Isolate Human C-reactive Protein And Preliminary Application Y

C-reactive protein(CRP) is one of the most important acute phase reactive protein in human,mainly synthesis in the liver.The normal synthesis rate of CRP is 1~10mg/d,the concentration of CRP in the normal population is lower,the newborn is 0.1~0.6mg/L,the adults is 0.4~5.2mg/L.When inflammation,infection,tissue damage,or malignant tumors occurred,the concentration of CRP will rapidly rise after 6~8h,reach the peak after 24~48h,even hundreds of times higher than normal in the acute phase.The growth of CRP was directly related with the severity of illness,and not be subject to radiotherapy,chemotherapy,drugs and other factors.Although CRP does not have an exclusive specificity of diseases,but more sensitive and reliable than some of the traditional test items such as WBC and IL-6.CRP has emerged as one of the routine clinical detection items,the detection of serum CRP have a great significance for the early diagnosis,auxiliary diagnosis,effect observation and prognosis of many diseases.The method for determination of CRP are mainly include latex agglutination test,radioimmunoassay,immunodiffusion,immunoelectrophoresis,gold mark method,enzyme linked immunosorbent assay,immune turbidimetric method,and so on.Among these method,the development of the immune transmission turbidity method is very rapid,it could achieve automatic detection analysis on the automated biochemical analyzer ,its application is more and more extensive.At present,the dometic usage of CRP immune transmission turbidity kit mainly rely on imported,the kit is very expensive,the testing cost are high,limited the promotion and application of CRP testing to the extent.Independently developed an economical,practical and reliable quantitative CRP immune transmission turbidity kit have a great significance.Compared with the monoclonal antibody,polyclonal antibody has higer affinity,lower cost and more simple preparation method,the weakness of polyclonal antibody is that prepare it need high purity antigen.Taking into account the cost of reagent,reagent performance and technical requirement,so the immune turbidimetric reagent uasually use polyclonal antibody.If using monoclonal antibody require matching and latex sensitization,the requirements of technology is higher.Commercial antigen often has a good performance,but the price is very expensive.In view of the importance of CRP,to satisfy the need of research and production,the purpose of this study is establish a economic and practical method which isolated and purified human CRP from clinical serum samples.The self-purified CRP preliminarily used to prepare polyclonal antibody,using the polyclonal antibody establish the CRP immune transmission turbidity method.Methods:1.Determining the protein concentration,the titer,the relative purity and the specificity of the CRP monoclonal antibody.2.Collected the high concentration (>100mg/L) CRP clinical serum samples from hospital,using the saturated ammonium sulfate solution precipitate the total protein of the samples,using the DEAE ion exchange chromatography preliminarily purified the target protein,further purified the target protein by the self-prepared monoclonal antibody affinity chromatographic column,and identify the target protein.3.Used the self-purified CRP as antigen,a goat was immunized to preparing polyclonal antibody,purified the polyclonal antibody by the octanoic acid-ammonium method,and identify the polyclonal antibody.4.Used the CRP polyclonal antibody to established the immune transmission turbidity method for quantitative determination of CRP,and the method was evaluated.5.Used the self-prepared reagent determining the clinical serum samples which collected from hospital.Results:1.The detected results of CPP monoclonal antibody as follows:its protein concentration is 0.5mg/mL,the ELISA titer is 1:2560,the gel scanning result shows that its relative purity is 96.2% after SDS-PAGE,and it could react specifically to the commercial CRP antigen.2.It is that isolated and purified 0.1mg human CRP from 2mL clinical serum sample,the SDS-PAGE result shows the purified product appeared bound with an apparent molecular weight of 23 kD,the gel scanning result shows that its relative purity is 93.1% after SDS-PAGE,and it could react specifically to the CRP monoclonal antibody.3.It is successfully prepared the CRP polyclonal antibody with the self-purified CRP,the total protein concentration of the purified polyclonal antibody is 36.3mg/mL,the ELISA titer is 1:256000,the relative purity is 91.4%,and it could react specifically to the CRP antigen.4.It is preliminarily established the immune transmission turbidity method for quantitative determination of CRP with the prepared CRP polyclonal antibody.The linear range is very good when the concentration of CRP is 3.1~150.0mg/L,the detection limit is 0.4mg/L,the repeatability , stability and anti-interference capacity are very good, and have a good commeasurability with the imported reagent.5.The results of clinical measurement are as follows: the CRP concentration of the normal group(n = 50) is 6.7±1.3mg/L,the rheumatisant group(n = 113) is 81.4±41.9mg/L,two group results exist significant difference.Conclusions:1.Successfully established a economic and practical method which isolate and purify human CRP from the clinical serum samples.2.The self-purified CRP could used to prepare antibody,successfully prepared the goat anti-huaman CRP polyclonal antibody with high titer and specificity.3.Used the CRP polyclonal antibody successfully established the immune transmission turbidity method for quantitative determination of CRP,the result of evaluation shows the properties of it is very good,can be use for clinical measurement.