Extraction of haemoglobin from human blood by affinity precipitation using a haptoglobin-based stimuli-responsive affinity macroligand

Affinity precipitation was compared to affinity chromatography and batch adsorption as the final purification step in a protocol for the isolation of haemoglobin from human blood. Haptoglobin was the affinity ligand. The first steps on the process were realized by traditional methods (lyses of red blood cells followed by ammonium sulphate precipitation). For affinity chromatography (and batch adsorption) the ligand was linked to Sepharose, for affinity precipitation to a thermoresponsive polymer, namely poly(N-isopropylacrylamide). Five haptoglobin-poly(N-isopropylacrylamide) bioconjugates (affinity macroligands) were constructed with different polymer: haptoglobin-coupling ratios. Conjugation of haptoglobin to the soluble poly(N-isopropylacrylamide) apparently does not change the interaction thermodynamics with haemoglobin, as the haemoglobin binding constants calculated by a Scatchard analysis for the affinity macroligand were of the same order of magnitude as those described in the literature for the haemoglobin-haptoglobin complex in solution. Two elution protocols were used for haemoglobin release from the various affinity materials, one at pH 2, the other with 5 M urea at pH 11. Both affinity chromatography and affinity precipitation yielded a pure haemoglobin of high quality. Compared to the affinity chromatography, affinity precipitation showed a significantly higher ligand efficiency (ratio of the experimental capacity to the theoretical one). The method thus makes better use of the expensive affinity ligands. As affinity precipitation only requires small temperature changes to bring about precipitation/redissolution of the affinity complexes and a centrifugation step for recovery of the precipitate, the method in addition has advantages in term of scalability and simplicity. (C) 2008 Elsevier B.V. All rights reserved

Screening of a Human Antibody Phage Display Library Against a Peptide Antigen Using Stimuli-Responsive Bioconjugates

A synthetic antibody phage library, (ETH-2) was screened against the MUC-1 peptide. Two standard methods were used, namely screening on immunotubes coated with the peptide antigen and on streptavidin-coated paramagnetic particles together with a biotinylated MUC-1 peptide. The results were compared with those obtained using a novel approach based on a stimuli-responsive bioconjugate of avidin and poly-(N-isopropylacrylamide), also in combination with the biotinylated peptide. The poly-(N-isopropylacrylamide)-based bioconjugate is soluble below a temperature of 30 degrees C in the aqueous solutions pertinent to this study, but precipitates rapidly once this temperature is surpassed. The process is reversible, and the bioconjugate redissolves once the temperature is lowered again. The stimuli-responsive bioconjugate was used to isolate specific phage from the library in several rounds of panning, consisting of repeated thermoprecipitation/redissolution cycles in a procedure known as "affinity precipitation." As Jar as we know, this is the first time a compound as big as a viral particle has been specifically captured by a stimuli-responsive bioconjugate. Compared to the two other investigated screening techniques, affinity precipitation of the phage led to a greater variety of genetically, different specific phage, while the process was easy to handle and required no dedicated equipment safe for a centrifuge and a thermostated water bath