Sensitive non-isotopic assays for autoantibodies to IA-2 and to a combination of both IA-2 and GAD65.
BACKGROUND
A sensitive ELISA for measurement of IA-2 autoantibodies has been developed and assessed. Also, a combination ELISA for detection of both GAD65 autoantibodies and IA-2 autoantibodies is described.


METHODS
The IA-2 autoantibody assay is based on the ability of IA-2 autoantibodies to form a bridge between IA-2 intracellular fragment coated onto ELISA plate wells and liquid-phase IA-2 labelled with biotin. The combination ELISA uses plates coated with both IA-2 and GAD65 and a mixture of IA-2-biotin and GAD65-biotin. Assay sensitivity was assessed using the WHO reference (NIBSC 97/550) for islet cell antibodies. IA-2 autoantibody measurements by ELISA were compared with measurements in immunoprecipitation assays (IPAs) based on 125I or 35S labelled IA-2. Combination ELISA results were compared with results obtained for individual autoantibodies.


RESULTS
As little as 15 units/mL of NIBSC 97/550 was detectable in the IA-2 autoantibody ELISA compared to 125 units/mL by 125I-IA-2 IPA. 110/216(51%) sera from patients with type 1 DM were positive in the IA-2 autoantibody ELISA while 97/216 (45%) and 91/216 (42%) were positive in the 125I-IA2 and 35S-IA-2 IPAs, respectively. The IA-2 autoantibody ELISA showed 100% specificity for type 1 DM. The combination ELISA was able to detect GAD65 and/or IA-2 autoantibodies in 183/216 (85%) diabetic sera and 183/216 (85%) were also found positive for autoantibodies to IA-2 and/or to GAD65 in the assays for individual antibodies. Autoantibody measurements in the individual autoantibody assays and in the combination ELISA showed good agreement by Pearson correlation (r=0.92, n=216, p<0.001) and by Bland and Altman analysis.


CONCLUSIONS
Sensitive and specific ELISAs for measurement of autoantibodies to IA-2 and to a combination of IA-2 and GAD65 have been developed. These assays are suitable for screening large numbers of samples in diabetes prediction and prevention trials.
Chen
Shu
S
FIRS Laboratories, RSR Ltd, Parc Ty Glas, Llanishen, Cardiff, CF14 5DU, UK. firs@rsltd.eclipse.co.uk
Willis
Jinny
J
Maclean
Clare
C
Ananieva-Jordanova
Rossitza
R
Amoroso
Marie-Andrée
MA
Brooking
Helen
H
Powell
Michael
M
Collins
Anne
A
Bennett
Stuart
S
Mitchell
Sarah
S
Burne
Peter
P
Furmaniak
Jadwiga
J
Smith
Bernard Rees
BR
D000918:Antibody Specificity; D001323:Autoantibodies / Q000097:blood* / Q000276:immunology; D001324:Autoantigens / Q000276:immunology* / Q000378:metabolism; D001710:Biotin / Q000737:chemistry; D003920:Diabetes Mellitus / Q000097:blood; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005968:Glutamate Decarboxylase / Q000276:immunology* / Q000378:metabolism; D006801:Humans; D007457:Iodine Radioisotopes; D007527:Isoenzymes / Q000276:immunology* / Q000378:metabolism; D008565:Membrane Proteins / Q000276:immunology* / Q000378:metabolism; D054562:Protein Tyrosine Phosphatase, Non-Receptor Type 1; D017027:Protein Tyrosine Phosphatases / Q000276:immunology* / Q000378:metabolism; D054635:Receptor-Like Protein Tyrosine Phosphatases, Class 8; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D001323:Autoantibodies; D001324:Autoantigens; D007457:Iodine Radioisotopes; D007527:Isoenzymes; D008565:Membrane Proteins; D001710:Biotin; C516862:PTPRN protein, human; D054562:Protein Tyrosine Phosphatase, Non-Receptor Type 1; D017027:Protein Tyrosine Phosphatases; D054635:Receptor-Like Protein Tyrosine Phosphatases, Class 8; D005968:Glutamate Decarboxylase; C401141:glutamate decarboxylase 2