eto ile-iwosan.