A sensitive non-isotopic assay for GAD65 autoantibodies.
BACKGROUND
A new ELISA for 65 kDa isoform of glutamic acid decarboxylase autoantibodies (GAD(65) Abs), which depends on GAD(65) Ab acting divalently and forming a bridge between immobilized GAD(65) and liquid-phase GAD(65)-biotin, is described.


METHODS
Sera (25 microl) were incubated in GAD(65)-coated ELISA plate wells followed by washing and incubation with GAD(65)-biotin. After a further wash step, GAD(65)-biotin bound was quantitated by addition of streptavidin peroxidase followed by tetramethylbenzidine. Assay calibration was with WHO reference preparation 97/550.


RESULTS
Using a cut-off for positivity of 5 units/ml, sera from 0.7% (2/300) healthy blood donors (HBDs), 100% (39/39) selected type 1 diabetes mellitus (DM) patients, 1.6% (1/62) type 2 diabetes mellitus patients and 3% (4/119) autoimmune disease controls were GAD(65) Ab positive in the ELISA. Levels of positivity in an immunoprecipitation assay (IPA) based on 125I-labelled GAD(65) (cut-off 25 units/ml) were 1%, 82%, 0% and 3%, respectively. ELISA inter-assay coefficients of variation (n=12) were 7.3%, 3.8%, 7.2% and 6.3% at 5.4, 40.8, 137 and 382 units/ml, respectively.


CONCLUSIONS
The ELISA we have described has sensitivity and specificity at least as high as current radioactive assays. It has good precision and handling making it suitable for routine use.
Brooking
Helen
H
FIRS Laboratories, RSR Limited, Parc Ty Glas, Llanishen, Cardiff, South Wales, CF14 5DU, UK.
Ananieva-Jordanova
Rossitza
R
Arnold
Clare
C
Amoroso
Marie
M
Powell
Michael
M
Betterle
Corrado
C
Zanchetta
Renato
R
Furmaniak
Jadwiga
J
Smith
Bernard Rees
BR
D001323:Autoantibodies / Q000032:analysis*; D001327:Autoimmune Diseases / Q000097:blood; D001710:Biotin / Q000737:chemistry; D003922:Diabetes Mellitus, Type 1 / Q000097:blood; D003924:Diabetes Mellitus, Type 2 / Q000097:blood; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005656:Fungal Proteins / Q000276:immunology; D005968:Glutamate Decarboxylase / Q000276:immunology* / Q000378:metabolism; D006801:Humans; D007118:Immunoassay / Q000379:methods; D007457:Iodine Radioisotopes; D007527:Isoenzymes / Q000276:immunology* / Q000378:metabolism; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D015003:Yeasts / Q000378:metabolism
D001323:Autoantibodies; D005656:Fungal Proteins; D007457:Iodine Radioisotopes; D007527:Isoenzymes; D001710:Biotin; D005968:Glutamate Decarboxylase; C401141:glutamate decarboxylase 2