Preparation Of Anti-cyclic Citrullinated Peptide Antibody Diagnostic Reagent And Research On Expression Of Apolipoprotein E

Objectives:(1)Rheumatoid arthritis(RA)is a common autoimmune disease.Considering the caused high disability rate and poor prognosis,early diagnosis and effective intervention are crucial.As a new specific serum marker of RA,anti-CCP antibody is routinely analyzed using chemiluminescence immunoassay at present.However,this detection method is strongly interfered by poor instrument and long time-consuming.More importantly,the detection reagent dependents on import so that it will cause the medical cost high.Therefore,it has great significance to develop a homemade immunodiagnostic reagent in China.The latex particle-enhanced immunoturbidimetry has advantages of high sensitivity,high specificity,wide linear range,and the capacity to be detected on ordinary biochemical analyzers.This study will develop an immunodiagnostic kit based on the principle of latex particle-enhanced immunoturbidimetry.(2)Apolipoprotein E is one of indicators for the detection of blood lipid.The apolipoprotein E standard used in existing kits is mainly dependents on import and difficult to obtain.In this study the apolipoprotein E is expressed n in E.coli and purified to obtain an independent standard.Methods:(1)Carboxylated polystyrene microspheres were prepared as carrier by emulsion polymerization,coupling with cyclic citrullinated peptide(CCP)and then determining whether the coupling is successful.After preparing the immunodiagnostic kit,repeatability,stability and linear range were evaluated,and the kit was used for measuring the content of anti-CCP antibodies in clinical serum samples on a large biochemical analyzer to confirm that the kit was successfully prepared.(2)A PET-28 a plasmid containing a target gene for apolipoprotein E was constructed and then the plasmid was introduced into E.coli.Apolipoprotein E peptide was expressed by proliferating E.coli,and then the optimal conditions for expression of apolipoprotein E were determined in this procedure.Then the activity of the expressed apolipoprotein E was evaluated by a kit for apolipoprotein E.Results:(1)By adjusting input amount of SDS,carboxylated microspheres of different particle sizes were successfully prepared.Then several coupling methods of CCP and carboxylated microspheres was investigated,and finally the layered coupling method was used to successfully prepare R2 solution in the immunodiagnostic kit.The cyclic citrullinated peptide was synthesized as antigen to immunize goats,and then the antiserum was successfully prepared by the immunoassay.The stability and repeatability tests showed that the prepared anti-CCP antibody immunodiagnostic kit had wide linear range,good stability and a good capacity to be used to detect expediently.In addition the high specificity and sensitivity of the kit were demonstrated by detecting of anti-CCP antibodies in serum samples.(2)After determining the optimal conditions,the activity of apolipoprotein E expressed in E.coli was determined.Results showed that the E.coli can successfully express activated apolipoprotein E.Conclusions:(1)The prepared anti-CCP antibody immunodiagnostic kit which based on the principle of latex particle-enhanced immunoturbidimetry had a wide linear range,good stability,high specificity and high sensitivity as well as easy to detect,especially can be used on ordinary biochemical analyzers.The kit can be used to detect relevant antibodies in patients with RA and it is an important significance for the early diagnosis,intervention and treatment of these people.(2)Apolipoprotein E expressed in E.coli had high activity and can be used as standard for diagnostic kit.