Murine high specificity/sensitivity competitive europium insulin autoantibody assay

Diabetes Technol Ther. 2009 Apr;11(4):227-33. doi: 10.1089/dia.2008.0072.

Abstract

Background: Most insulin autoantibody assays for both human and animal models are in a radioassay format utilizing (125)I-insulin, but despite the radioassay format international workshops have documented difficulty in standardization between laboratories. There is thus a need for simpler assay formats that do not utilize radioactivity, yet retain the high specificity and sensitivity of radioassays.

Methods: To establish an easier enzyme-linked immunosorbent assay (ELISA) for insulin autoantibodies of non-obese diabetic (NOD) mice, we used an ELISA format, competition with unlabeled insulin, europium-avidin, and time-resolved fluorescence detection (competitive europium insulin autoantibody assay).

Results: The competitive europium assay of insulin autoantibodies when applied to sera from NOD mice had high sensitivity and specificity (92% sensitivity, 100% specificity) compared to our standard insulin autoantibody radioassay (72% sensitivity, 100% specificity) in analyzing blind workshop sera. It is noteworthy that though the assay has extremely high sensitivity for murine insulin autoantibodies and utilizes human insulin as target autoantigen, human sera with high levels of insulin autoantibodies are not detected.

Conclusions: Our results clearly indicate that low levels of insulin autoantibodies can be detected in an ELISA-like format. Combining a europium-based ELISA with competition with fluid-phase autoantigen can be applicable to many autoantigens to achieve high specificity and sensitivity in an ELISA format.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Autoantibodies / blood*
  • Autoantigens / immunology
  • Biotinylation
  • Diabetes Mellitus, Type 1 / blood
  • Diabetes Mellitus, Type 1 / immunology*
  • Enzyme-Linked Immunosorbent Assay / methods
  • Europium
  • Humans
  • Insulin / deficiency
  • Insulin / genetics
  • Insulin Antibodies / blood*
  • Mice
  • Mice, Inbred BALB C
  • Mice, Inbred C57BL
  • Mice, Inbred NOD
  • Mice, Knockout
  • Sensitivity and Specificity
  • Streptavidin

Substances

  • Autoantibodies
  • Autoantigens
  • Insulin
  • Insulin Antibodies
  • Europium
  • Streptavidin