A simple 'free 3H-ligand' assay for rapid screening of hybridoma monoclonal antibodies against neurotransmitter analogs and anti-idiotype antibodies

Effective, rapid screening of hybridoma supernatants for monoclonal antibodies against the dopaminergic antagonists pimozide and haloperidol, and the serotonergic antagonist ketanserin was performed using a 'free 3H-ligand' assay. Anti-mouse Ig-coated microtiter plates were incubated with hybridoma supernatants prior to incubation with excess 3H-ligand. After removal of free 3H-ligand, bound 3H-ligand was eluted with acid for liquid scintillation counting. With minor modification, the assay can be used to screen hybridomas for anti-anti-ligand (anti-idiotypic) antibodies.     

Quantitative receptor autoradiography: tissue defatting eliminates differential self-absorption of tritium radiation in gray and white matter of brain

A four-fold greater absorption (quenching) of tritium emissions by white matter relative to gray matter produces a false ‘contrast’ effect in autoradiographs of³H-ligand binding to brain sections. The differential absorption is eliminated by tissue defatting prior to autoradiographic exposure.     

Association between glycoconjugate antibodies and Campylobacter infection in patients with Guillain-Barré syndrome

In a retrospective study we have analysed sera from a well-characterised Guillain-Barré syndrome (GBS) patient group for antibodies that react with gangliosides. Of 95 GBS patients and 85 control patients analysed, we found that 14 (15%) of GBS patients but only one control patient had antibodies that react with the gangliosides G_M1 and/or G_D1b but not G_M2, G_D1a and G_T1b using a sensitive enzyme-linked immunosorbent assay (ELISA). This pattern of reactivity suggests binding to the carbohydrate structure Gal(β1–3)GalNAc which is shared between some glycolipids and glycoproteins. Similar antibodies have been found previously in a subpopulation of patients with lower motor neuron disease. In the present study, the predominant immunoglobulin class of these anti-glycoconjugate antibodies was IgG rather than IgM. A correlation was found between the presence of these antibodies and prognosus in terms of disability at 3 and 12 months after presentation. Patients with anti-glycoconjugate antibodies also had a higher incidence of previous Campylobacter infections than the rest of the patient group, although the significance of this remains to be determined.     

Demonstration of the presence of a specific interferon-γ receptor on murine astrocyte cell surface

Interferon gamma (IFN-γ) is a pleiotropic lymphokine produced by T-lymphocytes which acts as a soluble mediator in immunological reactions. In addition to several immune target cells, such as monocytes and macrophages, it acts on the principal glial population, the astrocytes, inducing Ia antigen expression.We have developed a binding assay for ¹²⁵I-labeled recombinant murine IFN-γ, and show that, using this assay, IFN-γ interacts with a single specific receptor on the murine astrocyte cell membrane. The binding is specific and saturable and it takes place with a K_d = 1.64 × 10⁻⁹ M, with 11,100 receptor molecules per astrocytic cell. The binding shows, as for macrophages, species specificity.Using an immune assay including rabbit antibodies to IFN-γ and ¹²⁵I-labeled protein A, we have demonstrated an internalization of the ligand. This is an energy-dependent process, as around 50% of the bound IFN-γ is endocytosed after 4 h at 37°C when cultures are maintained in complete culture medium.     

Identification of sub-populations of chick neural retinal cells by monoclonal antibodies: a fluorescence activated cell sorter screening technique

An assay was developed which identifies monoclonal antibodies recognizing the cell surfaces of sub-populations of chick retinal cells which survive in culture. Antibodies from hybridoma culture supernatants were bound to monolayers of retina cells followed by a fluorescent secondary antibody. The quantitative fluorescence analysis ability of the fluorescence-activated cell sorter (FACS) was used to determine the fluorescence intensity associated with viable single cells and the frequency with which these cells appear in the total population. Hybridomas were generated which define both overlapping and non-overlapping retina cell populations. The cytotoxic activity of many of the monoclonal antibodies was determined on the FACS by propidium iodide exclusion, and the identity of various sub-populations was demonstrated by immunohistochemical staining of retina sections.     

