Immunoelectron microscopy: a new method for detection of insulin antibodies.

Abstract

The aim of the present study was to set up a sensitive technical alternative to the classical procedures for detection of human insulin antibodies. We developed a method of post-embedding immunoelectron microscopy (IEM) using as the substrate fresh human pancreas, embedded in acrylic resin to maintain its antigenic structure. The antigen was insulin within the mature secretory granule. Serum samples obtained from 10 patients with insulin antibodies detected at various titers by either radio binding assay (RBA) or enzymatic immunoassay (EIA) were incubated for 2 hr at 37 degrees C at dilutions of 1:25, 1:100, 1:400, 1:1600, 1:6400, 1:25,600, and 1:102,400. The electron microscope photographs were analyzed by computerized morphometry and the number of protein A-gold-IgG complexes was calculated per micron2 of insulin granule. IEM results were compared with those obtained with EIA. The specificity of both techniques towards insulin was assessed as the difference between the signals (number of gold particles per micron2 of insulin granule in IEM or optical density > or = 0.193 in EIA) with and without excess insulin. Sensitivity was defined as the detection limit of the assay. In all the 10 sera investigated, IEM was more sensitive, with a 12- to 40-fold lower detection limit than EIA. IEM, with native insulin granules as substrate, is a specific, reproducible, and sensitive method for detection of human serum insulin antibodies. These findings also suggest IEM as a procedure potentially suitable for identifying antigen specificity of autoantibodies circulating at low concentration.

Gene therapy of diabetes: glucose-stimulated insulin secretion in a human hepatoma cell line (HEP G2ins/g)

“…16 The the insulin antibody reaction has revealed the storage of insulin in discrete granules. These granules, although not presence of these granules, may imply that the HEP G2ins/g cell line stores insulin in a manner characteristic identical in appearance, are similar in size (300-350 nm) and resemble the secretory granules of a normal beta of the secretory granules of the islet beta cell and thus may explain how the HEP G2ins/g cell line exhibits regucell [13][14][15] and NIT-1 insulinoma cells used as controls in these experiments. Neither granules, nor the large vaculated insulin secretion in a fashion similar to the normal pancreatic beta cell.…”

“…17,18 Glucose had no These granules containing immunoreactive insulin were present in both the large vacuoles that have previously potentiating effect on 8-Br-cAMP-stimulated insulin release from parental HEP G2ins cells (Table 2). Howbeen described in the HEP G2ins cell line 3,16 and in the body of the cell, similar to that seen in a normal beta ever, glucose had a significant (P Ͻ 0.05) potentiating effect on 8-Br-cAMP-stimulated insulin release from cell [13][14][15] and NIT-1 insulinoma cells (Figure 4b), used as controls for the immunogold technique. No nonspecific GLUT 2-transfected HEP G2ins/g cells ( Table 2).…”

“…Albumin secretion of the transformed cells (5.1 g/10 5 cells per day ±0.9 s.e., n = 5) was beta cells. [13][14][15] These granules were previously described in the HEP G2ins cell line. 16 Large membrane-bound unaltered from untransformed cells (5.2 g/10 5 cells per day ± 0.4 s.e., n = 5).…”

Function of a genetically modified human liver cell line that stores, processes and secretes insulin

“…in a MiniSpin Plus Eppendorf centrifuge for the last embedding step only. Hardening was at 481C for 48 h. Sections were cut using a MT-1 microtome and labeling procedures were carried out immediately after sectioning as described by Saccomano et al, 51 with particular details as follows: non-specific binding was blocked using 1% BSA and 1% glycine in PBS at room temperature for 2 h. A goat anti-mouse IgG (EMGMHL 10) 10 nm gold probe 1:50 (BBInternational, Australian Laboratory Services Pty Ltd, Sydney, Australia) incubated for 2 h at room temperature was directed against a monoclonal mouse anti-human insulin (1:70) primary antibody (BioGenex, San Ramon, CA, USA) incubated overnight at 41C. Primary antibody was replaced by PBS to assess the level of non-specific binding of the gold probe.…”

Long-term in vitro exposure to high glucose increases proinsulin-like-molecules release by isolated human islets

“…Islets of Langerhans from six pancreata were processed for electron microscopy as previously described (Saccomanno et al 1993). Briefly, the islets were fixed for 2 h in 2·5% glutaraldehyde in 0·1 M cacodylate buffer.…”