Chymotrypsin-reactive antibodies in insulin-dependent diabetes mellitus

Abstract

In a previous study (Frazier et al., 1990), it was demonstrated that two patients with type 1 (insulin-dependent) diabetes mellitus had antibodies in their serum which reacted with four 29 kDa pancreas-specific proteins on two-dimensional immunoblots. This paper reports on the purification and identification of these pancreatic proteins. The protein with the pI closest to pH7 was purified through the use of ammonium sulfate fractionation and ion-exchange chromatography. Gel filtration chromatography established that the protein's molecular weight was closer to 25 kDa. Amino acid composition and sequence analyses demonstrated homology between the protein and chymotrypsin. It is suggested that an abnormal regulation of chymotrypsin activity might be related to antibodies formed in some diabetic patients.

Chymotrypsin gene expression in rat peripheral organs

“…Four different sources of antibodies generated against human chymotrypsin were used in the detection of chymotrypsin immunoreactivity: (1) rabbit polyclonal antiserum (Frazier et al 1992); (2) rabbit polyclonal antiserum (Vital Products, Inc., lot no. 10047);…”

“…Cryostat sections on chrome-alum slides were incubated in 0.3% Triton X-100 in PBS (1 h), rinsed briefly in PBS and incubated for 1 h in 5% normal goat serum in PBS. Slides were incubated in the rabbit polyclonal antisera (1:1000±1:20000) (Frazier et al 1992) or the mouse monoclonal antibody (QED) (1:1000±1:20000) in PBS containing 2% normal goat serum overnight at 4 C. Sections were washed and incubated at room temperature in biotinylated goat anti-rabbit IgG or goat antimouse IgG (both 1:400, Vector Laboratories) for 2 h, rinsed in PBS, and subsequently incubated for 2 h with streptavidin-peroxidase in PBS (1:2000, Kirkegaard and Perry Laboratories). Chymotrypsin immunoreactivity was visualized by immersion of the slides for 1±2 min in a solution containing 0.025% 3,3-diaminobenzidine, 0.6% nickel ammonium sulfate (Fisher), and 0.006% hydrogen peroxide in 50 mM TRIS (pH 7.6).…”

“…Pancreas Chymotrypsin immunoreactivity was detected by fluorescence microscopy in the glandular acinar cells using monoclonal (QED, 1:10000) and polyclonal (Frazier et al 1992, 1:1000) antibodies (Fig. 3).…”

“…C, D Rat pancreas sections. C Immunostained with a polyclonal antiserum (Frazier et al 1992, 1:1000, 2 days). Positive stain is seen in acini; an islet (arrow) is negative.…”

“…4A±D Chymotrypsin-positive endocrinelike cells of the stomach, fluorescence immunohistochemistry. A Cells observed primarily at the basal portion of the villi (Frazier et al 1992 polyclonal antiserum, 1:1000, 2 days). B High magnification revealed granulated cells among the basal epithelial cells of the gastric glands (QED monoclonal antibody, 1:10000, 3 days).…”