Detection of Autoantibodies to Protein Tyrosine Phosphatase-like Protein IA-2 with a Novel Time-resolved Fluorimetric Assay

Abstract

Background: Circulating autoantibodies to pancreatic glutamic acid decarboxylase (GAD65; the 65-kDa isoform of glutamic acid decarboxylase), protein tyrosine phosphatase-like protein IA-2, and insulin can be used as predictive markers of type 1 diabetes. We developed a novel assay for the detection of IA-2 autoantibodies (IA-2As) in serum based on time-resolved fluorimetry, hypothesizing that this kind of assay could provide several advantages over methods described to date, including radiobinding assays (RBAs) and ELISAs.

Evaluation of Two Nonisotopic Immunoassays for Determination of Glutamic Acid Decarboxylase and Tyrosine Phosphatase Autoantibodies in Serum

“…Autoantibodies against ␤-cells can be detected before clinical onset of DM1 (2,3 ) and can help to distinguish type 2 diabetes mellitus (DM2) from latent autoimmune diabetes of the adult (LADA) (4 ), which may represent one-half of cases of autoimmune diabetes (1 ). The major autoantibodies in DM1 are against islet cells, insulin, a protein tyrosine phosphatase-like protein (IA-2), and the 65-kDa isoform of glutamic acid decarboxylase (GAD65) (1 ).…”

“…The major autoantibodies in DM1 are against islet cells, insulin, a protein tyrosine phosphatase-like protein (IA-2), and the 65-kDa isoform of glutamic acid decarboxylase (GAD65) (1 ). Combined analysis of serum GAD65 and IA-2 autoantibodies (GADAs and IA-2As, respectively) may be able to replace the subjective and laborious immunofluorescent detection of islet cell autoantibodies (3,5,6 ) and, unlike insulin-autoantibody testing, help to confirm diabetes classification after insulin replacement therapy has begun.…”

“…RBAs, although cumbersome and requiring use of 35 S, reportedly provide more precise results and higher diagnostic sensitivity and specificity than ELISAs (2)(3)(4).…”

Time-Resolved Immunofluorometric Dual-Label Assay for Simultaneous Detection of Autoantibodies to GAD65 and IA-2 in Children with Type 1 Diabetes

“…The protein was expressed in Spodoptera frugiperda (Sf9) insect cells via baculoviral infection. IA-2 and glutathione S-transferase (GST)-IA-2 proteins were produced in Escherichia coli BL21 cells and purified as described previously (13 ).…”

“…We previously published single-label time-resolved immunofluorometric assay (TR-IFMA) methods for the detection of GADAs and IA-2As in a solid-phase streptavidin-coated 96-well microtiter plate format (12,13 ). The detection uses europium lanthanide chelate as a fluorescent label.…”