Myasthenia gravis sera containing antiryanodine receptor antibodies inhibit binding of [3H]-ryanodine to sacroplasmic reticulum

Myasthenia gravis (MG) patients with thymoma often have antibodies against the calcium-release channel of the sarcoplasmic reticulum (SR) in striated muscle, the ryanodine receptor (RyR). RyR function can be tested in vitro by measuring the degree of [³H]-ryanodine binding to SR. In this study, sera from 9 out of 14 MG patients containing RyR antibodies inhibited [³H]-ryanodine binding to SR membranes from rat skeletal muscle. The 9 patients with antibodies inhibiting ryanodine binding had more severe MG than those with noninhibiting antibodies (P = 0.006). Sera from MG patients with acetylcholine receptor and titin muscle antibodies but no antibodies against RyR and blood-donor sera did not have an inhibiting effect in the [³H]-ryanodine binding assay. The results show that RyR antibodies in MG patients have high affinity for the RyR, and that the binding of antibodies probably affects calcium release from SR by locking the RyR ion channel in a closed position. © 1998 John Wiley & Sons, Inc. Muscle Nerve 21:329–335.     

Anti-idiotypic antibodies: a useful alternative for studying the biochemical expression of μ/δ opioid binding sites in mammalian brain

A polyclonal antiserum was produced against opioid binding sites using an anti-idiotypic approach whereby antibodies directed against the opioid agonist DSLET were used as immunogen. The anti-idiotypic antiserum recognized specific brain proteins with molecular masses of 76 ± 4, 73 ± 4 and 59 ± 3 kDa, respectively. The immunolabeling of these proteins was mainly inhibited by μ, δ opioid agonists and a general antagonist, naloxone. The inhibition of immunoprecipitation by opioid agonists and antagonist and the developmental expression of these immunoreactive proteins found to occur during brain ontogeny strongly suggest that these three proteins were μ, δ but not κ opioid binding sites. The anti-idiotypic antiserum both inhibits ³H-DADLE binding and mimics the inhibitory agonist effects on the stimulated cAMP level of NG 108-15 cells which expressed 5 opiate receptors. Numerous mammalian brain opioid binding sites were labeled, due to the fact that the binding site was the epitope recognized by the anti-idiotypic antibodies. From the numerous studies performed with a view to characterizing the specificity of the anti-idiotypic antibodies, it was strongly suggested that the anti-idiotypic antibodies specifically recognize μ/δ opioid binding sites and they can therefore be powerful tools for studying the biochemical expression of these opioid binding sites in mammalian brains.     

Comparison of three immunoassays in the screening and characterization of monoclonal antibodies against arginine-vasopressin

A method for screening monoclonal antibodies (McAbs) to neuropeptides was evaluated using 8-arginine-vasopressin (AVP) as a model. Mice were immunized with AVP-thyroglobulin conjugate and their spleen cells were fused with X 63-Ag8.653 mouse myeloma cells. The resulting hybridoma supernatants were screened for specific antibody production using 3 different assays: solid phase enzyme radioimmunoassay in Terasaki plates (Ter-ELISA), liquid phase radioimmunoassay (LPRIA) and an immunohistochemical technique. From 2 independent fusions, 7 McAbs specific for AVP were obtained. They belonged to the IgG1 subclass and reacted more strongly to the ring part of the nonapeptide. The screening strategy proposed relies upon a crude selection of conjugate-reacting hybridomas, followed by neuropeptide-specific hybridoma identification using both LPRIA (with radioiodinated synthetic peptide) and an immunohistochemical technique (to detect natural neuropeptide). During subcloning steps Ter-ELISA is then chosen, to select for specific clones and to eliminate those reacting with the carrier thyroglobulin.     

