| The research background: Vitamin D(Vitamin D,VD)is a kind of sterol derived fat-soluble vitamins,there are mainly ergot calciferol(ergocalciferol,VD2)and bile calciferol(cholecalcif-erol,VD3)in two forms.These two forms of vitamin D had no biological activity,must be in the body by the liver into 25 25-hydroxyitamin D,then through the kidneys into 1,25(OH)D to biological effect into full play.Although 1,25-hydroxyl vitamin D is the main active form of vitamin D,but its short half-life(4-6 h),the relative content of volatile,and 25-hydroxy vitamin D is the main form of transportation in the vitamin D in the blood,a longer half-life(3-4 weeks),the range is small,so the clinical serum 25-25-hydroxyitamin D leels as a measure of vitamin D levels.Has always been people applied it to the prevention and treatment of rickets in children,adult rickets and osteoporosis.In recent years,a large number of studies have found that vitamin D is not only associated with bone disease,also with cancer,heart disease,neurological disease,immune function,important diseases such as infectious diseases have close relationship.Accurate to detect the contents of vitamin D can guide clinicians for bone disease and the disease diagnosis and treatment of skeletal system.Traditional testing method of 25-25-hydroxyitamin D has a high performance liquid chromatography and liquid chromatography-mass spectrometry(LCMS/MS)ia,competition in combination with protein,radiation immunoassay(RIA),enzyme-linked immunoassay,etc;But these methods exist vitamin D sample preparation step complicated,expensive equipment,radioactive pollution,affect the result of the temperature and humidity,and reaction time on the big drawback,etc.This research adopts the Light activation study luminescence immunoassay(Light initiated chemiluminescence assay,LiCA)technology to detect vitamin D,it is Light excitation produced by homogeneous chemiluminescence technology,is based on nanoscale microspheres new chemiluminescence detection technology.This technique compared with the traditional technique of heterogeneous immune analysis,realized no clean and high throughput detection,it has high sensitivity,low background,easy to use,the advantages of high throughput,easy operation,fast.Object:Set up light activation to learn technology to detect the contents of serum 25-25-hydroxyitamin D,the method of assessing methodology.Methods: 1、double antibody sandwich mode LiCA testing 25-hydroxy vitamin D Choose for 25-25-hydroxyitamin D antigen of a monoclonal antibody and polyclonal antibody,two with chessboard titration,the antibodies(a kind of package is luminous particles,another for biotin)two matching best double antibody combination,further to determine the optimum reaction double antibody density,so as to establish the double antibody sandwich model learning glowing light activation method to detect 25-25-hydroxyitamin D technology,making standard curve,the evaluation of this method.2、 the competition pattern LiCA testing 25-25-hydroxyitamin DSelection for 25-hydroxy living element D antigen of a monoclonal antibody and polyclonal antibody,two with chessboard titration,respectively the three antibodies after package is luminous microspheres with biotin 25-hydroxy raw D matching,and select the best antibody,antibodies to further determine the luminous microspheres with biotin,25-hydroxy reaction concentration,which thus establish competition pattern learning glowing light activation method to detect 25-25-hydroxyitamin D technology,making standard curve,the evaluation of this method.Result:1.The chessboard titration method is used to filter out the two optimal antibody respectively connected with luminous microspheres and biotin,successfully established a double antibody sandwich model LiCA quantitative detection method of 25-25-hydroxyitamin D.This test method of sensitivity for 3 ng/mL,within the scope of 3-152 ng/mL detection has good linear.Within the group of the average coefficient of variation was 4.40%,the day between the average coefficient of variation was 7.50%,in 25-hydroxy no Hook effect while higher concentration of vitamin D.Will be low,normal and high value 25-25-hydroxyitamin D a total of 110 specimens with double antibody sandwich model learning glowing light activation method and enzyme-linked immunoassay detection at the same time,we compare the test results of two methods,and shows that two methods have good correlation(r = 0.920),using this method to establish the reference range of 25-hydroxy raw D is 18.85 to 46.44 ng/mL.2.The chessboard titration method is adopted from the three kinds of antibody screening the optimal antibody package is a kind of luminous particles with biotinylated 25-25-hydroxyitamin D jointly established the competition pattern of LiCA quantitative detection method of 25-25-hydroxyitamin D.The sensitivity of the detection method for the 2 ng/mL,within the scope of 2-152 ng/mL detection has good linear.Within the group of the average coefficient of variation was 5.71%,the day between the average coefficient of variation was 7.43%,25-hydroxy no Hook effect while higher concentration of vitamin D.Will be low,normal and high value of 25 specimens of 25-hydroxyitamin D 110 USES the competition model of light activation study luminescence and enzyme-linked immunoassay detection at the same time,we compare the test results of two methods,and shows that two methods have good correlation(r = 0.947),using this method to establish D raw D reference range is 19.41 to 46.23 ng/mL.Conclusion : Successfully established the double antibody sandwich mode and competition mode of LiCA and measuring method of quantitative detection of vitamin D.LiCA quantitative detection of vitamin D in the two sort of modes have high sensitivity,good accuracy,good stability,disposable plates,wide linear range,high flux,reliable,fast,etc.By comparing this method with other vitamin D better correlation detection method,thus,light activation method has excellent optical detecting vitamin D potential clinical application. |
PDF Full Text Request