Murine monoclonal glutamic acid decarboxylase (GAD)65 antibodies recognize autoimmune-associated GAD epitope regions targeted in patients with type 1 diabetes mellitus and stiff-man syndrome.

To study the immune response to glutamic acid decarboxylase (GAD) in insulin-dependent diabetes mellitus, monoclonal GAD antibodies after fusion of splenocytes from a nondiabetes-susceptible BALB/c mouse immunized with human recombinant GAD65 were generated. Of the 44 monoclonals, 35 are specific for the GAD65 isoform, whereas 9 also react with GAD67. Some 37 monoclonals, including all GAD65/67 reactive antibodies, react with GAD by Western blot analysis.

Vector-derived expression of recombinant rat interleukin-6.

Rat interleukin-6 (IL-6) cDNA, coding for an important inflammation- and immune-regulatory polypeptide cytokine, was cloned into the novel expression vector pH6EX3 which directs the synthesis of inserted genes as a fusion protein with histidine hexapeptide (HH). The resultant vector (pRIL6.992) was shown to produce significant amounts of recombinant rat IL-6 fusion protein with HH at its N-terminus in various strains of Escherichia coli.

Mapping of an autoreactive epitope within glutamate decarboxylase using a diabetes-associated human monoclonal autoantibody and an epitope cDNA library.

Glutamate decarboxylase (GAD65) is a major autoantigen in insulin-dependent diabetes (IDDM) and the neurological disorder Stiff-Man-Syndrome (SMS). We derived a human monoclonal autoantibody (MICA 2) from peripheral blood of a patient newly diagnosed with IDDM, which reacted with GAD65 in Western blots. This indicated that a linear epitope is recognized by MICA 2.

Epitope mapping of a recombinant human TSH receptor extracellular domain: identification of a predominant epitope using animal sera.

The extracellular domain of the TSH receptor (TSHR-561, amino acids #78-389) was expressed as a hexa-histidine fusion protein in bacteria. The recombinant protein was purified to homogeneity and used to immunize porcine and ovine species. High titre antibodies were obtained from both species that recognized the recombinant protein in Western blot analysis but failed to interfere with the TSH radio receptor assay.

Autoreactive epitopes within the human alpha-enolase and their recognition by sera from patients with endometriosis.

Patients with endometriosis significantly develop autoantibodies directed against endometrial proteins, which may be involved in the aetiology of this gynaecological disease. Based on standard Western blot analysis, a 48 kDa protein was localized in the soluble protein extract of endometrial adenocarcinoma cells using sera from patients with clinically staged endometriosis and identified as the glycolytic enzyme alpha-enolase. The corresponding cDNA coding for the human alpha-enolase was isolated from a human endometrial cDNA library and cloned into the vector pH6EX3, allowing the efficient expression of recombinant human alpha-enolase with an N-terminal histidine-hexapeptide as affinity ligand in Escherichia coli.