| Objective:Guillain-Barre syndrome is a acute flaccid paralysis disorder of perpharal nervours system which often left severe residual neurologic deficits.It's pathogeny and pathogenesis is not fully understood yet. But the relation between infection of Campylobacter jejunum and GBS is formly conformed, the percentum of the relationship are quite different (7%~76%), most are around 30%.Researchers abord analyzed the chemical structure of Cj-LPS found that it has the same part as the gangliosides(Pennor O:19 serotyping mimic ganglioside GM1)Antibody to Cj-LPS is important to GBS. Reserch using serum of GBS patients both in tissue culture and injected under epineurium can lead injure of nervours. But in the serum of GBS patients there may have many antibodies, in order to slove this problem we purified LPS antibody in the serum of a patient with GBS use immunology affinity chromatography. We used the purified LPS antibody react with ganglioside GM1. And reacted with animals perpharal nervours to find out the reacted region on the nervours. This purified LPS antibody can be use in the research of the function of LPS antibody in occurring of GBS. In immunology network people think antibody molecule have both the character of react with antigen and the character of immunogenicity (idiotype, Id)which can activate the lymphocyte to produce anti-idiotypic antibody,AId. Autoimmune diseases are characted by having much autoimmune antibody. In the second part we prepare anti-idiotypic monoclonal antibody to LPS antibody, which can special recognize LPS antibody.We can use this monoclonal antibody detect LPS antibody in serum and tissue of patients with GBS. And use this monoclonal antibody to diagnosis and treatment Cj related GBS. Methods: Anaerobic culture Campylobacter jejunum , hydroxybenzene-water method extract LPS. DNase,RNase, Proteinase K digest DNA RNA and protein. SDS-PAGE identify it's purity. LPS was diluted to 10ug/ml with cover buffer. Microtiter plates were covered with LPS solution and blocked with PBS containing 2% BSA(blocking buffer). 5 GBS patients were test for the LPS-antibody titre. Selected the highest srea. CNBr-activated Sepharose 4 Fast Flow is washed initially with 10-15 medium volume of cold 1mM HCl. Use small wash portions. After the last wash the HCl was moved as much as possible to avoid affecting the coupling pH. Prepared the coupling solution , dissolved the LPS in coupling buffer. Mixed the washed medium and coupling solution immediately. The time interval between washing and coupling must be minimized. Stiring the medium up and down. At room temperature the reaction is usually completed after 2-4 hours. After coupling, non-reacted groups on the medium is blocked by keeping the coupled medium in Tris buffer for 2 hours at room temperature. Wash the coupled medium using alternate acetate buffer(pH 3-4) and Tris-HCl buffer(pH 8-9). Repeted this cycle 3-6 times. Filtrated the GBS patient's sera with 0.22um filter paper to move away granule matter. Fill the sera to the medium column and keep at slow flow rate. After running the sera washed the medium with PB buffer. Then the medium was eluted with 3M KSCN. Collected the elution. Dislyzed against PBS overnight at 4℃. ELISA assay was performed to test the cross react with the ganglionside GM1. Microtiter plates were coated with GM1. Immunhistochemistry was done to determine the locoation where the LPS-antibody bound the peripheral nerve. In the second part we prepare anti-idiotype monoclone antibody to LPS-antibody. 6-8 weeks old female BalB/C mice were immunized with LPS-antibody on day 1, day 14 and day 28 of the protocol. We fused mouse myeloma SP2/0 cells with spleen cells from BalB/C mice immunized with LPS-antibody using polyethylene glycol. And we did advance identification. Result: 1. LPS-antibody can cross react with ganglioside GM1. 2. LPS-antibody bounded to the myelin of the peripheral nerve, leave the axon unbounded. 3. Five hybridomas which secreate momoclone antibody were obtained, one reacted with human...
|
PDF Full Text Request