Anti-BSA antibodies are a major cause of non-specific binding in insulin autoantibody radiobinding assays.
Summary of "Anti-BSA antibodies are a major cause of non-specific binding in insulin autoantibody radiobinding assays."
Insulin autoantibodies (IAA) are usually the first risk-markers detected during the type 1 diabetes prodrome, but precise measurement is difficult as insulin binding is often low. Non-specific binding (NSB) of (125)I-labelled insulin necessitates competitive displacement with unlabelled insulin to demonstrate specificity. NSB varies with different batches of label, suggesting that it is caused by impurities in the label. Addition of bovine serum albumin (BSA) can reduce NSB, so we investigated whether BSA antibodies cause lack of specificity in IAA assays. Samples from patients with newly-diagnosed type 1 diabetes, healthy schoolchildren previously found to have raised (125)I-insulin binding (≥ 0.4 units) and IAA-negative schoolchildren were re-assayed for IAA by radiobinding microassay using commercial (125)I-insulin with and without 1g/dl BSA added to the buffer. Of 100 patients, 68 were IAA-positive on re-assay with BSA compared to 72 without BSA (p=0.125). Of 154 schoolchildren who previously had raised (125)I-insulin binding, only 45 had (125)I-insulin binding ≥ 0.4 units on re-assay with BSA compared to 90 without BSA (p<0.001). Following competitive displacement with unlabelled insulin, 40 were IAA-positive with BSA compared to 48 without BSA (p=0.02). No IAA-negative schoolchildren were IAA-positive on re-assay. Levels of NSB were associated with antibodies binding (125)I-BSA and purification of labelled insulin reduced NSB. Addition of BSA to assay buffer improves the screening efficiency of the IAA assay without reducing disease sensitivity in patients. High titre BSA antibodies interfere with IAA measurement because of (125)I-BSA present in some insulin labels. Improved purification of insulin labels should obviate the need for competitive displacement.
Affiliation
School of Clinical Sciences, University of Bristol, Bristol, UK. A.J.K.Williams@bristol.ac.uk
Journal Details
This article was published in the following journal.
Name: Journal of immunological methods
ISSN: 1872-7905
Pages: 199-203
Links
- PubMed Source: http://www.ncbi.nlm.nih.gov/pubmed/20833177
- DOI: http://dx.doi.org/10.1016/j.jim.2010.09.009
Medical and Biotech [MESH] Definitions
Insulin Antibodies
Antibodies specific to INSULIN.
Hyperinsulinism
A syndrome with excessively high INSULIN levels in the BLOOD. It may cause HYPOGLYCEMIA. Etiology of hyperinsulinism varies, including hypersecretion of a beta cell tumor (INSULINOMA); autoantibodies against insulin (INSULIN ANTIBODIES); defective insulin receptor (INSULIN RESISTANCE); or overuse of exogenous insulin or HYPOGLYCEMIC AGENTS.
Insulin Resistance
Diminished effectiveness of INSULIN in lowering blood sugar levels: requiring the use of 200 units or more of insulin per day to prevent HYPERGLYCEMIA or KETOSIS. It can be caused by the presence of INSULIN ANTIBODIES or the abnormalities in insulin receptors (RECEPTOR, INSULIN) on target cell surfaces. It is often associated with OBESITY; DIABETIC KETOACIDOSIS; INFECTION; and certain rare conditions. (from Stedman, 25th ed)
Immunoprecipitation
The aggregation of soluble ANTIGENS with ANTIBODIES, alone or with antibody binding factors such as ANTI-ANTIBODIES or STAPHYLOCOCCAL PROTEIN A, into complexes large enough to fall out of solution.
Insulin Receptor Substrate Proteins
A structurally-related group of signaling proteins that are phosphorylated by the INSULIN RECEPTOR PROTEIN-TYROSINE KINASE. The proteins share in common an N-terminal PHOSPHOLIPID-binding domain, a phosphotyrosine-binding domain that interacts with the phosphorylated INSULIN RECEPTOR, and a C-terminal TYROSINE-rich domain. Upon tyrosine phosphorylation insulin receptor substrate proteins interact with specific SH2 DOMAIN-containing proteins that are involved in insulin receptor signaling.
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