High-titer GAD65 autoantibodies detected in adult diabetes patients using a high efficiency expression vector and cold GAD65 displacement.
Adult type 2 diabetes patients with GAD65 autoantibodies (GADA) are known as latent autoimmune diabetes in adults (LADA). It has been suggested that GADA in LADA patients preferentially bind to the N-terminal end of GAD65. Using the N-terminal end extension of ³⁵S-GAD65 generated by the pEx9 plasmid, we tested the hypothesis that GADA in LADA patients preferentially react with ³⁵S-GAD65 from the pEx9 plasmid compared to the normal length pThGAD65 plasmid. Healthy control subjects (n = 250) were compared with type 1 (n = 23), type 2 (n = 290), and unspecified (n = 57) diabetes patients. In addition, radio-binding assays for GADA with ³⁵S-GAD65 generated from both the pEx9 and pThGAD65 plasmids were used in displacement assays with an excess of recombinant human GAD65 (2 μg/mL) to correct for non-specific binding. ³⁵S-GAD65 produced by either pEx9 or pThGAD65 did not differ in binding among the healthy controls and among the type 1 diabetes patients. Among the type 2 and unspecified patients, there were 4/290 and 3/57 patients, respectively, with binding to the pEx9 but not to the pThGAD65 generated ³⁵S-GAD65. In the displacement assay, we discovered 14 patients with very high-titer GADA among the type 1 (n = 3, 12,272-29,915 U/mL), type 2 (n = 7; 12,398-334,288 U/mL), and unspecified (n = 4; 20,773-4,053,580 U/mL) patients. All samples were fully displaced following appropriate dilution. We conclude that pThGAD65 is preferred for the coupled in vitro transcription translation of ³⁵S-GAD65 and that displacement with recombinant GAD65 may detect very high-titer GADA with possible clinical relevance.
Hansson
Ida
I
Department of Clinical Sciences, Lund University/CRC, Skåne University Hospital SUS, Malmö, Sweden. ida.hansson@med.lu.se
Lynch
Kristian F
KF
Hallmans
Göran
G
Lernmark
Åke
Å
Rolandsson
Olov
O
D000328:Adult; D000918:Antibody Specificity; D001323:Autoantibodies / Q000097:blood*; D003922:Diabetes Mellitus, Type 1 / Q000235:genetics / Q000276:immunology*; D003924:Diabetes Mellitus, Type 2 / Q000235:genetics / Q000276:immunology*; D005822:Genetic Vectors; D005968:Glutamate Decarboxylase / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D006801:Humans; D008715:Methionine; D008875:Middle Aged; D010957:Plasmids; D011863:Radioimmunoassay / Q000379:methods; D011994:Recombinant Proteins / Q000276:immunology / Q000378:metabolism; D013462:Sulfur Radioisotopes
D001323:Autoantibodies; D011994:Recombinant Proteins; D013462:Sulfur Radioisotopes; D008715:Methionine; D005968:Glutamate Decarboxylase; C401141:glutamate decarboxylase 2