Monoclonal Antibody Preparation And Electrochemical Immunosensor Preliminary Study Of High-sensitivity C-reactive Protein (hs-CRP)

CRP was first discovered by Tillet and Francis in1930, which is a kind of serum βglobulins and named by the precipitation reaction with pneumococcal cell wallC-polysaccharide, and its relative molecular weight is about115,000-140,000. CRP isconsidered to be an acute phase protein and often used as one of the diagnostic indicatorsfor acute inflammation and bacterial infection in clinical test. Previously CRP detectionmethods based on immunological turbidimetric or nephelometry were not effective andsensitive enough with detection limitation over5mg/L. With the development of detectingtechnology, high-sensitively detecting methods could measure lower concentration of CRP(0.1~10mg/L), and the detected CRP is called as the hs-CRP. Studies have shown thaths-CRP is closely related with AS and other cardiovascular diseases, and it is considered tobe an independent risk factor of cardiovascular diseases. Currently, hs-CRP test kit and itskey materials—specific and effective anti-CRP monoclonal antibodies mainly relied onimports, which is too expensive to clinical test. So preparation of specific and effectiveanti-hs-CRP monoclonal antibody and the establishment of a stable immunologicaldetection system has a good social value and a good economic prospect. In recent years,electrochemical detection sensors based on the combination of immune technique andelectrochemical detecting technology has been making a great progress, it is not only usedto quantitative analysis, but also has the potential for POCT test suitable for communityhospitals, which has drawn great attention. To improve the sensitivity of the biosensor,different markers were used to track the antigen or antibody molecules, beyond the mostcommonly used enzymes for tracking, G-quadruplex-hemin-DNA enzyme has attractedmore and more attention, a series of high sensitivity and high specificity of the new sensorshave been made by means of this enzyme. In this study, anti-CRP monoclonal antibodieswere prepared, its specificity and effectiveness for the antibodies has been checked by ELISA immunoassay system, and then the electrochemical immunosensor for hs-CRP hasbeen studiedMajor experiments work and results1. Prokaryotic expression and purification of recombinant CRPHuman CRP gene coding sequences was obtained from NCBI nucleotide database andsynthesized with full length. Firstly, recombinant vectors rhCRP28a and rhCRP22b wereconstructed by using the E.coli expression system, after that the protein concentrations ofthe recombinant CRP were detected, and then SDS-PAGE and Western-blot were appliedfor the identification of the recombinant protein. Results showed that the purity of theprepared recombinant CRP protein was more than90%and could bind commercial CRPantibody specifically, thus this recombinant CRP could be used as an immunogen for themonoclonal antibody preparation and for screening antigen and the standard ofimmunoassay.2. Preparation of anti-human CRP monoclonal antibodies and establishment of itsimmunoassaysBalb/c mice were immuned with the recombinant protein rhCRP22b, five specificmonoclonal antibodies for CRP were obtained. Among them2-A6and2-E11with titer over1:2.56×10~5and affinity more than1×10~9were sutible for establishing the immunoassayELISA test system. The linear range of our ELISA test system was7.2-166ng/ml, withsensitivity5.2ng/ml, intra CV≤6.43%, inter-assay CV≤5.93%, the average recovery was98.9%, and the hs-CRP test result of our immunoassay has a good comparability with OrionDiagnostica’s commercial hs-CRP detetion kit.3. Preliminary study of high-sensitivity CRP electrochemical immunosensorBased on the paired monoclonal antibodies2-A6and2-E11which can be used todetect CRP, the CRP sandwich-type electrochemical immunosensor has been successfullymanufactured with the G-quadruplex-hemin-DNA enzyme modified in2-E11and DNAenrichment step. The immune linear detection range and the specificity of the immunesensor were also studied, the results shown that the current signal was proportional to thelogarithm of CRP antigen concentration from0.1pg to100ng/ml with the linear equationIa=13.9-1.861lgC (R~2=0.99776), and there were no cross response between the selectedimmunosensor and the given disruptors (CEA and AFP). Those studies have shown that the immune sensor was with high sensitivity and specificity for hs-CRP test, which laid a solidfoundation for the further development of reliable hs-CRP immune sensor.