Iimveliso

Uvavanyo olutsha lwe-liquid chromatography entsha ephezulu yokusebenza kwe-glycosyltransferases esekwe kwi-derivatization kunye ne-anthranilic acid kunye nokufunyanwa kwe-fluorescence.

Ii-assays zaphuhliswa kusetyenziswa i-chemical label chemistry ye-2-aminobenzoic acid (2AA; i-anthranilic acid, i-AA) yokulinganisa imisebenzi ye-β1-4 galactosyltransferase (GalT-1) kunye ne-α2-6 sialyltransferase (ST-6) ngolwelo olusebenza kakhulu. chromatography (HPLC) kunye nobhaqo fluorescence (Anumula KR. 2006. Ukuqhubela phambili kwiindlela fluorescence derivatization ulwelo eliphezulu uhlalutyo chromatographic of glycoprotein carbohydrates. Anal Biochem. 350: 1-23).I-N-Acetylglucosamine (GlcNAc) kunye ne-N-acetyllactosamine isetyenziswe njengabamkeli kunye ne-uridine diphosphate (UDP) -galactose kunye ne-cytidine monophosphate (CMP) -N-acetylneuraminic acid (NANA) njengabanikezeli be-GalT-1 kunye ne-ST-6, ngokulandelanayo.Iimveliso ze-Enzymatic zibhalwe kwi-situ kunye ne-AA kwaye zahlulwa kwi-substrates kwikholamu ye-TSKgel Amide 80 usebenzisa iimeko zesigaba esiqhelekileyo.Iiyunithi ze-Enzyme zichongwe kwiindawo eziphakamileyo ngokuthelekisa kunye nemigangatho ekhutshwe ngokuhambelanayo ye-Gal-β1-4GlcNAc kunye ne-NANA-α2-6 Gal-β1-4GlcNAc.I-Linearity (ixesha kunye nokugxilwa kwe-enzyme), ukuchaneka (i-intra- kunye ne-interassay) kunye nokuveliswa kwakhona kwiimvavanyo zasekwa.Iimvavanyo zifunyenwe ziluncedo ekubekeni iliso kwimisebenzi ye-enzyme ngexesha lokuzihlukanisa kunye nokucoca.Uvavanyo lwalunovakalelo oluphezulu kwaye lwenziwa ngokulingana okanye ngcono kunemilinganiselo yeswekile ye-radioactive.Ifomathi yokuvavanya ingasetyenziselwa ukulinganisa umsebenzi wolunye udluliselo, ngaphandle kokuba abamkeli be-carbohydrate baqulethe isiphelo sokunciphisa ukulebula.Uvavanyo lwabamkeli be-glycoprotein lwaphuhliswa kusetyenziswa i-IgG.Indlela emfutshane ye-HPLC yeprofayili yaphuhliswa ukuhlukana kwe-IgG glycans (i-biantennary G0, G1, G2, i-mono- kunye ne-disialylated), eyaququzelela ukuzimisela kwe-GalT-1 kunye ne-ST-6 imisebenzi ngokukhawuleza.Ngaphaya koko, le ndlela yokubhala iprofayile kufuneka ingqineke iluncedo ekubekeni iliso kutshintsho kwi-IgG glycans kwiisetingi zeklinikhi.