Polyclonal anti-idiotypic antibodies as internal images of dopamine. Applications for biochemical and morphological studies of DA receptors in the rat brain.

Polyclonal anti-idiotypic antiserum raised against both rabbit and monoclonal anti-dopamine (DA) antibodies was produced in rabbits. It was characterized for its specificity and was shown to (1) inhibit the binding of both polyclonal and monoclonal idiotypic anti-DA antibodies directed to immobilized DA conjugates; (2) inhibit the binding of (3H) DA to rat brain membranes; (3) to cross-react with a peptide extracted from a neuroblastoma cell line (NCB-20), known to express functional DA receptors. Finally, immunocytochemical studies were performed on paraformaldehyde-fixed rat brain. Anti-idiotypic antibodies were used to visualize the cellular and subcellular distribution of DA receptor binding sites in the striatum, a region that contains both D1 and D2 receptors subtypes. Under the electron microscope, the immune reaction product was observed to be concentrated in postsynaptic sites belonging mainly to dendritic spines, while presynaptic structures were sparsely labeled.     

Identification of a common idiotype on myelin oligodendrocyte glycoprotein-specific autoantibodies in chronic relapsing experimental allergic encephalomyelitis

The myelin/oligodendrocyte glycoprotein (MOG) is a target antigen for autoantibody-mediated demyelination in chronic relapsing experimental allergic encephalomyelitis (CREAE) in the Strain 13 guinea pig. Anti-idiotypic antibodies directed against a demyelinating MOG-specific monoclonal antibody (8-18C5) have identified a cross-reactive idiotype in 10/10 CREAE sera. In nine of these sera inhibition studies demonstrated that this idiotype was at or in close proximity to the combination site of guinea pig anti-MOG autoantibodies. This cross-reactive anti-MOG idiotype may represent an important target for the anti-idiotypic regulation of demyelinating antibody responses in CREAE.     

Neural membrane glycoproteins associated with chicken Thy-1: an anti-idiotypic antibody study

In order to investigate the possible binding of Thy-1 to other neuronal cell surface proteins, anti-idiotypic antibodies were raised using a panel of anti-Thy-1 monoclonal antibodies. Anti-idiotypic antibodies were selected for their ability to bind to day-old chick brain membrane components in enzyme-linked immunosorbent assays (ELISA), and to bind to membrane glycoproteins as determined by Western transfer immunoblotting assays. The 5 monoclonal anti-idiotypic antibodies bind to a membrane glycoprotein component of 70 kDa, and one of the antibodies also binds to 3 higher molecular weight components of 160 kDa, 120 kDa and 90 kDa. These antibodies bind to areas of the chicken cerebellum known to be rich in Thy-1. It is postulated that these molecules are associated with Thy-1, and that the role of Thy-1 on the neuronal cell surface, may be to form complexes with, and/or to stabilise these higher molecular weight glycoproteins during synaptic development.     

Human monoclonal IgM autoantibodies with restricted antigenic specificity for myelin express unrelated idiotypes

Idiotype-specific polyclonal antisera were prepared against myelin-binding human IgM paraproteins with specificity for the myelin-associated glycoprotein (MAG). Eight anti-idiotypic sera (6 against one monoclonal IgM and 2 against another) were tested by gel precipitation, immunoradiometric and ELISA assays for binding to the myelin-binding IgM paraproteins from 12 patients with demyelinating peripheral neuropathy. Anti-idiotypic antibodies bound only to the MAG-specific IgM used for immunization; there was no evidence of idiotypic cross-reactivity between the different IgM paraproteins.     

Use of monoclonal antiacetylcholine receptor antibodies to investigate the macrophage inflammation of acute experimental myasthenia gravis: refractoriness to a second episode of acute disease

The acute phase of experimental autoimmune myasthenia gravis (EAMG) is characterized by macrophage inflammation of muscle endplates and by muscle fiber necrosis. We induced acute EAMG by passive transfer of monoclonal antibodies (mAbs) directed against the acetylcholine receptor (AChR) to investigate this brief and self-limited disorder. After the initial acute phase, animals were refractory to induction of a second episode by subsequent injection of the same mAb, or another anti-AChR mAb of different idiotype and which binds to a separate epitope. Therefore, the refractory state was not caused by an anti-idiotypic response or epitopic modulation. Adoptive transfer of spleen cells from refractory animals had no effect, excluding a role of suppressor lymphocytes, and there was no evidence from experiments involving adoptive transfer of spleen cells from naive animals to refractory animals that refractory animals lacked effector cells.     

