Abstract
Immunology and Infection
Aktivering og bøjning af opløselige polysaccharider ved hjælp af 1-Cyano-4-Dimethylaminopyridine Tetrafluoroborat (CDAP)
ERRATUM NOTICE
Important: There has been an erratum issued for this article. Read more …Konjugatvacciner er bemærkelsesværdige fremskridt inden for vaccinologi. Til fremstilling af polysaccharidkonjugatvacciner kan polysaccharider bekvemt funktionaliseres og forbindes med vaccinebærerproteiner ved hjælp af 1-cyano-4-dimethylaminopyridin tetrafluoroborat (CDAP), et cyanylende reagens, der er let at håndtere. CDAP aktiverer polysaccharider ved at reagere med kulhydrathydrathydrxylgrupper ved pH 7-9. Stabiliteten og reaktiviteten af CDAP er meget pH-afhængige. Reaktionens pH-fejl falder også under aktiveringen på grund af hydrolysen af CDAP, hvilket gør god pH-styring af nøglen til reproducerbar aktivering. Den oprindelige CDAP-aktiveringsprotokol blev udført ved stuetemperatur i ikke-byggede pH 9-løsninger.
På grund af den hurtige reaktion under denne tilstand (<3 min) og det medfølgende hurtige pH-fald fra den hurtige CDAP-hydrolyse var det udfordrende hurtigt at justere og vedligeholde målreaktions-pH'en inden for den korte tidsramme. Den forbedrede protokol, der er beskrevet her, udføres ved 0 °C, hvilket bremser CDAP-hydrolyse og forlænger aktiveringstiden fra 3 min til ~ 15 min. Dimethylaminopyridin (DMAP) blev også brugt som buffer til at justere polysaccharidopløsningen til målaktiverings-pH'en, før CDAP-reagenset blev tilføjet. Den længere reaktionstid kombineret med den langsommere CDAP-hydrolyse og brugen af DMAP-buffer gør det lettere at opretholde aktiverings-pH'en i hele aktiveringsprocessens varighed. Den forbedrede protokol gør aktiveringsprocessen mindre hektisk, mere reproducerbar og mere modtagelig for opskalering.
Erratum
Erratum: Activation and Conjugation of Soluble Polysaccharides using 1-Cyano-4-Dimethylaminopyridine Tetrafluoroborate (CDAP)An erratum was issued for: Activation and Conjugation of Soluble Polysaccharides using 1-Cyano-4-Dimethylaminopyridine Tetrafluoroborate (CDAP). A figure was updated.
Figure 4 was updated from:
Figure 4: Representative results for CDAP activation of dextran. Typical standard curves for the (A) resorcinol/sulfuric acid and (B) TNBS assays. The assay results for dextran activated with 0.25 and 0.5 mg CDAP/mg dextran are shown. Glucose was used as the standard for the resorcinol assay. Dextran, in mg/mL, is divided by 100 kDa to give a molar concentration. The hydrazide concentration is determined using ADH as the standard and the results expressed as µM Hz. (C) Calculation of hydrazide: dextran ratios.The level of derivatization was calculated as hydrazides per 100 kDa of dextran to facilitate the comparison between polymers of different average molecular weights. The % weight ratio of g ADH/g dextran was calculated using a MW of 174 g/mole for ADH. Please click here to view a larger version of this figure.
to:
Figure 4: Representative results for CDAP activation of dextran. Typical standard curves for the (A) resorcinol/sulfuric acid and (B) TNBS assays. The assay results for dextran activated with 0.25 and 0.5 mg CDAP/mg dextran are shown. Glucose was used as the standard for the resorcinol assay. Dextran, in mg/mL, is divided by 100 kDa to give a molar concentration. The hydrazide concentration is determined using ADH as the standard and the results expressed as µM Hz. (C) Calculation of hydrazide: dextran ratios.The level of derivatization was calculated as hydrazides per 100 kDa of dextran to facilitate the comparison between polymers of different average molecular weights. The % weight ratio of g ADH/g dextran was calculated using a MW of 174 g/mole for ADH. Please click here to view a larger version of this figure.
ABOUT JoVE
Copyright © 2024 MyJoVE Corporation. All rights reserved