Fibrillar Alzheimer’s amyloid peptide A β1–42stimulates low density lipoprotein binding and cell association, free radical production and cell cytotoxicity in PC12 cells

Amyloid forming peptides are known to disturb vital cellular functions and induce cell death. However, the mechanisms by which fibrillogenic peptides induce cytotoxic effects in various cells has not been established. In this study the effects on low density lipoprotein binding and uptake of fibrils of the Alzheimer’s amyloid β-peptide (Aβ_1–42), which is known to play a central role in the pathogenesis of Alzheimer’s disease, were investigated in pheochromocytoma PC12 cells. Fibrillar Aβ_1–42at μmol concentrations increased low-density lipoprotein (LDL) binding and cell asociation by 460% and 200% respectively, and LDL degradation by about 62%. Approximately 49% and 34% of Aβ fibril stimulated LDL cell association and degradation was inhibited by anti-LDL receptor antibodies. The soluble form of Aβ had no effect on any of these measures of LDL metabolism. The observed increased glutathione reductase activity, DNA fragmentation (TUNEL assay) and decreased DNA synthesis ([³H] thymidine incorporation assay) in cells treated with Aβ_1–42fibrils alone or together with LDL relative to controls, suggests that the interaction of fibrils with LDL receptors might be one possible pathway which contributes to fibril cytotoxicity.     

Transforming growth factor-β1 inhibits tumor necrosis factor-α/lymphotoxin production and adoptive transfer of disease by effector cells of autoimmune encephalomyelitis

We previously reported that the CD4⁺ suppressor cells (Ts) that regulate recovery of Lewis rats from experimental autoimmune encephalomyelitis (EAE) produce transforming growth factor-β (TGF-β). We also reported that TGF-β downregulates interferon-γ (IFN-γ), but not interleukin-2 (IL-2) production, by the CD4⁺ effector T cells (Te) that mediate EAE. We now report that TGF-β also inhibits the production of tumor necrosis factor/lymphotoxin (TNF/LT) by EAE effector cells. When activated in vitro with myelin basic protein (MBP), Te produced TNF/LT, as measured using a WEHI 164 cytotoxicity assay. The specificity of cytokine action was demonstrated using neutralizing antibodies to TNF/LT. When added to the Te + MBP cultures, TGF-β inhibited TNF/LT production in a dose-dependent fashion. Moreover, neutralizing anti-TGF-β antibodies augmented TNF/LT production in the Te + MBP cultures. We also confirm that TGF-β inhibits adoptive transfer of EAE. In contrast, murine IL-10 only partially inhibited TNF/LT and IFN-γ production by Te. We conclude that TGF-β production by Ts plays a major role in recovery from EAE in the Lewis rat by inhibiting TNF/LT and IFN-γ production by the effector cells that mediate EAE.     

Characterizing κ3 opioid receptors with a selective monoclonal antibody

To help characterize κ₃ receptors and establish their relationship to traditional μ and δ receptors, we have generated a κ₃-selective monoclonal antibody. Monoclonal antibodies were raised against BE(2)-C cells, a human neuroblastoma cell line containing μ, κ₃, and δ opioid receptors. Of the 5,000 hybridoma cell lines screened, approximately 2,000 hybridomas tested positive against BE(2)-C membranes by ELISA, but only 98 of these were negative against a different neuroblastoma cell line lacking opioid receptors. Supernatants from one hybridoma, 8D8, inhibited up to 90% of ³H-NalBzoH (κ₃) binding without affecting ³H-DAMGO (μ) or ³H-naltrindole (δ) binding in BE(2)-C membranes. The selectivity of the antibody was further demonstrated by its blockade of the inhibition of cAMP accumulation in BE(2)-C cells by the κ₃ agonist NalBzoH but not the μ agonist morphine. Monoclonal antibody 8D8 (mAb8D8) also recognizes κ₃ receptors from mouse, rat, and calf brain. Administered intracerebroventricularly, mAb8D8 blocked κ₃ but not morphine (μ) analgesia in vivo. On Western blots, mAb8D8 recognized a protein with a molecular mass of approximately 70 kilodaltons in BE(2)-C. These studies demonstrate the selectivity of mAb8D8 for κ₃ receptors and provide additional support for the existence of this unique opioid receptor subtype. © 1996 Wiley-Liss, Inc.     

