A New Luminescence Assay for Autoantibodies to Mammalian Cell–Prepared Insulinoma-Associated Protein 2

In the LIPS assay, IA-2 (amino acids 601–979) was cloned into the pREN2 mammalian expression vector downstream of the Renilla luciferase (Ruc) reporter (3–5). Monkey kidney cells (Cos1) in a 100 mm² dish were transfected, and 48 h later, the cells were sonicated, clarified by centrifugation, and the extract (enough for ∼1,500 assays in duplicate) used without further purification. Total luciferase activity in the extract was measured in a luminometer and adjusted so that each reaction contained ∼1.0 × 10⁷ light-forming units per 0.1 μl of extract. The extract was then incubated with 1.0 μl of sera for 1 h at room temperature in a total volume of 100 μl, transferred to 96-well filtration plates containing 7 μl of 30% suspension of protein A/G beads (Pierce Biochem), and incubated for an additional hour. The A/G beads with the captured antibody–luciferase labeled IA-2 complexes were washed using a BioMek-FX workstation, Renilla luciferase substrate (Promega, Madison, WI) was added, and the light-forming units were determined in a Microplate Luminometer (Berthold Technologies, Bad Wildbad, Germany).

In the RIP assay, performed as previously described (6), the same intracellular portion of IA-2 used in the LIPS assay was cloned into a pGBKT7 vector (Clontech, Mountain View, CA), and the IA-2 protein was produced by in vitro transcription/translation in the presence of ³⁵S-methionine. IA-2 autoantibodies were detected by liquid-phase radioimmunoprecipitation using ∼20,000 cpm of radiolabeled IA-2.

A total of 100 sera from patients with newly diagnosed type 1 diabetes who had been previously screened for IA-2 autoantibodies by RIP were selected on the basis of the counts precipitated, from very high to moderate and low to negative. A total of 100 sera from age-matched nondiabetic individuals served as the control (6). A serum was scored as positive if the precipitated cpm exceeded the mean + 3 SD of the control subjects.

Sera from diabetic (n = 100) and control subjects (n = 100) were screened for IA-2 autoantibodies by RIP assay. Only one of the control subject sera fell outside the 3 SD range, whereas 90% of the diabetic subject sera exceeded the 3 SD range of the control subject sera (Fig. 1A). The coefficient of variation (CV) for duplicate samples of the diabetic subject sera was 5.0%.

The same sera were screened for IA-2 autoantibodies by LIPS assay. None of the control subject sera fell outside the 3 SD range, whereas 85% of the diabetic subject sera exceeded the 3 SD range of the control subject sera (Fig. 1B). The CV of the diabetic subject sera was 9.3%.

Comparison of the RIP and LIPS assays revealed a high correlation, with a 0.805 coefficient of determination (R²) (Fig. 1C). Evaluation of the 5 diabetic subject sera that were negative by the LIPS assay but positive by the RIP assay showed that 3 of the 5 negative sera were at the very borderline of positivity (Fig. 1D).

Receiver operating characteristic analysis showed that the areas under the curves for IA-2 by RIP and LIPS were not statistically different (P = 0.120) (Fig. 1E).

From these experiments, we concluded that there was no significant difference in sensitivity or specificity between the LIPS and RIP assays. However, the LIPS assay offers several potential advantages. First, because it uses luminescence rather than radioactivity, it lends itself better to most clinical laboratories and can be easily automated. Second, because the preparation of the protein does not require in vitro transcription/translation, the protein can be prepared at a fraction of the cost of the RIP assay. Third, because the recombinant protein is fused with a luminescence marker, it does not need to be externally labeled or laboriously separated from the other proteins in the mammalian cell lysate, thereby eliminating the purification steps required for both bacteria-prepared proteins (7) used in solid-phase enzyme-linked immunosorbent assays. Fourth, because the proteins are made in mammalian cells, they may undergo a variety of processing and posttranslational modifications that do not occur when they are prepared via in vitro transcription/translation and, therefore, may more truly reflect the natural state of the autoantigen. It will be of interest to see if this increases detection and/or sensitivity when other autoantigens, prepared in mammalian cells, are screened for autoantibodies.

Our findings suggest that the LIPS assay may be of practical value at the clinical level for the detection of autoantibodies not only for diabetes, but also for other autoimmune diseases. However, as with all new assays, LIPS requires validation with a large number of sera and comparison with RIP in a Diabetes Autoantibody Standardization Program workshop.

This research was supported by the Intramural Research Program of the National Institute of Dental and Craniofacial Research, National Institutes of Health.