Ahmed Jehanli

A rapid procedure for the isolation of human IgM myeloma proteins

A two-step rapid procedure for the isolation of human IgM paraproteins from plasma samples is described. It involves gel filtration on Ultrogel AcA 34 followed by anion exchange chromatography on DEAE-Sepharose CL6B. The overall yield of IgM is greater than 70% and the final product is pure as judged by immunoelectrophoresis and SDS-polyacrylamide gel electrophoresis. The procedure is applicable to both euglobulin and cryoglobulin IgM proteins of a wide range of electrophoretic mobilities.

An ELISA for the detection of anti-acetylcholine receptor antibodies using biotinylated α-bungarotoxin

An antibody-capture enzyme immunoassay has been developed for the detection of anti-acetylcholine receptor (AChR) antibodies in tissue culture supernatants using biotinylated alpha-bungarotoxin (B alpha BGT). Immunoglobulins in culture supernatants were bound indirectly to microtitre plates via an anti-globulin antibody already coupled to polyvinyl plates. Anti-AChR antibodies were then detected by incubation with AChR crude extract. Bound AChR was revealed by incubation with B alpha BGT followed by horseradish peroxidase-conjugated avidin. This assay is specific, more sensitive than the commonly used double antibody radioimmunoassay, avoids the use of radioactive material, is practical for large numbers of samples and is particularly suitable for detecting anti-AChR antibodies in tissue culture supernatants.

Stimulation by mitogens and neuronal membranes of lymphocytes from patients with motor neurone disease.

Stimulation of lymphocytes from motor neurone disease patients by either concanavalin A or PHA was shown to be significantly depressed relative to that from normal controls, as assayed by incorporation of [3H]thymidine or [3H]leucine or by glucose uptake. Corresponding significant differences were not shown by assays based upon incorporation of [3H]uridine or of lactate release. Lymphocytes from 4 out of 14 motor neurone disease patients showed a blastogenic response to membranes from rat spinal cord cells, compared with those from 0 out of 9 normal controls. These results not only suggest the possibility of an impaired cellular immune control in MND patients but also indicate the presence of lymphocytes sensitised specifically to neuronal membrane components.

Immunological Factors in Neuronal Degeneration with Particular Reference to Motor Neurone Disease

Cellular and humoral immunoreactivity to neuronal antigens was investigated in patients with motor neurone disease (MND). Lymphocytes from patients with MND and normal healthy controls were cultured with a membrane fraction prepared from cultured spinal cord neurones. 4 out of 14 patients with MND and 0 out of 9 normal controls showed a significantly increased stimulation index. An enzyme-linked immunoabsorbent assay (ELISA) was established to detect antibodies to synaptic membrane fraction prepared from human motor cortex. Sera from MND patients showed a significantly increased immunoglobulin binding with respect to normal control sera. Antineuronal antibody production by MND lymphocytes was studied by using Epstein-Barr virus transformation followed by fusion with a mouse myeloma cell line. Antibody-producing clones were isolated. This procedure would allow a more detailed analysis of the antineuronal antibody production in MND.

Raised levels of maternal serum secretory acetylcholinesterase may be indicative of fetal neural tube defects in early pregnancy

BACKGROUND To investigate the levels of maternal serum secretory acetylcholinesterase from a sample of pregnancies involving fetal neural tube defects and compare those results with alphafetoprotein levels. METHODS Secretory acetylcholinesterase levels were measured using a new Enzyme Capture Immunoassay, in a small blind prospective study. The study group comprised pregnancies covering a gestational age range of 13-24 weeks where 98 had normal fetuses, 21 suffered from neural tube defects, and 15 had other complications. RESULTS Maternal serum secretory acetylcholinesterase levels were found to be low and independent of gestational age between 14-20 weeks in a sample of normal pregnancies with normal alphafetoprotein levels. Raised levels of maternal serum secretory acetylcholinesterase were found in 100% of pregnancies involving spina bifida (17/17) and three of four anencephalics compared with raised alphafetoprotein levels found in 88% (15/17), and 100% (4/4) of the same samples. Only seven of 13 maternal serum samples from pregnancies with a normal outcome and none of the four twin pregnancies, all with raised alphafetoprotein levels, had raised secretory acetylcholinesterase levels. CONCLUSIONS Raised levels of maternal serum secretory acetylcholinesterase may provide a useful indicator of neural tube defects in early pregnancy.