Whole blood impedance aggregometry detects heparin-induced thrombocytopenia antibodies

Heparin-induced thrombocytopenia (HIT) is a serious complication of heparin use. IgG antibodies to complexes of platelet factor 4 (PF4) and heparin trigger the clinical manifestations of HIT. Only a subset of these antibodies will activate platelets and these can only be identified with platelet aggregation (functional) assays. Heparin-induced platelet aggregation (HIPA) and ¹⁴C-serotonin release (SRA) assays for HIT are time-consuming and complex to perform. We have developed a whole blood impedance (WBI) test using the new Multiplate^® analyser.All samples referred to our laboratory over a 10 month period were screened for heparin-PF4 antibodies by an ELISA method (Zymutest HIA IgG). The 4T's score was used to assess HIT pretest probability.Twenty antibody positive samples were further tested by all three functional assays: light transmission aggregometry (LTA), SRA and WBI. Thirteen out of twenty samples were positive by LTA (10 patients) and 15 by WBI (11 patients). SRA, considered to be the gold standard, was used as a confirmatory test and 11 were found to be positive (10 patients); four discrepant samples were weakly positive by WBI. The prevalence of a positive functional test was strongly correlated with the 4T's clinical risk score, but a small number of low-risk patients had positive functional assays.In this study, the WBI assay detected all SRA positive patients and was positive for two others suggesting greater sensitivity. The rapid and easy to perform assay may be a useful tool for haematology laboratories to detect platelet-activating HIT antibodies.     

Induction of antimyelin and antioligodendrocyte antibodies by vaccinia virus An experimental study in the mouse

The appearance of serum antibodies binding to specific brain antigens was monitored in mice inoculated intracerebrally with a dermotropic or a neurotropic strain of vaccinia virus. Antibodies were measured with a binding assay using [¹²⁵I]protein A. Inoculation with the neurotropic strain caused an induction of serum antibodies binding to the myelin membrane, the myelin basic protein and oligodendrocytes while no induction of binding antibodies to neurons was observed. The dermotropic strain failed to elicit the formation of binding antibodies to brain antigens.     

β2-glycoprotein I, anti-β2-glycoprotein I, and fibrinolysis

β₂-glycoprotein-I (β₂GPI) is a phospholipid-binding plasma protein that consists of five homologous domains. Domain V is distinguished from others by bearing a positively charged lysine cluster and hydrophobic extra C-terminal loop. β₂GPI has been known as a natural anticoagulant regulator. β₂GPI exerts anticoagulant activity by inhibition of phospholipid-dependent coagulation reactions such as prothrombinase, tenase, and factor XII activation. It also binds factor XI and inhibits its activation. On the other hand, β₂GPI inhibits anticoagulant activity of activated protein C. According to the data from knockout mice, β₂GPI may contribute to thrombin generation in vivo. Phospholipid-bound β₂GPI is one of the major target antigens for antiphospholipid antibodies present in patients with antiphospholipid syndrome (APS). Binding of pathogenic anti-β₂GPI antibodies increases the affinity of β₂GPI to the cell surface and disrupts the coagulation/fibrinolysis balance on the cell surface. These pathogenic antibodies activate endothelial cells via signal transduction events in the presence of β₂GPI. Impaired fibrinolysis has been reported in patients with APS. Using a newly developed chromogenic assay, we demonstrated lower activity of intrinsic fibrinolysis in euglobulin fractions from APS patients. Addition of monoclonal anti-β₂GPI antibodies with β₂GPI also decreased fibrinolytic activity in this assay system. β₂GPI is proteolytically cleaved by plasmin in domain V (nicked β₂GPI) and becomes unable to bind to phospholipids, reducing antigenicity against antiphospholipid antibodies. This cleavage occurs in patients with increased fibrinolysis turnover. Nicked β₂GPI binds to plasminogen and suppresses plasmin generation in the presence of fibrin, plasminogen, and tissue plasminogen activator (tPA). Thus, nicked β₂GPI plays a role in the extrinsic fibrinolysis via a negative feedback pathway loop.     

Anti-ganglioside complex antibodies in Miller Fisher syndrome

Background

Some ganglioside complexes (GSCs) are target antigens for serum antibodies in patients with Guillain–Barré syndrome (GBS). Anti‐GSC antibodies may be associated with particular clinical features of GBS.

