Linked Life Data

9367509

Source:http://linkedlifedata.com/resource/pubmed/id/9367509

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
2
pubmed:dateCreated
1997-12-16
pubmed:abstractText
Antibodies are routinely purified by acid/salt elution from antigen affinity columns. The antibodies recovered with this procedure are active, but the recovery of protein is often low. We investigated the effect of acid and other denaturing or chaotropic solvents on the conformation of monoclonal antibodies (mAbs) made against the extracellular region of Her2 receptor (sHer2) derived from Chinese hamster ovary cells. The mAb remain almost completely folded in the 0.1 M glycine, pH 2.9, commonly used for elution, with the beta-sheet secondary structure intact, and only very small changes detected in the environment of the tryptophans. In 7 M urea, 50 mM NaAc pH 4.0, the antibody was partially unfolded, with the Trp environment further perturbed and some of the beta-sheet structure converted to disordered structure. In 6 M guanidine HCl, 50 mM NaAc, pH 4.0, the antibody is completely unfolded, with no secondary or tertiary structure present. The antibodies exposed to glycine or urea were refolded by dialysis into phosphate-buffered saline (PBS), while the guanidine HCl-denatured antibodies were refolded by dialysis into 7 M urea, pH 4.0, followed by dialysis into PBS. The refolded antibodies were capable of forming antigen-antibody complexes which could be isolated by gel filtration chromatography. Two different mAbs were subjected to immunoaffinity chromatography on sHer2-Sepharose. mAb86 was eluted by 0.1 M Gly, pH 2.9, while mAb52 was eluted with the 7 M urea, 50 mM NaAc, pH 4.0. The isolated antibodies were refolded by dialysis into PBS, analyzed for their ability to recognize native sHer2 by immunoprecipitation, and denatured sHer2 by Western blot analysis. Both preparations recognized the native protein, but precipitated slightly different forms of sHer2, indicating that they might recognize different epitopes. The mAb52 is a more sensitive reagent for Western blot analysis. Thus, this procedure can be used to recover antibodies which would not be recovered with glycine as the only eluate. It is also possible that the antibodies can be fractionated by the different eluants into populations which can be used for different applications.
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Nov
pubmed:issn
0003-2697
pubmed:author
pubmed:copyrightInfo
Copyright 1997 Academic Press.
pubmed:issnType
Print
pubmed:day
15
pubmed:volume
253
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
236-45
pubmed:dateRevised
2009-11-19
pubmed:meshHeading
pubmed:year
1997
pubmed:articleTitle
Effect of three elution buffers on the recovery and structure of monoclonal antibodies.
pubmed:affiliation
Amgen Inc., Thousand Oaks, California, 91320-1789, USA.
pubmed:publicationType
Journal Article