Research paper
Species and epitope specificity of two 65 kDa glutamate decarboxylase time-resolved fluorometric immunoassays

https://doi.org/10.1016/j.jim.2006.11.007Get rights and content

Abstract

The 65 kDa isoform of human glutamate decarboxylase (GAD65) is a major autoantigen in type 1 diabetes (T1D). In the present study, we have developed a sensitive sandwich time-resolved fluorescence immunoassay (TRFIA) for the quantification of GAD65 in cell extracts, cell media and serum. The monoclonal antibody GAD-6 is used to selectively capture GAD65 but not the slightly larger isoform GAD67, and the utilization of different detecting antibodies with distinct GAD65 epitope specificity allows modulating the specificity of the assay. To this effect we have biotinylated a recombinant antigen-binding fragment (rFab) with epitope specificity for the N-terminal region of rat and human GAD65 (rFab N-GAD65) and another rFab that selectively binds to the middle part of human GAD65 (rFab b96.11). In the assay the biotinylated rFabs are recognized by Europium labeled streptavidin. The obtained time-resolved fluorescence (TRF) is directly proportional to the concentration of GAD65 over a large measuring range (0.1 to > 100 ng/mL). Based on total error estimation including both bias and imprecision, the lower limit of quantitation (LLOQ) of GAD65 in cell extracts is 0.33 ng/mL with the N-GAD65 TRFIA, and 0.10 ng/mL with the b96.11 TRFIA, but the latter is suitable for human GAD65 only, whereas the N-GAD65 TRFIA has equal sensitivity with rat and human GAD65. Specificity was further checked with GAD65/67 fusion proteins, confirming that the presence of intact capture as well as detection epitope on the analyte is a prerequisite for recognition in both assays. We show that the beta cell-specific marker GAD65 can be quantified in pancreatic cell extracts and in serum, allowing studies on discharge during cell death in vitro as well as in vivo.

Introduction

The 65 kDa isoform of human glutamate decarboxylase (GAD, EC 4.1.1.15) is a key autoantigen in type 1 diabetes (T1D) (Baekkeskov et al., 1990). GAD65 autoantibodies (GADA) precede or accompany clinical onset, and their presence in circulation is considered predictive of overt disease or later insulin needs (Leslie et al., 1999, Gorus and Pipeleers, 2001, Pihoker et al., 2005). GADA are considered to represent a humoral immune reactivity against the GAD65 protein of pancreatic beta cells, which carry specificity in terms of its localization outside the nervous system.

In normal beta cells, GAD65 is reversibly anchored to the membrane of small synaptic-like microvesicles within the pancreatic beta cells by means of either palmitoylation in the NH2-terminal domain (Christgau et al., 1992), specific N-terminal sequences (Solimena et al., 1994) or both (Shi et al., 1994). When isolated beta cells are destroyed by the toxin streptozotocin, the GAD enzymatic and immunochemical activity was found to be released in the medium (Smismans et al., 1996, Hao et al., 1999). The mechanism and kinetics of this discharge has not yet been clarified. It is not known whether it occurs in other conditions of beta cell death and whether it is also observed in vivo. In order to investigate these questions, a quantitative assay is needed with adequate sensitivity and specificity. A method to measure the enzyme activity has first been described (Okada et al., 1976), followed by direct quantification of the GAD65 protein with a competitive RIA (Hao et al., 1999) or ELISA (Schlosser et al., 1997). The radioimmunoassay is hampered by its sub-optimal sensitivity (e.g. 1–3 ng/mL detection limit in serum), and the use of a polyclonal antiserum. The ELISA uses two monoclonal antibodies (mAb) that simultaneously bind to GAD65 with minimal mutual competition, reaching a lower detection limit of 0.03 ng/mL in rat brain extract. This ELISA is however potentially limited by the epitope specificity of the used mAbs, and its narrow dynamic range. The aim of the present work was to develop a sensitive immunoassay for GAD65 using a set of recombinant antigen-binding fractions (rFabs) with known and distinct – including human – epitope specificities (Madec et al., 1996, Schwartz et al., 1999, Padoa et al., 2003). The assay uses a sandwich immunoassay format and time-resolved fluorescence detection (TRFIA), a technique securing a broad dynamic range (Hemmila et al., 1984, Lövgren et al., 1985). It offers the advantage of varying the detecting Fabs in order to analyze epitopes that become exposed following shedding of GAD65 from damaged or dying cells.

Two GAD65 assays are now reported with different species and epitope specificities, each providing a large range of detection and quantification in human as well as rat beta cell extracts, culture media and serum.

Section snippets

Antibodies

The capture mAb GAD-6 was a generous gift from Dr. D. Gottlieb, St Louis, MO, USA. It exclusively binds to the 65 kDa isoform of GAD (Gottlieb et al., 1986) and in particular to the C-terminal region of the protein (Ujihara et al., 1994), where rat and human GAD65 have 100% similarity (Butler et al., 1993). Two recombinant antigen-binding fractions were chosen as detecting antibodies. The rFab b96.11 with middle part epitope specificity for human GAD65 was cloned (Padoa et al., 2003) from the

Properties of the biotinylated rFabs

Based on testing with streptavidin-coated microtiterplates, the biotinylation degree was estimated to average 6.2 and 6.4 molecules of biotin per rFab molecule for B-rFab N-GAD65 and B-rFab b96.11 respectively (not shown). Radiobinding experiments indicated similar 35S-rhGAD65 binding affinity of biotinylated antigen-binding fragments B-rFab N-GAD65 and B-rFab b96.11 as compared to their non- biotinylated whole mAbs (Fig. 1). The unbiotinylated rFabs could compete with their biotinylated

Discussion

This study reports on the development of a novel sensitive GAD65 TRFIA. It is based on a sandwich-type format where the capture antibody is specific for the 65 kDa isoform of GAD, and further epitope specificity can be modulated by selecting the rFab that serves as detecting antibody. The chosen rFab is biotinylated in order to be recognized by Europium labeled streptavidin, of which the time-resolved fluorescence is measured.

In the case of GAD65, a series of rFabs (Padoa et al., 2003) are

Acknowledgements

We thank Dr. I. Weets of the Belgian Diabetes Registry (BDR) for providing human sera, and P. Goubert for his technical assistance in producing rhGAD65. The beta cell bank of the JDRF center for beta cell therapy provided the human pancreatic tissues and cells, and FACS-sorted human and rat cell preparations were kindly provided by the Functional Cytomics facility of the Diabetes Research Center (DRC).

The Eli Lilly and Company provided rat and human insulin standards. This study was supported

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