cobrotoxin and Myasthenia-Gravis

Topics: Amino Acid Sequence; Animals; Autoantibodies; Binding Sites; Bungarotoxins; Cobra Neurotoxin Proteins; Epitopes; Erabutoxins; Humans; Immune Tolerance; Mice; Mice, Inbred C57BL; Models, Molecular; Molecular Sequence Data; Myasthenia Gravis; Neurotoxins; Peptide Fragments; Protein Binding; Receptors, Antigen, T-Cell; Receptors, Nicotinic; Structure-Activity Relationship; T-Lymphocytes; Torpedo

Topics: Acetylcholine; Animals; Autoimmune Diseases; Cobra Neurotoxin Proteins; Disease Models, Animal; Humans; Membrane Potentials; Motor Endplate; Myasthenia Gravis; Rats; Receptors, Cholinergic; Synaptic Transmission

Autoantibodies against nicotinic acetylcholine receptor (nAChR) in myasthenia gravis (MG) patients are usually detected by radioimmunoprecipitation assays using extracted acetylcholine receptors labeled irreversibly with 125I-alpha-bungarotoxin (alpha-BuTx). To provide a nonradioactive immunoassay, we established an assay using nAChRs labeled with Eu(3+)-alpha-cobratoxin (alpha-CTx).. We derivatized alpha-CTx with a diethylenetriaminepentaacetate moiety and formed a complex with Eu(3+). The complex was purified by HPLC, and the fractions were tested for binding to Torpedo and human nAChRs. The most active fractions were used to label nAChRs for the immunoprecipitation assay, and the bound Eu(3+) was quantified by time-resolved fluorescence.. Eu(3+)-labeled alpha-CTx competed with 125I-alpha-BuTx for binding to Torpedo nAChRs and saturated the binding sites of human nAChRs, with a K(d) of 7.2 x 10(-9) mol/L. Results of the immunoassay performed with Eu(3+)-labeled alpha-CTx were similar to those obtained with 125I-alpha-BuTx, with a slightly higher limit of detection [0.3 nmol/L (n = 6) vs approximately 0.1 nmol/L for isotopic assay]. None of 34 negative sera tested (16 healthy controls, 10 patients with nonmyasthenia-related disease, 8 patients seronegative for MG) gave a value >0.3 nmol/L. Of the 35 positive myasthenic sera (with antibody values, previously determined by isotopic assay, of 0.4-1290 nmol/L) compared in the two assays, 32 tested positive with the Eu(3+) assay. Linear regression analysis yielded the equation: y = 1.035x - 0.013 nmol/L; S(y:x) = 0.172 nmol/L; r(2) = 0.977.. The new time-resolved fluorescence method for quantification of antibodies to nAChRs in MG patients provides a performance similar to that of the widely used isotopic assay and could be used in laboratories with restricted use of isotopes.

Topics: Adult; Aged; Aged, 80 and over; Animals; Autoantibodies; Chromatography, High Pressure Liquid; Cobra Neurotoxin Proteins; Electric Organ; Europium; Female; Fluoroimmunoassay; Humans; Male; Middle Aged; Myasthenia Gravis; Receptors, Nicotinic; Sensitivity and Specificity; Torpedo; Tumor Cells, Cultured

Serotherapy, an approach currently used to protect humans against animal bites or stings, is often too specific. To broaden antiserum paraspecificity, use of antibodies directed against areas shared by all members of a toxin family was previously proposed. MST2 is a mAb that recognizes all long-chain curaremimetic toxins (Charpentier et al. (1990) J. Mol. Recog. 3, 74-81). It binds to toxin residues that make contact with the toxin's target, e.g., the nicotinic acetylcholine receptor (AcChoR). We now show that MST2 also recognizes (-) nicotine, an agonist of AcChoR. Binding properties of MST2 therefore mimick, at least partially, binding properties of AcChoR. Injection in rabbits of MST2 mixed with adjuvant, elicited anti-idiotypic (anti-Id) antibodies that inhibited binding of the toxin to AcChoR. A proportion of these anti-Id antibodies specifically bound AcChoR and thereby mimicked the toxin. Furthermore, rabbits immunized with MST2 elicited auto-anti-anti-Id antibodies capable of binding the toxin. Our data provide a molecular explanation for the previously reported signs of myasthenia gravis as triggered by antibodies raised against cholinergic antagonists. Implications in the design of antisera to toxic proteins are discussed.

