A recent report described serum antiGQlb ganglioside antibodies in Miller Fisher syndrome (MFS), a clinical variant of Guillain-Barre syndrome (GBS). Four consecutive cases ofMFS all had high titre anti-GQlb antibodies which were absent from all control sera including those of patients with GBS. (i Neurol Neurosurg Psychiatry 1993;56:204-206) Antibodies directed against gangliosides are being increasingly identified in patients with a variety of acute and chronic neuropathies. ' Patients and methods All patients were diagnosed and treated in this institute and represent the consecutive cases of MFS and GBS admitted over the last 18 months. MFS was defined as a clinical syndrome comprising ataxia, areflexia and ophthalmoplegia with or without bulbar or facial muscle involvement and with clinically insignificant limb weakness. Serum samples were collected at the time of admission (as close to the onset of symptoms as possible) and stored in aliquots at -70°C until assayed. Sera from four patients with typical MFS and 20 patients with GBS were tested. Control sera comprised randomly selected patients with multiple sclerosis (n = 10), other neurological diseases including myasthenia gravis, myopathies and motor neuron disease (n = 14) and normal volunteers from the local community (n = 13).Anti-ganglioside antibody assays were performed using standard enzyme linked immunosorbent assay (ELISA) and thin layer chromatography (TLC) overlay techniques as previously described.8 Briefly, ELISAs were performed by coating microtitre trays with 100 ng purified GQlb (Novabiochem, Nottingham, UK) or 100 ng of GM1, GD1a, GDlb, GD3 and GTlb (Sigma Chemicals, Poole, UK) in 50,u1 methanol per well and evaporating to dryness. Sera were serially diluted from 1/100 in fivefold dilutions in phosphate buffered saline (PBS) containing 0-1% bovine serum albumin (BSA), pH 7*4 and incubated overnight at 4°C. CSF was assayed neat and serially diluted in twofold dilutions as necessary. After washing the plates 10 times with PBS, a 1/3000 dilution of peroxidase labelled anti-human IgM or IgG (Dako) was added and incubated for 4 hours at 4°C. Plates were developed by adding substrate solution comprising 20 mg o-phenylenediamine (Sigma) in 60 mls 0-1 M citrate buffer pH5-5 with 20 pul of 30% hydrogen peroxide.The reaction was stopped with 20,ul H2S04 after 20 minutes and the optical densities (OD) read in an MR5000 plate-reader (Dynatech). All samples were assayed in duplicate on at least two separate occasions. Both antigencoated wells and blank wells were incubated with test serum and the OD values and titres calculated after subtracting the blank well OD from the coated well OD.For the TLC overlay, gangliosides were separated on aluminium backed Kieselgel 60 WF254 S HPTLC plates (Merck) in the solvent system chloroform:methanol:0 25% KCI in a volume ratio of 50:40:10. The chromatograms were air-dried, dipped in 0 1% polyisobutylmethacrylate (PIMBS) beads for 20 seconds at 40°C, air dried, blocked with 1% BSA in PBS pH 7-5 for 1 h...

IgM paraproteins associated with autoimmune peripheral neuropathy and anti-Pr cold agglutinins react with sialic acid epitopes present on disialylated gangliosides including GD1b, GT1b, GQ1b, and GD3. A causal relationship between the paraprotein and the neuropathy has never been proven experimentally. From peripheral blood B cells of an affected patient, we have cloned a human hybridoma secreting an antidisialosyl IgM mAb, termed Ha1, that shows identical structural and functional characteristics to its serum counterpart. Variable region analysis shows Ha1 is encoded by the same V H 1 family heavy chain gene, V1-18, as the only other known anti-Pr antibody sequence and is somatically mutated, suggesting that is arose in vivo in response to antigenic stimulation. In the rodent peripheral nervous system, Ha1 immunolocalizes to dorsal root ganglia, motor nerve terminals, muscle spindles, myelinated axons, and nodes of Ranvier. After intraperitoneal injection of affinity-purified antibody into mice for 10 d, electrophysiological recordings from the phrenic nerve-hemidiaphragm preparation demonstrated impairment of nerve excitability and a reduction in quantal release of neurotransmitter. These data unequivocally establish that an antidisialosyl antibody can exert pathophysiological effects on the peripheral nervous system and strongly support the view that the antibody contributes to the associated human disease. ( J. Clin. Invest. 1996. 97:1155-1164.)

