title: Antibodies against insulin measured by electrochemiluminescence predicts insulitis severity and disease onset in non-obese diabetic mice and can distinguish human type 1 diabetes status.
abstract: BACKGROUND
  The detection of insulin autoantibodies (IAA) aids in the prediction of autoimmune diabetes development. However, the long-standing, gold standard 125I-insulin radiobinding assay (RBA) has low reproducibility between laboratories, long sample processing times and requires the use of newly synthesized radiolabeled insulin for each set of assays. Therefore, a rapid, non-radioactive, and reproducible assay is highly desirable.
  
  
  METHODS
  We have developed electrochemiluminescence (ECL)-based assays that fulfill these criteria in the measurement of IAA and anti-insulin antibodies (IA) in non-obese diabetic (NOD) mice and in type 1 diabetic individuals, respectively. Using the murine IAA ECL assay, we examined the correlation between IAA, histopathological insulitis, and blood glucose in a cohort of female NOD mice from 4 up to 36 weeks of age. We developed a human IA ECL assay that we compared to conventional RBA and validated using samples from 34 diabetic and 59 non-diabetic individuals in three independent laboratories.
  
  
  RESULTS
  Our ECL assays were rapid and sensitive with a broad dynamic range and low background. In the NOD mouse model, IAA levels measured by ECL were positively correlated with insulitis severity, and the values measured at 8-10 weeks of age were predictive of diabetes onset. Using human serum and plasma samples, our IA ECL assay yielded reproducible and accurate results with an average sensitivity of 84% at 95% specificity with no statistically significant difference between laboratories.
  
  
  CONCLUSIONS
  These novel, non-radioactive ECL-based assays should facilitate reliable and fast detection of antibodies to insulin and its precursors sera and plasma in a standardized manner between laboratories in both research and clinical settings. Our next step is to evaluate the human IA assay in the detection of IAA in prediabetic subjects or those at risk of type 1 diabetes and to develop similar assays for other autoantibodies that together are predictive for the diagnosis of this common disorder, in order to improve prediction and facilitate future therapeutic trials.
authors: Laboratory of Immunology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA.
authors: Bernice
authors: B
authors: Lo
authors: Austin D E
authors: AD
authors: Swafford
authors: Kimberly A
authors: KA
authors: Shafer-Weaver
authors: Lawrence F
authors: LF
authors: Jerome
authors: Luba
authors: L
authors: Rakhlin
authors: Douglas R
authors: DR
authors: Mathern
authors: Conor A
authors: CA
authors: Callahan
authors: Ping
authors: P
authors: Jiang
authors: Lucy J
authors: LJ
authors: Davison
authors: Helen E
authors: HE
authors: Stevens
authors: Carrie L
authors: CL
authors: Lucas
authors: Jill
authors: J
authors: White
authors: Reid
authors: R
authors: von Borstel
authors: John A
authors: JA
authors: Todd
authors: Michael J
authors: MJ
authors: Lenardo
mesh_terms: D000818:Animals; D001323:Autoantibodies / Q000097:blood; D003922:Diabetes Mellitus, Type 1 / Q000097:blood* / Q000175:diagnosis* / Q000473:pathology; D018450:Disease Progression; D004563:Electrochemistry / Q000379:methods*; D005260:Female; D006801:Humans; D007330:Insulin Antibodies / Q000097:blood*; D050417:Insulin-Secreting Cells / Q000378:metabolism / Q000473:pathology*; D008163:Luminescent Measurements / Q000379:methods*; D051379:Mice; D016688:Mice, Inbred NOD; D012372:ROC Curve; D011869:Radioligand Assay; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
chemical_list: D001323:Autoantibodies; D007330:Insulin Antibodies