Topics: Animals; Antibodies, Anti-Idiotypic; Antibodies, Monoclonal; Autoimmune Diseases; Binding Sites; Cobra Neurotoxin Proteins; Immunization; Myasthenia Gravis; Neuromuscular Nondepolarizing Agents; Nicotine; Protein Conformation; Rabbits; Receptors, Cholinergic

Topics: Animals; Autoantibodies; Chromatography, Affinity; Cobra Neurotoxin Proteins; Humans; Immunosorbent Techniques; Myasthenia Gravis; Nylons; Rabbits; Receptors, Cholinergic; Serum Albumin, Bovine; Torpedo

Cobrotoxin-binding protein was isolated by affinity chromatography from human thymoma which had been surgically removed from patients with myasthenia gravis. The protein was composed of polypeptides with a molecular mass of 40, 51, 65, and 74 kilodaltons as determined by polyacrylamide gel electrophoresis in the presence of sodium dodecylsulphate. Isoelectric focusing of the protein gave pI values of 5.2-5.6 and 11. This is the first report of the isolation of the protein from human thymoma. These findings suggest that the cobrotoxin-binding protein from human thymoma patients with myasthenia gravis has subunits similar to those of fish electric organs or mammalian muscles.

Topics: Adult; Chromatography, Affinity; Cobra Neurotoxin Proteins; Electrophoresis, Polyacrylamide Gel; Female; Humans; Male; Middle Aged; Molecular Weight; Myasthenia Gravis; Receptors, Cholinergic; Thymoma; Thymus Neoplasms

Using the immunohistochemical technique, it was revealed that serum immunoglobulins of patients with myasthenia gravis (Ig) were irreversibly attached to the myoneuronal connections of the rat intercostal muscles like the marker of the nicotin cholinireceptors--the cobra venom neurotoxin (CT). In addition, Ig differs from CT in the binding to nervous cells of the claustrum and diencephalon reticular formation and with certain cells of the nucleus caudatus and hemispheric cerebral cortex. It is suggested that the autoimmune processes in patients with myasthenia gravis do not only involve myoneuronal connection but also participate in central mechanisms of the disease genesis.

Topics: Animals; Binding Sites; Binding Sites, Antibody; Brain; Cobra Neurotoxin Proteins; Elapid Venoms; Humans; Immunoglobulin G; Muscles; Myasthenia Gravis; Neuromuscular Junction; Rats

To determine the nature of the cellular immune response directed against acetylcholine receptor in myasthenia gravis, we compared lymphocyte stimulation by eel receptor with clinical factors. The mean (+/- SEM) stimulation index was 4.5 +/- 0.9 for 39 myasthenic patients and 0.97 +/- 0.18 for 48 controls (p less than 0.001). Positive response in patients was associated with disease onset after 50 years (10 of 13 patients) and presence of thymoma (seven of eight patients), but not with HLA type. Stimulation index correlated with disease activity (rs = 0.63, p less than 0.01). Blocking the active portion of the receptor molecule with naja toxin resulted in 68 percent diminution of the response, suggesting that this site plays a significant role in the cellular immune response in myasthenia gravis.

Topics: Adult; Antigen-Antibody Reactions; Binding Sites; Binding Sites, Antibody; Cobra Neurotoxin Proteins; Female; Histocompatibility Antigens; HLA Antigens; Humans; Immunity, Cellular; Lymphocytes; Male; Middle Aged; Myasthenia Gravis; Receptors, Cholinergic