A patient with a chronic, large fibre sensory neuropathy had an immunoglobulin MB monoclonal paraprotein reactive at titres in excess of 1/105 with NeuNAc(a2-8)NeuNAc(a2-3)Gal configured disialosyl groups present on the gangliosides GDlb, GTlb, GQlb, and GD3. The paraprotein showed weaker reactivity with GDla, GM3, and LM1 but no reactivity with GM2, GM1, or asialo-GM1. In addition, the paraprotein had cold agglutinating activity with anti-Pr2 specificity, Pr2 being an antigenic determinant on membrane glycoproteins or glycolipids in erythrocytes or both. A large fibre sensory neuropathy with monoclonal anti-disialosyl antibodies is an increasingly recognised form of paraproteinaemic neuropathy.

Patients with multifocal motor neuropathy frequently have elevated titers of serum antibodies reactive with GM1 ganglioside. Although these antibodies may cause the syndrome, this has yet to be proven directly. As part of our studies on the nature and pathogenic potential of anti-GM1 antibodies, we have cloned B cells from the peripheral blood of 3 patients with multifocal motor neuropathy and generated four stable heterohybridoma cell lines secreting human monoclonal IgM anti-GM1 antibodies. In this report we describe the basic properties of these monoclonal antibodies in comparison with the patient's sera from which they were derived. The antibodies all differ in their pattern of reactivity with GM1 and other Gal(beta 1-3)GalNAc-containing glycoconjugates. They have widely varying thermal ranges and their reactivities are strongly influenced by the presence of accessory lipids. Affinity purification of the patient's sera with GM1 led to the identification of previously unrecognized paraproteins that were resolvable above the background of polyclonal anti-GM1 IgM. Our data demonstrate considerable heterogeneity in the immune response to GM1 both within individual sera and between different patients, which is likely to be of importance to their role in disease pathogenesis.

Anti-GM1 and anti-GQ1b ganglioside antibodies are found in association with acute and chronic peripheral neuropathies, including Guillain-Barré syndrome. They are believed to arise as a result of molecular mimicry with immunogenic microbial polysaccharides. Although anti-ganglioside antibodies are suspected to play a causal role in neuropathy pathogenesis, the details of this have yet to be proven. The approach in this laboratory to solving this issue has been to generate anti-GM1 and anti-GQ1b monoclonal antibodies from peripheral blood lymphocytes of affected patients and to study their immunolocalization in peripheral nerve and their electrophysiologic effects in animal models in which peripheral nerve sites are exposed to anti-ganglioside antibodies. These data show that anti-ganglioside antibody-reactive epitopes are widely distributed in peripheral nerve and can cause electrophysiologic abnormalities in a variety of model systems; thus, these data support the view that anti-ganglioside antibody-reactive epitopes may directly contribute to neuropathy pathogenesis.

A series of monoclonal IgM anti-GM1 ganglioside antibodies has been cloned from peripheral blood lymphocytes of patients with multifocal motor neuropathy and Guillain-Barré syndrome. In solid-phase immunoassay, the antibodies react with GMI, and also in differing degrees to the structurally related glycolipids asialo-GM1 (GA1) and GD1b. Here we describe the binding patterns of six human anti-GM I antibodies to epitopes within the human nervous system. Antibodies were observed to bind to motor neurons and spinal grey matter, dorsal and ventral spinal roots, dorsal root ganglion neurons, nodes of Ranvier, neuromuscular junctions and skeletal muscle. The distribution of immunoreactive epitopes, which included sensory structures, extended beyond those sites conventionally regarded as pathologically affected in anti-GM1 antibody-associated motor nerve syndromes. This undermines a model of disease pathogenesis based solely on antigen distribution. Factors other than the presence or absence of antigen, such as the local ganglioside topography, antibody penetration into, and pathophysiological vulnerability of a particular site may also influence the clinicopathological outcome of anti-GM1 antibody-mediated autoimmune attack.

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High expression and secretion of recombinant ovine beta-lactoglobulin has been achieved in the yeast Kluyveromyces lactis. The yield of beta-lactoglobulin is 40-50 mg per litre of culture supernatant and accounts for approx. 72% of the total secreted protein. Constitutive expression is under the control of the Saccharomyces cerevisiae phosphoglycerate kinase promoter from an intronless version of the beta-lactoglobulin gene. Secretion is specified by the ovine protein's own signal sequence. this system, coupled to an efficient and novel recovery protocol, allows 30 mg of pure protein to be isolated from a typical 1 litre culture. The protein is virtually indistinguishable from beta-lactoglobulin conventionally purified from sheep milk by its behaviour in native PAGE and SDS/PAGE, reactivity to antibodies, CD, fluorescence spectroscopy and N-terminal sequencing. Attempts to achieve a similar expression and secretion system in the yeast S. cerevisiae met with only limited success, although it was found that heat-shock treatment modestly increased the yield up to approx. 3-4 mg per litre of culture supernatant. Site-directed mutagenesis showed that secretion in S. cerevisiae depended upon correct formation of the two disulphide bonds present in beta-lactoglobulin.