Expression of recombinant human glutamic acid decarboxylase (GAD) in myeloma cells and enzyme-linked immunosorbent assay (ELISA) for autoantibodies to GAD.
Detection of serum autoantibodies to glutamic acid decarboxylase (GAD) is a new method to differentiate insulin-dependent diabetes mellitus (IDDM) and non-insulin-dependent diabetes mellitus (NIDDM). We established a transformed mouse myeloma cell line, SPG14, which expresses recombinant human GAD65, a major isomer of 65 kDa, inside the cells. GAD65 was partially purified by affinity chromatography using the mouse anti-GAD antibody (Ab). We also established a sandwich ELISA for anti-GAD Ab of the IgG class using GAD65 for coating and the anti-human IgG for detection and examined 54 sera of the IDDM patients and 45 sera of normal individuals. When the mean +2 SD of the color development of the sera of normal individuals was used as a cut-off level, 59.2% of patients with IDDM were positive. This indicates that the ELISA was effective to differentiate IDDM and NIDDM. The result also indicates that the autoantibody to GAD does not dissociate from the recombinant GAD rapidly, even when unbound anti-GAD Ab was fully removed. By using a perfusion culture system, we obtained as many as 4.2 x 10(10) SPG14 cells, from which 5 mg of GAD65 could be obtained; this is sufficient for 5,000 assays. This system could be clinically useful for large-scale diagnosis of IDDM.
Matsuba
T
T
Tokyo Research Center, Tosoh Corporation, Kanagawa.
Yano
M
M
Abiru
N
N
Takino
H
H
Akazawa
S
S
Nagataki
S
S
Yasukawa
K
K
D000818:Animals; D001323:Autoantibodies / Q000097:blood*; D003922:Diabetes Mellitus, Type 1 / Q000097:blood / Q000201:enzymology / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005285:Fermentation; D005968:Glutamate Decarboxylase / Q000235:genetics / Q000276:immunology* / Q000378:metabolism*; D006801:Humans; D051379:Mice; D009101:Multiple Myeloma / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D014407:Tumor Cells, Cultured
D001323:Autoantibodies; D011994:Recombinant Proteins; D005968:Glutamate Decarboxylase