Autoimmune Involvement in Motor Neurone Disease

The possibility of an autoimmune pathogenesis in motor neurone disease (MND) has been debated for many years with little consensus. However, recent evidence from different sources has served to redirect attention towards such an involvement. Thus, early findings of a serum-borne neurotoxic factor in MND patients[l] have been confirmed by Roisen et al.[2], while Gurney and his colleagues[3,4] have reported the presence in MND sera of autoantibodies to a muscle-derived growth factor for spinal neurones.

Pronase and proteinase K digestion of human immunoglobulin M

Human 19S IgM was digested with pronase and proteinase K. Proteolysis was relatively fast, producing Fab2 mu-like fragments (approx. mol. wt 114,000) and Fab mu-like fragments (approx. mol. wt 46,500) as major products. Immunochemical analysis indicated that the fragments produced by either enzyme are very similar and that they are produced by cleavage at the C mu 2-C mu 3 and C mu 1-C mu 2 domain junctions respectively. An intermediate species of mol. wt 74,300, immunologically identical to F(ab)2 mu, was also identified. This is thought to represent an F(ab)2 mu fragment with one Fab mu fragment removed. Fc mu-related fragments were not identified in the digestion mixture with either enzyme. Covalently linked and non-covalently linked 7S human IgM (IgMs and IgMr respectively) were digested with pronase and proteinase K. IgMs was degraded very rapidly by either enzyme, producing relatively stable F(ab)2 mu- and Fab mu-like fragments. These fragments were similar in mol. wt and immunochemical properties to those produced from 19S IgM. IgMr was also degraded rapidly by either enzyme, in this case producing Fab mu-like fragments with no detectable F(ab)2 mu-like fragments. The kinetics of digestion and nature of the products suggest that cleavage of 19S IgM by pronase or proteinase K proceeds via an initial attack at the C mu 2-C mu 3 junction, followed by further degradation at the Cmu 1-C mu 2 junction. The results obtained using 7S IgM show that the intersubunit disulphide bonds, and the associated pentameric structure, are responsible for the relative resistance of 19S IgM to proteolysis. The inter-heavy-chain disulphide bonds, in particular the bond at cys 337, are responsible for the limited susceptibility of F(ab)2 mu-like fragments to proteolysis.

A passive haemagglutination test for the detection of anti-nicotinic acetylcholine receptor antibodies

A detergent-solubilised membrane protein (the nicotinic acetylcholine receptor) was coupled to red blood cells by the chromic chloride method. The bound receptor retained both antigenic activity and the ability to bind alpha neurotoxins. Coated cells were successfully used in a haemagglutination test to determine antibody titre and cross-reactivity of polyclonal and monoclonal anti-acetylcholine receptor antisera.

Autoantibodies to neuronal membranes in motor neurone disease

To evaluate effects of learned helplessness on natural k i l l e r (NK) cells, several genetic strains of mice have been subjected to daily six minute swimming t r i a l s in a small space for varying time periods. After i n i t i a l vigorous act iv i ty , the animals become conditioned to immobility (behavioral despair) and stop swimming. Duration of immobility varies among different mouse strains and is reversed by imipramine in a dose dependent manner. Splenic NK act iv i ty (SICr release assay) following despair conditioning in presence and absence of imipramine was compared to cage and drug controls. NK cell act iv i ty is signif icantly lower after behavioral conditioning for >22 and <35 days (p<.O01). Up to 10 ug/kg imipramine daily for 7 days does not ~ffect N~ac t i v i t y ,bu t 20 ~g/kg augments the NK response of normal (not conditioned) CBA/N mice. When imipramine (20 ug/kg) is administered concomitant with the swimming stress, NK act iv i ty is not restored to normal, but remains low as in behavioral despair conditioning in absence of drug. Administration of imipramine (20 ug/kg daily for 7 days) following 22 to 30 days of behavioral conditioning signif icantly lowers NK cell act iv i ty compared to controls (p_<.O01) although the conditioned despair behavior (immobility) is reversed. (Supported by the American Osteopathic Association Grant #86-11-215).