Murine high specificity/sensitivity competitive europium insulin autoantibody assay

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.

FIG. 1.

The newly developed CE-IAA.

FIG. 2.

(A) Insulin autoantibodies measured by our standard mIAA assay. The horizontal line gives the cutoff (0.01). (B) Standard E-IAA. The horizontal line gives the cutoff (0.01). (C) Competitive CE-IAA. The horizontal line gives the cutoff (0.002). B6, C57BL/6 mice (n = 4–13); Balb, BALB/c mice (n = 20); NZB mice (n = 4–13); NOD (F) and NOD (M), female and male NOD mice (n = 53–57 and n = 10–20, respectively); 2KO(F) and 2KO(M), female and male mice with 2KO (n = 9–17 and n = 6–13, respectively). The samples were randomly selected and not identical in each group (mIAA assay, E-IAA, and CE-IAA).

FIG. 3.

(A and B) Noncompetitive and (C and D) competitive europium assays using (A and C) NOD mice or mice with 2KO and (B and D) control (C57BL/6 [B6], BALB/c [Balb], or NZB) mice.

FIG. 4.

CE-IAA inhibition by different amounts of competing fluid-phase insulin. The initial competitive insulin concentration was 2.2 × 10⁻⁵ M, which is the same as that used in our standard CE-IAA, and was then diluted. The results are shown twice with different scales (A and B) to highlight the full range of values. Autoantibodies determined with the mIAA radioassay are shown in parentheses.

FIG. 5.

(A) Correlation between results of the CE-IAA assay and the mIAA radioimmunoassay (RIA) with data presented for NOD mice (solid circles, n = 68), 2KO mice (open squares, n = 16), and control strains (open triangles, n = 46). (B–D) Coded serum samples from an international animal models workshop (Second IDS Animal Models Workshop, October 2002, provided by Dr. Cliver Wasserfall). (B) Correlation between results of the CE-IAA and the mIAA radioassay with data presented for NOD (solid circles, n = 25) and C57BL/6 (open triangles, n = 22) mice. Sensitivity and specificity are given for (C) the CE-IAA and (D) the mIAA RIA.

FIG. 6.

Sera of BALB/c mice immunized with B:9–23 insulin peptide in complete Freund's adjuvant were measured with the CE-IAA. The horizontal line gives the cutoff for the CE-IAA (0.002). The difference between the two groups was tested by Mann-Whitney's U test.