Production and characterization of polyclonal and monoclonal antibodies to the zeta (ξ) opioid receptor

The opioid growth factor, [Met⁵]-enkephalin, is an inhibitory agent of cell proliferation and maturation that interacts with the zeta (ξ) opioid receptor to modulate growth. In order to learn more about this receptor, polyclonal and monoclonal antibodies were raised against binding subunits identified on two-dimensional gels by ligand blotting. Using Western blotting, the polyclonal antibodies and some of the monoclonal antibodies recognized all 4 binding polypeptides (32, 30, 17, and 16 kDA) in developing rat cerebellum; no reaction was recorded in adult cerebellum. In addition, other monoclonals were able to distinguish only certain subunits (e.g. 17 kDa). The monoclonal antibodies and their F(ab′)₂ fragments, as well as the polyclonal antibodies, blocked the binding of [³H][Met⁵]-enkephalin to preparations of developing cerebellum. Both the polyclonal and monoclonal antibodies immunoprecipitated ξ opioid binding polypeptides from 6-day-old cerebellar homogenates solubilized by the zwitterionic detergent, CHAPS. Immunocytochemistry performed with polyclonal antibodies showed immunoreactivity associated with proliferating and differentiating cerebellar cells, but no specific staining was detected in the adult cerebellum. These results have identified and characterized antibodies to the ξ opioid receptor, and the antibodies were used to localize this receptor; these antibodies will be valuable to further cellular and molecular studies.     

Anti-acetylcholine receptor antibodies in myasthenia gravis: Part 1. Relation to clinical parameters in 250 patients

We examined the significance of the presence or absence of anti-acetylcholine receptor (anti-AChR) antibodies in 250 myasthenia gravis (MG) patients and the relation between clinical features and anti-AChR levels.We found high anti-AChR levels in 2 out of 11 thymoma patients without MG, while 37 out of 250 MG patients had no detectable anti-AChR. The absence of these antibodies was related to purely ocular disease and to steroid therapy and/or thymectomy.Differences in anti-AChR levels did not correspond significantly to differences in disease activity when single measurements in patients were analysed. However, the results were influenced by both the presence or absence of a thymoma, the age at onset of disease and by steroid therapy. The thymic pathology and age at onset seemed to act independently. Early onset of disease was associated with high anti-AChR levels and absence of antibodies to striated muscle (anti-SM), whereas late onset was associated with low anti-AChR and the presence of anti-SM. Thymomas both have high anti-AChR and high anti-SM. The effect of steroid therapy on antibody levels was seen in all patient groups but was strongest in thymoma patients with early onset of disease.     

The species distribution of nervous system antigens that react with anti-myelin-associated glycoprotein antibodies

The reactivity of monoclonal and polyclonal antibodies directed against human central nervous system (CNS) myelin-associated glycoprotein (MAG) was investigated in a number of animal species. The antibodies included mouse monoclonal antibodies obtained by immunization with human MAG; HNK-1, a mouse monoclonal antibody raised against a human lymphoblastoma and used to identify a subset of lymphocytes with natural killer function; human IgM paraproteins associated with neuropathy; and polyclonal antibodies obtained from rabbits immunized with rat or human MAG. Following polyacrylamide gel electrophoresis of CNS and peripheral nervous system (PNS) tissue from human, bovine, cat, rabbit, guinea pig, rat, mouse, frog, gold fish and chicken, proteins were electrophoretically transferred onto nitrocellulose. The immune-staining of electroblots showed distinct interspecies variation in the reactivity of the antibodies with MAG. In addition, the species distribution of several low molecular weight glycoproteins present in PNS tissue that cross-react with anti-MAG antibodies was determined. These low molecular weight antigens are not present in CNS homogenates or in purified human CNS myelin. It was also shown that IgM from a patient with peripheral neuropathy and paraproteinemia associated with anti-MAG antibodies recognized these low molecular weight antigens. The results suggest that IgM paraproteins, HNK-1 and some mouse monoclonal antibodies react with carbohydrate determinants shared by MAG and several lower molecular weight glycoproteins present only in human, bovine, cat and chicken PNS. Rabbit polyclonal anti-rat MAG antisera and mouse monoclonal antibodies reacting with peptide epitopes of MAG are much more specific for detecting MAG than antibodies reacting with carbohydrate epitopes of human MAG. The results are discussed in relation to human demyelinating peripheral neuropathy associated with IgM paraproteinemia.     

