Detection of glutamic acid decarboxylase (GAD) autoantibodies by indirect immunofluorescence using CHO cells expressing recombinant human GAD65.
The reaction between human glutamic acid decarboxylase (GAD65) expressed in CHO cells and GAD antibodies was studied by indirect immunofluorescence (IIF). The monoclonal antibodies GAD1 and GAD6, which recognize conformational and continuous GAD epitopes respectively, yielded distinct staining patterns. Twelve of 26 sera from newly-diagnosed insulin-dependent diabetes mellitus (IDDM) patients displayed a variety of anti GAD specific IIF images encompassing the two extremes observed with the monoclonal antibodies. None of 21 normal sera tested positive in this assay. As a control, the sera were tested by a reference immunoprecipitation (IP) assay using in vitro produced, folded 35S-GAD65. Only one of the patient sera reacted by IP using heat- and detergent-denatured 35S-GAD65 indicating that most of the auto-antibodies recognized only a folded antigen. Eleven patient sera were both IIF and IP anti-GAD-positive. The IIF reactivity of these sera was blocked by soluble GAD from brain extracts. One serum was positive only by IIF, and its reactivity was not blocked by soluble GAD. Eight sera were positive only by IP. Our results established differences in anti GAD antibodies in terms of their capacity to recognize human GAD65 in the context of transformed CHO cells compared with conventional IP assays. These differences should be considered in future attempts to improve the available assays for the detection of IDDM autoantibodies.
Papouchado
M L
ML
Cátedra de Immunología-Instituto de Estudios de la inmunidad Humoral, Universidad de Buenos Aires-CONICET, Argentina.
Ermácora
M R
MR
Poskus
E
E
D000293:Adolescent; D000818:Animals; D001323:Autoantibodies / Q000032:analysis*; D016466:CHO Cells; D002648:Child; D002675:Child, Preschool; D006224:Cricetinae; D005260:Female; D019084:Fluorescent Antibody Technique, Indirect; D005968:Glutamate Decarboxylase / Q000276:immunology*; D006801:Humans; D007223:Infant; D008297:Male; D011233:Precipitin Tests; D051381:Rats; D017208:Rats, Wistar; D011994:Recombinant Proteins / Q000276:immunology
D001323:Autoantibodies; D011994:Recombinant Proteins; C024211:islet cell antibody; D005968:Glutamate Decarboxylase