Objective

To investigate antibodies to GSCs in the sera of patients with Miller Fisher syndrome (MFS) characterised by elevation of the IgG anti‐GQ1b antibody.

Results

In all, 7 of 12 (58%) consecutive patients with MFS were found to have IgG antibodies to GSCs containing GQ1b, of whom 5 had IgG antibodies to GQ1b‐GM1 complex (GQ1b/GM1) and 2 had antibodies to GQ1b/GD1a; 4 of 5 patients without sensory symptoms had anti‐GQ1b/GM1 antibodies.

Conclusions

At least three different specificities in MFS‐associated antibodies, GQ1b‐specific, anti‐GQ1b/GM1‐positive and anti‐GQ1b/GD1a‐positive, were observed. In patients with MFS not only GQ1b itself but also clustered epitopes of GSCs, including GQ1b, may be considered to be prime target antigens for serum antibodies. A tendency to escape sensory disturbances is shown by anti‐GQ1b/GM1‐positive MFS.We recently reported that some ganglioside complexes (GSCs) are target antigens for serum antibodies in patients with Guillain–Barré syndrome (GBS), an acute immune‐mediated polyradiculoneuropathy, and suggested that anti‐GSC antibodies may be associated with particular clinical features of GBS.¹ Because glycolipids including gangliosides tend to form clustered complexes with cholesterols in lipid rafts in the plasma membrane,² anti‐GSC antibodies are likely to cause nerve dysfunction through binding to GSCs in lipid rafts in neuronal membranes.Miller Fisher syndrome (MFS) is characterised by a clinical triad of ophthalmoplegia, ataxia and areflexia, and is considered to be a variant of GBS.³ The presence of the IgG anti‐GQ1b antibody in serum is an excellent diagnostic marker for MFS.⁴ This antibody often cross reacts with GT1a^4,5 and is pathophysiologically associated with ophthalmoplegia or ataxia in MFS and GBS.^5,6,7 Thus, MFS is a clinically and serologically well‐defined syndrome with a pathophysiological mechanism similar to that of GBS, which suggests that patients with MFS may also have anti‐GSC antibodies. Here, we examined the serum samples of patients with MFS and found antibodies specific for a mixture of two gangliosides, including GQ1b or GT1a.     

Generation of monoclonal antibodies specific for developmentally regulated antigens of the chicken retina

In order to raise monoclonal antibodies specific for neural antigens that might be involved in regulatory events during histogenesis of the chick visual system, 5 different immunogen fractions were employed in conjunction with 4 immunization regimes. By screening 17,000 hybridoma supernatants from 58 fusion experiments, empirical guidelines were formulated for generation of antibodies with distinct specificities. A number of positive and negative results would not have been predicted from theoretical considerations and emphasize the relevance of empirical data. To substantiate our observations we present 10 monoclonal antibodies that bind to distinct cell types or layers of the retina. Single cell as well as explant cultures in conjunction with Western blot analysis were employed to determine subcellular localization and cell type specificity of selected antibodies. Six antibodies bind specifically to the inner limiting membrane or the optic fiber layer or the inner plexiform layer or the outer plexiform layer. Four recognize selectively ganglion cells or Müller glial cells or presumably subpopulations of amacrine cells. These antibodies promise to be helpful tools to evaluate mechanisms of neural differentiation.     

Autoradiographic localization and quantification of dopamine D2 receptors in normal human brain with [H]N-n-propylnorapomorphine