Clonotypic analysis of the antibody response to the acetylcholine receptor in experimental autoimmune myasthenia gravis

Autoreactive B cells reactive with the acetylcholine receptor (AChR), and the antibodies produced by them, are proposed to play a primary role in the immunopathology of myasthenia gravis and its animal models. Therefore, the anti-AChR antibody response induced in rats was characterized for the clonotypic heterogeneity, isotype distribution, and affinity by isoelectric focusing (IEF) and affinity immunoblotting. It was determined that the rat anti-AChR serum antibody was relatively heterogeneous, reflecting the oligoclonality of the response. Furthermore, isotypic dominance by IgG2a was observed in that the majority of clonal products detected by IEF were of this isotype in both primary and secondary responses. Lastly, the clonotypic anti-AChR antibodies were of relatively low affinity (avidity) when compared to antibodies reactive with the highly immunogenic protein antigen, keyhole limpet hemocyanin; anti-AChR antibody avidity did not appear to increase when the antibodies in the secondary response were compared to antibodies in the primary response. These antibody characteristics are discussed in terms of their role in disease induction.     

Cross-reactivity of anti-acetylcholine receptor autoantibodies

The heterogeneity of the specificities of anti-acetylcholine receptor (anti-AChR) antibodies of myasthenia gravis (MG) patients has been demonstrated by comparing reactions against a panel of xenogeneic AChR. For each patient there was a more or less unique cross-reactivity profile. Such heterogeneity emphasizes the need to use human AChR for the routine detection of anti-AChR. In vitro cross-reactivity was important in predicting the effect of anti-AChR after passive transfer to rats. Specificity may influence the outcome in human neonates receiving maternal anti-AChR via the placenta. In contrast to the extreme heterogeneity seen in spontaneous MG, the antibodies associated with D-penicillamine–induced MG were more homogeneous.     

Anti-idiotypic opioid receptor antibodies

Anti-idiotypic antibodies were raised against the monoclonal antibody 3-E7. One of these anti-idiotypic antibodies interferes with the binding of 3H-diprenorphine at solubilized opioid receptors, and exhibits opioid agonistic activity on NG108CC15 hybrid cells.     

Neuroimmunology of myasthenia gravis

Myasthenia gravis (MG) is an autoimmune disease in which the immune system attacks the nicotinic acetylcholine receptors (AChRs) of the neuromuscular junction. Anti-AChR antibodies are present in 85% of patients and bind to distinct epitopes on the surface of the AChR alpha subunits, as defined by competition with monoclonal anti-AChR antibodies. There are at least three types of the disease, defined by thymic histology, age of onset, and HLA associations, and anti-AChR antibodies show some differences in fine specificity between those with thymic hyperplasia and those with thymic tumors. Peripheral blood lymphocytes from MG patients contain T lymphocytes specifically sensitized to AChR. These are stimulated by purified Torpedo AChR and some human alpha subunit synthetic peptides. The T and B cell epitopes on the primary sequence of the alpha subunit are currently being mapped using recombinant human AChR subunit fragments.     

Development of an anti-idiotypic antibody that blocks substance P primary antibodies and substance P membrane binding

Polyclonal antibodies against substance P were raised in rabbits and partially purified. This apparently homogeneous immunoglobulin fraction was used to immunize other rabbits. The anti-idiotypic antibodies derived from these rabbits were substantially more effective in competing with substance P than in competing with beta-endorphin for binding to their respective primary antibodies. The anti-idiotypic antibody was also 50 times more potent in competing with substance P binding than in competing with substance K binding to rat duodenal membranes, a tissue containing receptors for substance P and substance K. The anti-idiotypic antibodies exhibited significant enhancement of substance P induced spasmogenic response on the rat uterus and guinea pig ileum (GPI). The results indicate that it is possible to develop anti-idiotypic antibodies that recognize substance P receptors. These antibodies will be of value in studies of the physiological roles of the neuropeptide, substance P.     

