Non-specific binding to protein A Sepharose and protein G Sepharose in insulin autoantibody assays may be reduced by pre-treatment with glycine or ethanolamine

AJK Williams, AJ Norcross, MA-M Chandler, PJ Bingley

Research output: Contribution to journalArticle (Academic Journal)peer-review

16 Citations (Scopus)

Abstract

Insulin autoantibody (IAA) microassays are widely used for predicting type 1 diabetes. As levels of IAA are often low in type 1 diabetes, non-specific binding (NSB) needs to be minimised if assays are to achieve high analytical sensitivity. IAA microassays use protein A Sepharose (PAS) or protein G Sepharose (PGS) to isolate the antibody-bound label, but NSB by the gel can differ between commercially-produced batches. We investigated whether pre-incubation of gel with glycine or ethanolamine could overcome this problem. Batches of PAS/PGS shown to have high NSB (0.3-3.2%) were incubated with glycine or ethanolamine at various pHs between 8 and 10.6 for 2-18 h at 4 [degree sign]C or room temperature. Treating PAS at pH 10.6 with 0.2 M glycine overnight at room temperature reduced NSB by > 84%, with minimal reduction in specific binding (<5%). Treating PGS at pH 10.6 with 0.2 M ethanolamine overnight at 4 [degree sign]C reduced background by > 95%, with minimal reduction in specific binding by most sera. Treatment at high pH was critical in reducing NSB to both PAS and PGS, with slight reduction at pH 8, but a major reduction at pH 10.6. Pre-treatment with glycine or ethanolamine allows "poor" batches of PAS or PGS to be used in sensitive IAA assays, improving both consistency and performance.
Translated title of the contributionNon-specific binding to protein A Sepharose and protein G Sepharose in insulin autoantibody assays may be reduced by pre-treatment with glycine or ethanolamine
Original languageEnglish
Pages (from-to)170 - 173
Number of pages4
JournalJournal of Immunological Methods
Volume314 (1-2)
DOIs
Publication statusPublished - Jul 2006

Bibliographical note

Publisher: Elsevier

Fingerprint Dive into the research topics of 'Non-specific binding to protein A Sepharose and protein G Sepharose in insulin autoantibody assays may be reduced by pre-treatment with glycine or ethanolamine'. Together they form a unique fingerprint.

Cite this