Prolonged incubation in the two-colour immunofluorescence test increases the prevalence and titres of islet cell antibodies in Type 1 (insulin-dependent) diabetes mellitus

Abstract

The conventional indirect immunofluorescence test of islet cell antibodies was recently improved by the development of a two-colour immunofluorescence assay using a monoclonal proinsulin antibody to detect islet B cells. The aim of this study was to test whether in this new assay the prevalence and titre of ICA were affected by the time of incubation carried out in the presence of aprotinin (Trasylol) as an inhibitor of proteolysis. The end-point titre of ICA was therefore determined in sera from 70 children aged 0.6 to 15 years with recent onset Type 1 (insulin-dependent) diabetes mellitus, 50 healthy control subjects and 97 non-diabetic siblings of Type 1 diabetic children. In the conventional twocolour assay, ICA was positive in 53/70 (76%) Type 1 diabetic patients, 1/50 control subjects and 2/97 siblings after 30 min incubation. Prolonged incubation for 18 h increased the prevalence of ICA positive samples to 62/70 (89%) in the diabetic patients and to 2/50 in the control subjects, while the prevalence among the siblings was unchanged. Of the ICA positive non-diabetic subjects, one control child has a father with Type 1 diabetes, and one of the siblings subsequently developed Type 1 diabetes. In the diabetic patients the median titre was 1:32 for the 30 min incubation, and it increased to 1:64 for the 18 h incubation (p<0.001). A marked prozone effect was seen; 16% of the samples from the Type 1 diabetic children sera were negative at a 1:2 dilution, but were found positive at higher dilutions. In conclusion, an 18 h incubation increases the end point titres and the prevalence of ICA in the two-colour ICA assay in Type 1 diabetic children of recent onset. The prevalence and levels of ICA among these patients may be larger than hitherto expected.