發(fā)布時間：2018-06-02 23:58

  本文選題：降鈣素原 + 銪離子熒光微球�。� 參考：《安徽醫(yī)科大學(xué)》2017年碩士論文

【摘要】：背景:降鈣素原(Procalcitonin,PCT)是診斷和監(jiān)測細(xì)菌性炎癥感染的一個重要臨床診斷標(biāo)志物,在全身炎癥綜合反應(yīng)征和膿毒癥患者的治療和監(jiān)測中具有極大的應(yīng)用價值。目前臨床上PCT有多種檢測方法:膠體金層析檢測靈敏度低、特異性不強;酶聯(lián)免疫檢測耗時耗力,操作繁瑣;均相熒光檢測也存在著操作時間長、檢測成本高的缺點。目的:本研究用銪離子熒光微球作為抗體標(biāo)記物,旨在建立一種快速、高靈敏度的降鈣素原免疫層析方法,文中對建立的層析方法進行優(yōu)化并對其性能進行評價;同時本研究初步探討了銪離子熒光微球在磁分離均相免疫檢測中的應(yīng)用。方法:將PCT單克隆抗體(檢測抗體)和雞Ig Y標(biāo)記上銪離子熒光微球(Europium(III)chelated microparticle,簡稱Eu-CM),然后處理在熒光結(jié)合墊上;另一株P(guān)CT單克隆抗體(包被抗體)包被于硝酸纖維素膜的檢測線T位置;山羊抗雞Ig Y抗體包被于硝酸纖維素膜質(zhì)控線C位置,熒光墊和硝酸纖維素膜組裝組成免疫層析試劑條。血清中的降鈣素原與Eu-CM標(biāo)記的檢測抗體特異性結(jié)合形成抗原抗體反應(yīng)復(fù)合物,反應(yīng)復(fù)合物沿著硝酸纖維素膜前移,在硝酸纖維素膜T位置形成包被抗體-PCT-檢測抗體-Eu-CM復(fù)合物,在C線位置形成山羊抗雞Ig Y-雞Ig Y-Eu-CM復(fù)合物,通過免疫熒光檢測儀檢測T、C位置的熒光信號。用本研究研制的免疫層析試劑條檢測PCT重組抗原,用Sigma Plot 12.5分析該方法的標(biāo)準(zhǔn)曲線,空白檢測限,最低檢測限和功能性靈敏度;檢測60份臨床樣本,Deming’s線性回歸法分析該方法與法國梅里埃全自動酶聯(lián)免疫熒光分析系統(tǒng)的相關(guān)性。在磁分離均相系統(tǒng)中,以PCT單克隆抗體標(biāo)記Eu-CM作為檢測抗體,另一株P(guān)CT單克隆抗體標(biāo)記生物素作為固相抗體,偶聯(lián)親和素的磁微球用來分離Eu-CM-檢測抗體-PCT-固相抗體-生物素復(fù)合物,初步探索Eu-CM在磁分離均相免疫檢測中檢測PCT重組抗原的最低檢測限。結(jié)果:本研究建立了一種基于Eu-CM的降鈣素原免疫層析檢測方法,方法的最佳檢測時間為15 min,免疫層析方法中PCT標(biāo)準(zhǔn)品空白檢測限為0.01 ng/ml,最低檢測限為0.02 ng/ml,檢測范圍0.02-25 ng/ml,功能性檢測靈敏度為0.05 ng/ml(CV10%)。同法國梅里埃全自動酶聯(lián)免疫熒光分析系統(tǒng)相比,兩種檢測方法間的線性相關(guān)系數(shù)為0.9864(n=60),兩種方法學(xué)具有高度的相關(guān)性。Eu-CM應(yīng)用于磁分離均相免疫檢測中,標(biāo)準(zhǔn)曲線匹配二次函數(shù),函數(shù)方程為y=19170.12+75493.74*X+(-26.00)*X2(R2=0.9986),PCT重組抗原最低檢測限為0.04 ng/ml。綜上所述,本研究為臨床檢測血清中PCT提供一種快速、準(zhǔn)確的免疫層析方法;同時本研究中的Eu-CM也可以應(yīng)用于磁分離均相免疫檢測中,其在PCT重組抗原的檢測中有較高的檢測靈敏度,將來有可能成為臨床PCT檢測的又一種高靈敏度的檢測方法。
[Abstract]:Background: Procalcitonin (PCT) is an important clinical diagnostic marker for the diagnosis and monitoring of bacterial inflammatory infection. It is of great value in the treatment and monitoring of systemic inflammatory syndrome and sepsis. At present, there are a variety of clinical detection methods for PCT: colloidal gold chromatography detection sensitivity is low, specificity is not strong; enzyme-linked immunosorbent assay (Elisa) time-consuming and labor-consuming, complicated operation; homogeneous fluorescence detection also has the shortcomings of long operation time and high detection cost. Objective: to establish a rapid and highly sensitive immunochromatographic method for calcitonin with europium ion fluorescent microspheres as antibody marker. At the same time, the application of europium ion fluorescent microspheres in magnetic separation homogenous immunoassay was discussed. Methods: PCT monoclonal antibody (detection antibody) and chicken IgY were labeled with Europium II Ichelated microparticle (Eu-CMN), and then treated on fluorescent binding pad. Another PCT monoclonal antibody (coated antibody) was coated in the T position of the detection line of the nitrocellulose membrane, and the goat