Affinity chromatography on immobilized "biomimetic" ligands. Synthesis, immobilization and chromatographic assessment of an immunoglobulin G-binding ligand.
A synthetic bifunctional ligand (22/8) comprising a triazine scaffold substituted with 3-aminophenol (22) and 4-amino-1-naphthol (8) has been designed, synthesised, characterised and immobilized on agarose beads to create a robust, highly selective affinity adsorbent for human immunoglobulin G (IgG). Scatchard analysis of the binding isotherm for IgG on immobilized 22/8 (90 micromol 22/8/g moist weight gel) indicated an affinity constant (Ka) of 1.4 x 10(5) M(-1) and a theoretical maximum capacity of 151.9 mg IgG/g moist weight gel. The adsorbent shows similar selectivity to immobilized protein A and binds IgG from a number of species. An apparent capacity of 51.9 mg human IgG/g moist weight gel was observed under the experimental conditions selected for adsorption. Human IgG was eluted with glycine-HCl buffer with a recovery of 67-69% and a purity of 97.3-99.2%, depending on the pH value of the buffer used for elution. Preparative chromatography of IgG from human plasma showed that under the specified conditions, 94.4% of plasma IgG was adsorbed and 60% subsequently eluted with a purity of 92.5%. The immobilized ligand was able to withstand incubation in 1 M NaOH for 7 days without loss of binding capacity for IgG.
Teng
S F
SF
Institute of Biotechnology, University of Cambridge, UK.
Sproule
K
K
Husain
A
A
Lowe
C R
CR
D000818:Animals; D002645:Chickens; D002846:Chromatography, Affinity / Q000379:methods*; D006801:Humans; D007074:Immunoglobulin G / Q000302:isolation & purification / Q000378:metabolism*; D008024:Ligands; D051379:Mice; D008958:Models, Molecular; D018716:Molecular Mimicry; D009284:Naphthols / Q000138:chemical synthesis* / Q000737:chemistry*; D011817:Rabbits; D051381:Rats; D012756:Sheep; D013045:Species Specificity; D014227:Triazines / Q000138:chemical synthesis* / Q000737:chemistry*
C409762:2-(3-aminophenol)-6-(4-amino-1-naphthol)-4-chloro-triazine; D007074:Immunoglobulin G; D008024:Ligands; D009284:Naphthols; D014227:Triazines