Glutamic acid decarboxylase autoantibody assay using 125I-labelled recombinant GAD65 produced in yeast.
We describe a new method for measuring autoantibodies (Ab) to the 65 kDa isoform of glutamic acid carboxylase (GAD65). In particular, GAD65 without the hydrophobic N-terminal region has been produced in yeast, purified, labelled with 125I and reacted with GAD65 Ab. Antibody bound 125I-GAD65 is then precipitated by the addition of solid phase protein A. With the assay, GAD65 Ab were detected in 59 of 71 (83%) islet cell antibody (ICA) positive IDDM patients and in 8 of 23 (35%) ICA negative IDDM patients (overall 67 of 94 (71%) of IDDM patients). Low concentrations of GAD65 Ab were also detected in 2/98 (2%) healthy blood donors and 1/27 (4%) Graves' disease patients had a high level of antibody. GAD65 Ab were not detected in any of 10 Hashimoto's thyroiditis, 20 Addison's disease or 19 myasthenia gravis sera. There was good agreement between the 125I assay and the current reference method based on 35S-labelled full-length GAD65 (produced by in vitro transcription/translation reaction) and solid phase protein A (r = 0.91, n = 108). Overall, our 125I assay showed sensitivity, precision and disease group specificity at least as good as any assay so far described. These features, combined with a simple assay protocol and the convenience of 125I counting and handling indicate that the method is suitable for routine GAD65 Ab measurements.
Powell
M
M
FIRS Laboratories, RSR Ltd., Llanishen, Cardiff, UK.
Prentice
L
L
Asawa
T
T
Kato
R
R
Sawicka
J
J
Tanaka
H
H
Petersen
V
V
Munkley
A
A
Morgan
S
S
Rees Smith
B
B
Furmaniak
J
J
D000328:Adult; D000368:Aged; D001323:Autoantibodies / Q000032:analysis*; D002648:Child; D002675:Child, Preschool; D003922:Diabetes Mellitus, Type 1 / Q000175:diagnosis; D005656:Fungal Proteins / Q000276:immunology*; D005968:Glutamate Decarboxylase / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D006801:Humans; D007118:Immunoassay; D007223:Infant; D007457:Iodine Radioisotopes; D008875:Middle Aged; D011994:Recombinant Proteins / Q000276:immunology*; D012680:Sensitivity and Specificity; D013379:Substrate Specificity
D001323:Autoantibodies; D005656:Fungal Proteins; D007457:Iodine Radioisotopes; D011994:Recombinant Proteins; D005968:Glutamate Decarboxylase