Anticorps dans la myasthénie

In autoimmune myasthenia gravis, 75 to 80% of patients have antiacetylcholine receptor antibodies (anti-AChR abs) quantified by immunoprecipitation. Anti-AChR abs are polyclonal, directed against all AChR subunits, with a major fraction against the main immunogenic region, (alpha-subunit, aminoacids 67-76); they cause AChR loss by three mechanisms: blocking of acetylcholine binding; accelerated degradation or AChR due to bridging of two adjacent AChR molecules (antigenic modulation); lysis of postsynaptic membrane induced by complement. Neither anti-AChR ab level nor antigenic repertoire are correlated with disease severity. Studies performed on rat and human myotubes have shown that capacity of myasthenic patients's sera or immunoglobulins to induce AChR loss was correlated with anti-AChR ab titer but not with severity. Highest anti-AChR ab titers are found in young women with hyperplastic thymus, lowest in older patients and atrophic thymus. Ten to fifteen percent of babies born to myasthenic mothers suffer from a transitory neonatal myasthenic syndrome due to passive transfer of maternal anti-AChR abs. There is no correlation between clinical condition of the baby (presence and severity of neonatal myasthenia) and severity of maternal myasthenia. The risk of neonatal myasthenia is high when maternal ab titer is elevated (≥ 100 nM). Very rare and severe cases of foetal neonatal myasthenia gravis with arthrogryposis, hypomobility are due to presence in maternal serum of anti-AChR ab directed against foetal (gamma) AChR. In generalized myasthenia without anti-AChR ab, antibodies directed against MuSK, a postsynaptic molecule involved in AChR aggregation, are detected in around 40% of patients. Features of MuSK+ myasthenia are the following: strong female preponderance, severity (respiratory and bulbar) requiring immunosuppressants, facial and tongue atrophy, poor response to anticholinesterase inhibitors, atrophic thymus and poor response to thymectomy. Low-affinity anti-AChR abs have been recently reported in myasthenia gravis without anti-AChR and anti-MuSK ABS.     

Diagnostic significance of IgG, C3, and C9 at the limb muscle motor end-plate in minimal myasthenia gravis

We detected deposits of IgG, C3, and C9 (immune complexes) at the limb muscle motor end-plates (biceps brachii muscle) in 16 of 19 patients who exhibited only ocular signs and symptoms of myasthenia gravis that were improved by intravenous injections of edrophonium chloride. Circulating anti-acetylcholine receptor (anti-AChR) antibodies were negative in 6 of the 16 patients, but the motor end-plate fine structure in the postsynaptic regions was abnormal in all 16. Single-fiber EMG revealed no abnormalities in 8 of 13 patients studied. Our results indicate that the detection of immune complexes at the limb muscle end-plate provides a highly sensitive and confirmative method for diagnosing patients with minimal or atypical myasthenia gravis who have no detectable anti-AChR antibodies in their serum.     

Preparation and characterization of antisera and monoclonal antibodies to serotonergic and dopaminergic ligands

In an attempt to produce polyclonal antisera and monoclonal antibodies to serotonin, SKF 38393 (D-1 agonist), dopamine, and haloperidol (D-2 antagonist) several procedures for the preparation of immunogenic ligand-protein carrier conjugates were investigated. The Mannich reaction utilizing formaldehyde as the chemical linker was used to prepare serotonin-protein conjugates; antibodies raised to this conjugate reacted specifically to the conjugated serotonin moiety but did not react to native serotonin. Chemical conjugations involving dimethylpimelylimidate or N-carboxymethyl derivatives for the coupling of serotonin, dopamine and SKF 38393 to carrier proteins produced antibodies primarily directed against the 'chemical coupling arm' and very little antibody activity against the ligand itself could be detected. Synthesis of a haloperidol derivative suitable for chemical coupling to a protein carrier via oxobutyric acid produced an immunogen which was capable of eliciting both polyclonal and monoclonal antibodies specific for the hapten. The pitfalls of the various chemical conjugation procedures and the difficulties of producing antibodies to free ligands are discussed.     

