Method Description

Radioimmunoassay:

(125)I-labeled recombinant human antigen is incubated with patient sample. Anti-human IgG is then added to form an immunoprecipitate. After washing the immunoprecipitate, the amount of (125)I-labeled antigen in the immunoprecipitate is measured using a gamma-counter. The amount of gamma emission in the precipitate is proportional to the amount of antigen-specific IgG in the sample. Results are reported as units of precipitated antigen (nMol) per L of patient sample.(Masuda M, Powell M, Chen S, et al. Autoantibodies to IA-2 in insulin-dependent diabetes mellitus. Measurements with a new immunoprecipitation assay. Clin Chim Acta. 2000;291(1):53-66; Walikonis JE, Lennon VA. Radioimmunoassay for glutamic acid decarboxylase [GAD65] autoantibodies as a diagnostic aid for stiff-man syndrome and a correlate of susceptibility to type 1 diabetes mellitus. Mayo Clin Proc. 1998;73[12]:1161-1166; Horta ES, Lennon VA, Lachance DH, et al. Neural autoantibody clusters aid diagnosis of cancer. Clin Cancer Res. 2014;20[14]:3862-9386)

Enzyme-Linked Immunosorbent Assay

Zinc transporter 8 (ZnT8) antibodies are principally directed against the C terminal domain of ZnT8. The ZnT8 autoantibody enzyme-linked immunosorbent assay is based on the bridging principle that employs the ability of divalent ZnT8 autoantibodies to bind to ZnT8 coated on to the plate well with one arm, and to liquid ZnT8-biotin with the other arm. Calibrators or undiluted samples in duplicate are added to ZnT8 coated plate wells and incubated overnight. ZnT8-biotin is added to each well and plates. After further incubation, aspiration, and wash, streptavidin-peroxidase is added to each well. After further incubation, aspiration, and wash, peroxidase substrate is added. After further incubation, 0.5 mol/L sulfuric acid stop solution is added to each well. Absorbance is measured at 450nm, blanked against wells containing peroxidase substrate and sulfuric acid only.(Petruzelkova L, Ananieva-Jordanova R, Vcelakova J, et al. The dynamic changes of zinc transporter 8 autoantibodies in Czech children from the onset of Type 1 diabetes mellitus. Diabet Med. 2014;31(2):165-171)