The precise distribution of dopamine receptors has been studied autoradiographically in the normal human brain using [³H]N-n-propylnorapomorphine ([³H]NPA) as a ligand. Preliminary experiments aimed at optimizing the binding assay conditions revealed that preincubation washing of caudate nucleus sections was a prerequisite to obtain a good ratio of specific to non-specific binding. The binding of [³H]NPA to caudate-putamen sections was saturable, stereospecific, reversible, of high affinity (K_d = 0.27–0.35 nM) and occurred at a single population of sites. Competition experiments with various drugs indicated that in the caudate-putamen the specific [³H]NPA binding sites possess the pharmacological features of the dopamine D₂ receptor. The highest levels of [³H]NPA binding sites were found in the caudate nucleus, putamen, globus pallidus and nucleus accumbens. There were also intermediate to low concentrations of the ³H-ligand in the hippocampus, the insular and cingular cortices and in the occipito-temporal gyrus, while almost undetectable levels of binding were found in the anteior frontal cortex. Thorough examination of the subregional distribution of [³H]NPA binding sites in the caudate-putamen-pallidum complex revealed heterogenous patterns of radioactivity. In these brain regions, the distribution of autoradiographic grains was punctate and islands of high and low densities were observed. Moreover, in the caudate nucleus, there was a subtle high lateral to low medial gradient in the topography of the [³H]NPA binding sites and a more pronounced gradient along the rostrocaudal axis; the highest levels of binding being located at the midbody of the nucleus. No gradients of [³H]NPA binding were observed in the putamen. The present data indicate that [³H]NPA is a suitable ligand for accurate autoradiographic labeling of dopamine D₂ receptors in human postmortem brain tissue and that dopamine receptors are heterogeneously distributed and topographically organized in patches and gradients in the basal ganglia regions.     

Non-uniform expression of α subunit isoforms of the Na/K pump in rat dorsal root ganglia neurons

Tissue sections and antibodies selectively recognizing isoforms of the α subunit of the Na⁺/K⁺ pump were used to determine the expression of α₁, α₂ and α₃ pump isoforms in the plasma membrane of adult rat dorsal root ganglia (DRG) neurons. There was no detectable membrane signal from DRG neurons that were probed with antibodies to the α₂ isoform of the Na⁺/K⁺ pump. The α₁ isoform of the Na⁺/K⁺ pump was found in most (77±4%) studied DRG neurons, regardless of cell size. Only 16±7% of the neurons expressed a detectable level of the α₃ Na⁺/K⁺ pump and all were apparently from a subpopulation of large DRG neurons. Comparison of cell size distributions and a study of neurons identified in serial sections suggested that of the α₃ positive DRG neurons about 75% coexpressed the α₁ isoform of the Na⁺/K⁺ pump. These data show that the expression of the protein of the α subunit isoforms of the Na⁺/K⁺ pump is not uniform throughout the population of DRG neurons and that α₁ is the predominant isoform in the plasma membrane of these neurons.     

Cell-mediated autoimmunity in paraneoplastic neurological syndromes with anti-Hu antibodies

Paraneoplastic encephalomyelitis or subacute sensory neuronopathy associated with small-cell lung cancer (SCLC) and high titers of anti-HuD antibodies, also called the “anti-Hu syndrome,” is believed to result from an immune response triggered by tumor antigens and misdirected to the neurons. To further assess the issue of cell-mediated immunity in this disease, the peripheral blood lymphocyte surface phenotype was studied in 15 patients suffering from the anti-Hu syndrome (seropositive group) and in two control groups consisting of 12 seronegative SCLC patients without neurological syndrome and 15 healthy volunteers. In addition, the recombinant human HuD protein was used to stimulate in vitro peripheral blood mononuclear cells of 10 seropositive patients and of 10 patients from each control group. Phenotypic analysis of the peripheral blood lymphocytes revealed a significant increase of the memory helper (CD45RO⁺CD4⁺) T cells in the seropositive group in comparison with the two control groups. Antigen-specific proliferation of peripheral blood mononuclear cells, measured by [³H]thymidine uptake after HuD antigen stimulation, was much higher in the seropositive group than in the two control groups, and phenotypic analysis of proliferating cells revealed a significant expansion of the CD45RO subpopulation of T cells in the seropositive group. Furthermore, after HuD stimulation, a significant increase of the interferon-γ/interleukin-4 ratio was found in culture supernatants of the seropositive group compared with seronegative SCLC patients and normal controls. Taken together, these results indicate that HuD protein is an antigenic target for autoreactive CD4⁺ T cells, presumably of the Th1 subtype, which could therefore be directly involved in cell-mediated injury of the nervous system as well as in antitumoral immunity. Ann Neurol 1999;45:162–167