Fusion proteins for combined analysis of autoantibodies to the 65-kDa isoform of glutamic acid decarboxylase and islet antigen-2 in insulin-dependent diabetes mellitus.
BACKGROUND
Prediction, risk assessment, and diagnosis of autoimmune diseases often rely on detection of autoantibodies directed to multiple target antigens, such as the 65-kDa isoform of glutamic acid decarboxylase (GAD65-abs) and the tyrosine phosphatase-like protein islet antigen-2 (IA2-abs), the two major subspecificities of islet cell antibodies (ICAs) associated with insulin-dependent diabetes mellitus. We hypothesized that a combination of autoantigens in a fusion protein unifying the important immunodominant epitopes could provide an efficient target for cost-effective, one-step screening of sera.


METHODS
Chimeric proteins composed of GAD65 and IA2 residues were constructed, analyzed for their immune reactivity with monoclonal antibodies and sera, and used in a diagnostic assay with (35)S-labeled protein as antigen.


RESULTS
Length and order of GAD65 and IA2 sequences were critical for conservation of the conformational epitopes in the fusion protein. Among four chimera tested, only IA2((606-979))/GAD65((1-585)) retained wild-type-like folding of GAD65 and IA2 domains and yielded a stable protein after baculovirus expression. Reactivity of GAD65 antibody- and IA2 antibody-positive sera from patients newly diagnosed with insulin-dependent diabetes mellitus, from ICA-positive prediabetics, and from ICA-positive first-degree relatives demonstrated conservation of the relevant autoreactive epitopes. The assay based on the in vitro translated fusion antigen had a sensitivity and specificity identical to those for detection of GAD65- and IA2-abs based on the two separate GAD65 and IA2 proteins.


CONCLUSIONS
Autoantigens such as GAD65 and IA2 can be combined successfully in a fusion protein of similar immune reactivity. This allows simultaneous detection of GAD65- and IA2-abs in a one-step screening assay and cost-effective identification of positive individuals at risk of diabetes or at onset of disease.
Rickert
M
M
Department of Orthopedic Surgery, University of Heidelberg, D-96118 Heidelberg, Germany.
Seissler
J
J
Dangel
W
W
Lorenz
H
H
Richter
W
W
D000818:Animals; D000911:Antibodies, Monoclonal; D001323:Autoantibodies / Q000097:blood* / Q000235:genetics; D002460:Cell Line; D003922:Diabetes Mellitus, Type 1 / Q000175:diagnosis / Q000276:immunology*; D000939:Epitopes; D005968:Glutamate Decarboxylase / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D006801:Humans; D015151:Immunoblotting; D007313:Insecta / Q000166:cytology; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D008970:Molecular Weight; D014176:Protein Biosynthesis; D012372:ROC Curve; D011863:Radioimmunoassay; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D012306:Risk; D014158:Transcription, Genetic
D000911:Antibodies, Monoclonal; D001323:Autoantibodies; D000939:Epitopes; C096322:ICA512 autoantibody; D007527:Isoenzymes; D011993:Recombinant Fusion Proteins; D005968:Glutamate Decarboxylase; C401141:glutamate decarboxylase 2