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

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Species and epitope specificity of two 65 kDa glutamate decarboxylase time-resolved fluorometric immunoassays. / Rui, M; Hampe, CS; Wang, C; Ling, Z; Gorus, FK; Lernmark, Åke; Pipeleers, DG; De Pauw, PE.

In: Journal of Immunological Methods, Vol. 319, No. 1-2, 2007, p. 133-143.

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Rui, M ; Hampe, CS ; Wang, C ; Ling, Z ; Gorus, FK ; Lernmark, Åke ; Pipeleers, DG ; De Pauw, PE. / Species and epitope specificity of two 65 kDa glutamate decarboxylase time-resolved fluorometric immunoassays. In: Journal of Immunological Methods. 2007 ; Vol. 319, No. 1-2. pp. 133-143.

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TY - JOUR

T1 - Species and epitope specificity of two 65 kDa glutamate decarboxylase time-resolved fluorometric immunoassays.

AU - Rui, M

AU - Hampe, CS

AU - Wang, C

AU - Ling, Z

AU - Gorus, FK

AU - Lernmark, Åke

AU - Pipeleers, DG

AU - De Pauw, PE

PY - 2007

Y1 - 2007

N2 - 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.

AB - 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.

KW - GAD65

KW - Immunoassay

KW - Type 1 diabetes

KW - Pancreatic beta cells

U2 - 10.1016/j.jim.2006.11.007

DO - 10.1016/j.jim.2006.11.007

M3 - Article

C2 - 17210161

VL - 319

SP - 133

EP - 143

JO - Journal of Immunological Methods

JF - Journal of Immunological Methods

SN - 1872-7905

IS - 1-2

ER -