Islet-cell antibodies (ICA) were measured in 198 patients with insulin-dependent diabetes mellitus (IDDM) and 177 non-diabetic controls using a standard indirect immunofluorescence method (IF method) and a newly established three-layer immunofluorescence method applying the biotinavidin system (BAS method) as described below.
All sera were diluted 1: 2 in 10mM phosphate-buffered saline (PBS), pH7.2, applied to unfixed human pancreatic sections (5μm), and incubated at room temperature for 30min in a moist chamber. The slides were washed over 5-min intervals in PBS 3 times. Biotinated goat anti-human IgG (H+L) serum (Vector Labs., Burlingame, California) diluted 1: 150 in PBS, was then applied to the tissue sections which were then incubated at room temperature for 30min. The sections were then washed for 5-min intervals 3 times in PBS. Fluorescein-isothiocyanate (FITC)-conjugated avidin D (Vector Labs., Burlingame, California) diluted 1: 150 in PBS was then applied to the sections which were incubated at room temperature for 30 min. The sections were then washed for 5min 3 times in PBS.
ICA titers detected by the BAS method correlated well with those determined by the standard IF method (rs=0.987, p<0.01). The BAS method had a sensitivity for ICA about eight times higher than that of the IF method.
The overall prevalence of ICA detected by the BAS method (ICA-BAS) vs that by the IF method (ICA-IF) was 41%(82/198) vs 28%(56/198) in patients with IDDM. All 177 non-diabetic controls except one showed negative for ICA-BAS. ICA-IF was detected in none of these non-diabetic controls.
In IDDM patients, ICA-BAS was positive in all cases less than 1 month after the onset of diabetes, while the prevalence of ICA-IF was 83%(20/24) during the same period. The prevalence of ICA-IF decreased rapidly with the duration of disease, reaching a value of 6%(3/55) in patients with a disease duration of 10 years or more. The incidence of ICA-BAS also decreased, although to a lesser extent than ICA-IF, with the duration of disease.
These results suggest that in almost all Japanese IDDM patients pancreatic autoimmune processes occur immediately after the onset of disease. The higher sensitivity of the BAS method compared with the standard IF method was of significant diagnostic value, especially in diabetic patients with a long duration of disease.