anti-chicken IgY antibody was coated in the C-position of the nitrocellulose membrane quality control line. The fluorescent pad and nitrocellulose membrane were assembled to form immunochromatographic reagents. The serum procalcitonin specifically binds to the Eu-CM labeled antibody to form antigen-antibody reaction complex, which moves forward along the nitrocellulose membrane and forms a coated antibody -PCT- test antibody -Eu-CM complex at the T position of the nitrocellulose membrane. Goat anti-chicken Ig Y-Eu-CM complex was formed at the C-line position, and the fluorescence signal of TG-Y- chicken Ig Y-Eu-CM was detected by immunofluorescence detector. The recombinant PCT antigen was detected by immunochromatographic reagent strip. The standard curve, blank detection limit, minimum detection limit and functional sensitivity of the method were analyzed by Sigma Plot 12.5. 60 clinical samples were detected by linear regression method to analyze the correlation between the method and the automatic enzyme-linked immunofluorescence (Elisa) system of Merier in France. In the magnetic separation homogenous system, Eu-CM was labeled with PCT monoclonal antibody and biotin was labeled with another PCT monoclonal antibody as solid phase antibody. Magnetic microspheres of conjugated avidin were used to isolate Eu-CM-detection antibody -PCT- solid phase antibody biotin complex, and to explore the minimum detection limit of Eu-CM for detection of PCT recombinant antigen in magnetic separation homogenous immunoassay. Results: a method of procalcitonin proimmunochromatography based on Eu-CM was established in this study. The best detection time was 15 mins, the blank detection limit of PCT standard product was 0.01 ng / ml, the lowest detection limit was 0.02 ng / ml, the detection range was 0.02-25 ng / ml, and the sensitivity of functional detection was 0.05ng / ml CV10g / ml. The linear correlation coefficient between the two detection methods is 0.9864 (0.9864). Eu-CM is used in magnetic separation homogeneous immunoassay, and the standard curve matches quadratic function, compared with the French Merriere automatic enzyme-linked immunofluorescence analysis system, and the linear correlation coefficient between the two methods is 0.9864. The minimum detection limit of PCT recombinant antigen was 0.04 ng / ml. In conclusion, this study provides a rapid and accurate immunochromatographic method for clinical detection of PCT in serum, and the Eu-CM in this study can also be used in magnetic separation homogeneous immunoassay. It has high sensitivity in the detection of PCT recombinant antigen, and may become another high sensitivity detection method for clinical PCT detection in the future.
【學(xué)位授予單位】：安徽醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R446.6

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本文編號：1970674