The Establishment Of Light Initiated Chemiluminescence Assay For The Quantitative Determination Of Plasma Erythropoitin

Erythropoietin(erythropoietin,EPO)is the first hematopoietic growth factor discovered and used in clinical,its biological role is to promote the red bone marrow cells growth,differentiation and maturation.Lack of oxygen is an important factor to stimulate the secretion of EPO.EPO combined with bone marrow erythroid progenitor cells and erythroblast surface erythropoietin receptor(EPOR)after secreted by the body,regulate the proliferation and differentiation of erythroid kinase,to increased red blood cell count and hemoglobin content,to stabilize the red cell membrane and then improve the function of red cell membrane antioxidant enzymes to correct anemia.On the one hand accurately detect erythropoietin content can reflect the stress response of the body in a variety of physiological and pathological conditions,to guide clinicians on the diagnosis and treatment of certain diseases;on the other hand,can also prevent EPO abused in competitive sports.The EPO detection technology are development.In clinical we detect EPO content in the serum or urine to determine the body is normal EPO level.Light initiated chemiluminescence assay(LiCA)technology is a new generation of chemiluminescence technology based on nanoscale polymer particles,is generated by the homogeneous chemiluminescence technology.As a new quantitative immunoassay technology rised in recent years,it more sensitivie,more time resolution ability,more wider linear range,more lower background fluorescence,disposable plates,more stability,more faster and more easier miniaturization automation than the traditional non-homogeneous immunoassay.Object Establish quantitative light initiated chemiluminescence assay of plasma EPO content and assess its methodologyMethods Checkerboard titration with one EPO antigen monoclonal antibodies and two polyclonal antibodies,filter out the best double-antibody combination pair.One coated to the light emitting particles,the other biotinylated.And then to determine the the double antibodies optimal reaction concentration,and then establishment of the double antibody sandwich mode LiCA which can be used to test plasma EPO,establish a standard curve,and evaluation of this method.We use one antigen monoclonal antibody and two polyclonal antibodies for EPO binded to light-emitting particles respectively,and paired with the biotinylated EPO,filter out the best antibody to establish a competitive mode LiCA which can be used to test plasma EPO.And then to determine the optimal reaction concentration and standard curve,evaluation of this method.Statistical analysis was performed by using the software SPSS 19.0 and ELISA Calc.Resultl.We select a pair of optimal antibody,connected with the light emitting particles and biotin respectively and then successfully established a quantitative the sandwich mode LiCA method which can be used to test plasma EPO.The results showed that the analytical sensitivity was 0.09 ng/ml and has a good linear in 0.09-30ng/ml range;the intra-and inter-assay CV of EPO-LiCA were 2.93%and 3.86%respectively.No high-dose hook effect was observed at EPO concentrations up to 30ng/ml.There is a has a good correlation(p<0.01,r=0.957)between our method and radioimmunoassay of EPO with measurement 51 cases of EPO increased and 51 cases of healthy subjects;the reference range is between 0.76 to 6.80ng/ml.2.We select an optimal antibody,connecting it with the light emitting particles,then with biotinylated EPO establishing a competition mode LiCA method which can be used to test plasma EPO.The assay sensitivity is 0.03ng/ml and has a good linear in 0.03-30ng/ml range;the intra-and inter-assay CV of this method were 3.66%and 4,13%respectively.No high-dose hook effect was observed at EPO concentrations up to 30ng/ml.There is a has a good correlation(p<0.01,r=0.913)between our method and radioimmunoassay of EPO with measurement 51 cases of EPO increased and 51 cases of healthy subjects.the reference range is between 0.77 to4.88ng/ml.Conclusion:Successfully established a double-antibody sandwich and a competition two modes of LiCA that can be used to test plasma EPO.Both mathods have a high sensitivity,disposable plates,wide linear range,low background fluorescence,disposable plates,quick and easy use feature.Have excellent prospect in the EPO detection.