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.
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 degrees 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 degrees 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.
Williams
Alistair J K
AJ
Clinical Science at North Bristol, University of Bristol, UK. A.J.K.Williams@bristol.ac.uk <A.J.K.Williams@bristol.ac.uk>
Norcross
Alastair J
AJ
Chandler
Kyla A
KA
Bingley
Polly J
PJ
D001323:Autoantibodies / Q000276:immunology; D001667:Binding, Competitive; D019856:Ethanolamine / Q000494:pharmacology*; D005998:Glycine / Q000494:pharmacology*; D006801:Humans; D007330:Insulin Antibodies / Q000097:blood*; D009419:Nerve Tissue Proteins / Q000276:immunology*; D011863:Radioimmunoassay / Q000379:methods*; D012680:Sensitivity and Specificity; D012685:Sepharose / Q000031:analogs & derivatives* / Q000276:immunology; D013205:Staphylococcal Protein A
D001323:Autoantibodies; D019856:Ethanolamine; D005998:Glycine; D007330:Insulin Antibodies; D009419:Nerve Tissue Proteins; D012685:Sepharose; C029152:G-substrate; C042030:Staphylococcal protein A-sepharose; D013205:Staphylococcal Protein A