Particle Enhanced Immunoturbidimetric Assay For Serum Homocysteine

In recent years, a large number of studies have demonstrated thathyper-homocysteinemia plays an important role in pathogenesis of theatherosclerosis and thromboembolic diseases such as cerebrovasculardisease, coronary heart disease and venous thrombosis, etc. and has beenconsidered as one of independent risk factors for prediction of stroke. Ithas been confirmed by epidemiological and clinical studies that thepathogenesis by homocystein mainly includes damaging vascularendothelial cells, promoting the cell proliferation of vascular smoothmuscle, destructing the balance between blood coagulation and fibrinolysis,resulting in a pre-thrombosis state. The homocystein also affect lipidmetabolism, and methyl transfer reactions, promoting formation of arterialsclerosisOBJECTIVE:To preparation of SAH monoclonal antibody by purified S-adenosylhomocysteine (SAH) Immune Balb/c mice. With which SAH monoclonalantibodies produced in mice ascites were prepared. The particle enhancedimmunoturbidimetric assay ware establishment of clinical immunoassaysfor human serum Hcy. According to the standards and guidelines set by theClinical and Laboratory Standards Institute (CLSI), the analyticperformances of the particle enhanced immunoturbidimetric assay forhomocystein were evaluated.METHODS:1. Use of lymphocytes prepared by hybridoma technique SAH monoclonal antibody to SAH-BSA binding material immune Balb/C micespleen cells with Sp2/0myeloma cells using cell fusion technologypreparation of SAH of mAb. To establish a stable secretion of hybridomacell lines anti-SAH, and a large number of monoclonal antibodies to detectin vivo induced ascites monoclonal antibody titer and their specific2. The use of saturated ammonium sulfate precipitationchromatography SAH monoclonal antibody purification.3. Separation of the purified S-adenosyl homocysteine hydrolase(SAH hydrolase SAHase), and purification of SAHase of verified.4. Using SAH antibody coated latex particles, and the establishmentof the particle enhanced immunoturbidimetric assay reaction system.5. According to the standards and guidelines set by the Clinical andLaboratory Standards Institute (CLSI), the analytic performances of theparticle enhanced immunoturbidimetric assay for homocystein wereevaluated.RESULTS:1. Three hybridoma obtained by the PEG cell integration technology(respectively of name SAH-M1,SAH-M2,SAH-M3), the culturesupernatant, respectively, reaction with SAH, adenosine, methionine, S-adenosylmethionine, adding to the package SAH-BSA ELISA plate,ELISA results showed that SAH-M3specifically bind to SAH.2. The selected hybridoma cell lines were used to prepare ascites.Protein A chromatography was used to purify IgG. Protein concentrationwas determined by Lowery method and was adjusted to1.0mg/ml forfurther use.3. Purification of SAHase to do Western blot validation, proved to berequired for S-adenosyl homocysteine hydrolysis enzyme, compared withthe Western Blot and Coomassie blue staining on Mark’s S-adenosyl thesame type of cysteine acid hydrolase monomer molecular weight for the 190kD.4. Using SAH antibody coated latex particles, the determination ofHcy in the automatic biochemical analyzer to establish the method ofparticle enhanced immunoturbidimetric assay, after a systematic approachto evaluation and obtained the establishment of the performance indicatorsand imported goods of reagents is consistent with this act and EnzymaticCycling Assay method, The correlation with the for HPLC methodHomocystein is excellent, the serum showed no significant difference.CONCLUSIONS:1. Using Lymphocyte hybridoma technique was successfully preparedSAH monoclonal antibody.2. Separated purified preparation of S-adenosyl homocysteinehydrolase (SAHase).3. Using SAH antibody, the establishment of the particles can bedetermined by the automatic biochemical analyzer Hcy enhancedimmunoturbidimetric assay, with imported goods reagents in goodagreement, The correlation with the for HPLC method Homocystein isexcellent, can be used in routine clinical laboratory, Satisfies the needs ofroutine clinical.