IgG paraproteins in neurological diseases: lack of association with neurotropic viral/bacterial antigens

In this study we tested the hypothesis that IgG paraproteins in neurological diseases might be regarded as an anti-idiotypic response raised by a nervous system antigen associated with certain neurotropic agents. After initial ELISA screen, positive samples from 34 neurological patients who had paraproteins in their CSF and serum on routine IEF investigation, were tested by immunoblot technique for the presence of specific monoclonal immunoglobulin G (IgG) against eight different neurotropic antigens. Only one patient had specific monoclonal IgG against herpes simplex virus. The results of this study did not confirm our hypothesis. Further study of IgM/IgA paraproteins is indicated.     

Arginine Vasopressin-Anti-Idiotypic Immunostaining of Human Brain Cells

Polyclonal anti-idiotypic antibodies, generated against the IgG fraction of antisera to arginine vasopressin (AVP), were shown to recognize two proteins in rat brain and bovine pituitary associated with [³ H]AVP binding. Immunochemical analyses with these antisera revealed reactivity in paraventricular and supraoptic nucleus neuronal elements and in terminals of the posterior pituitary in the human central nervous system. With the use of a dual immunocytochemical staining technique employing both the anti-idiotype and idiotype for AVP it was possible to demonstrate a pattern of AVP-anti-idiotypic-immunoreactivity on AVP neuronal elements which suggests the existence of autoreceptors.     

Detection of anti-acetylcholine receptor antibody by an ELISA using human receptor from a rhabdomyosarcoma cell line

A simple and reliable enzyme-linked immunosorbent assay (ELISA) has been developed for the measurement of anti-acetylcholine receptor (AChR) antibodies. The test utilizes a membrane-bound AChR obtained from a human rabdomyosarcoma cell line (TE671) as antigen and employs an affinity-purified rabbit anti-human immunoglobulin G alkaline phosphatase-conjugated antibody as labelled antibody. To assess the sensitivity and the specificity of our assay we tested serum samples from 13 anti-AChR antibody-positive myasthenia gravis (MG) patients known to contain between 2 and 120 nmol/l of anti-AChR antibody, three anti-AChR antibody-negative MG patients, and 70 control subjects including patients with other neurological and autoimmune diseases. A panel of six different anti-AChR monoclonal antibodies and membranes from a AChR-negative rat adrenal pheochromocytoma cell line (PC 12) were also used in competitive studies. The test showed to be specific and able to detect as low as 2.0 nmol/l of anti-AChR antibodies. Moreover, we found a good correspondence between anti-AChR antibody levels measured in the serum samples tested by our assay and levels measured by the routinely adopted radioimmuno assay (RIA) using human-AChR (r = 0.96). Cross-reaction phenomena were observed only using serum samples containing high-titer anti-DNA antibodies. The proposed ELISA, circumventing the limitation of the commonly used RIA (radioactivity and amputated legs as source of human antigen), can be considered as an useful screening test for the diagnosis of myasthenia gravis.     

Antimuscle and antiacetylcholine receptor antibodies in myasthenia gravis

The sera of 134 patients were examined for antimuscle antibodies by immunofluorescence (IF). These derived from 77 myasthenics, 30 myasthenics with thymoma, 6 patients with thymoma and no clinical evidence of myasthenia, and 21 patients with other autoimmune or neuromuscular diseases. Three separate patterns of antimuscle antibodies could be identified in the myasthenic sera by examination of the relaxed glycerinated myofibrils by both IF and phase-contrast optics: A-band (9 with thymoma, 1 without), I-band (11 with thymoma, 17 without), and a mixed A plus I pattern (5 with thymoma, 3 without). Seventy-seven myasthenic serum samples (24 with thymoma, 53 without) were available for evaluation of antibodies to acetylcholine receptor (anti-AChR) by radioimmunoassay. Ninety-one percent reacted with crude human receptor extract and 80% with receptor extracted from denervated rat muscle. There was no correlation between the titers of anti-AChR and the presence or staining patterns of antimuscle antibodies, but patients without anti-AChR did not have antimuscle antibodies. Myasthenics with thymoma had the highest prevalence of anti-AChR (23/24) and of antimuscle antibodies (25/30), and 15 of the 20 positives stained A-bands alone or with I-band, as compared to 4 of 21 positive reactions in those without tumor. Immunoabsorption, which removed or significantly reduced anti-AChR, did not alter antimuscle reactivity. The discrepancies between anti-AChR levels and the presence and types of antimuscle antibodies suggest that these are independent autoantibodies. Current theories of immunopathogenesis implicate altered thymic antigens or a major breakdown in immune regulation, either of which could explain their production.