时间分辨荧光免疫分析技术检测免疫球蛋白

发布时间：2018-07-03 07:38

本文选题：BHHCT + BBCAP　；参考：《华中科技大学》2012年硕士论文

【摘要】：作为一种新型免疫分析方法，时间分辨荧光免疫分析法(TRFIA)选用稀土络合物作为标记物，这类标记物荧光寿命长、Stokes位移大、特征峰尖锐，因此可以通过延迟测量时间的方法，有效地消除背景荧光的干扰，从而具备了相比一般免疫分析方法更高的灵敏度。本工作的主要目的是建立基于AP酶的ELISA和荧光酶免疫分析(FEIA)，以及时间分辨荧光免疫分析方法，其中涉及到稀土螯合物BBCAP、BHHCT和抗体的偶联，以及使用时间分辨荧光免疫分析方法进行免疫球蛋白抗体的测定。本论文研究的主要内容有：（1）以BBCAP作为配体，与羊抗小鼠IgG偶联，测定其免疫特性。（2）合成BHHCT-羊抗小鼠IgG偶联物，并对Eu~(3+)标记抗体技术进行了实验研究，讨论了Eu~(3+)浓度、稀释后的偶联物与Eu~(3+)使用液混合后放置时间、Eu~(3+)放置时间、偶联物稀释倍数、光致漂白效应、温度、Wallac洗液对免疫反应的影响。优化后采取的实验条件为：Eu~(3+)使用浓度为1*10-5M，偶联物稀释倍数为20倍、偶联物与Eu~(3+)使用液混合后放置时间为60min。（3）以小鼠IgG为抗原，羊抗小鼠IgG为抗体，建立了竞争酶联免疫吸附测定（ELISA）、荧光酶免疫分析（FEIA）和时间分辨荧光免疫分析（TRFIA）体系，ELISA、FEIA、TRFIA达到的灵敏度分别为IC(20)=2.477μg/mL，IC(50)=21.115μg/mL；IC(20)=2.522μg/mL，， IC(50)=30.723μg/mL； IC(20)=0.113μ g/mL， IC(50)=1.131μg/mL。
[Abstract]:As a new immunoassay method, time resolved fluorescence immunoassay (TRFIA) uses rare earth complexes as markers. The fluorescence lifetime of these markers has long Stokes shift and sharp characteristic peaks, so they can be measured by the method of delayed measurement of time. The interference of background fluorescence is eliminated effectively and the sensitivity is higher than that of general immunoassay. The main purpose of this work is to establish an AP enzyme based Elisa and fluorescence enzyme immunoassay (FEIA), and a time resolved fluorescence immunoassay method involving the coupling of rare earth chelates BBCAPHHCT and antibodies. Time-resolved fluorescence immunoassay was used to detect the immunoglobulin antibody. The main contents of this thesis are as follows: (1) using BBCAP as ligand, coupling with goat anti-mouse IgG to determine its immunological characteristics. (2) synthesizing BHHCT- goat anti-mouse IgG conjugate, and studying the technique of Eu3 labeled antibody. The effects of EU ~ (3) concentration, the time of depositing EU ~ (3) after mixed with EU ~ (3), dilution multiple, photobleaching effect and temperature Wallac lotion on the immune reaction were discussed. The optimized experimental conditions were as follows: the concentration of 10 ~ (-3) was 1 ~ 10 ~ (-5) M, the dilution multiple of the conjugate was 20 times, and the time of storage was 60 min after mixing the conjugate with EU ~ (3). (3) the mouse IgG was used as antigen and sheep anti-mouse IgG as antibody. Competitive enzyme linked immunosorbent assay (Elisa), fluorescence enzyme immunoassay (FEIA) and time resolved fluorescence immunoassay (TRFIA) were established. The sensitivity of ELISAA FEIAA TRFIA was 2.477 渭 g / mL IC (50) 21.115 渭 g / mL IC (20) 2.522 渭 g / mL, IC (50) 30.723 渭 g / mL; IC (20) 0.113 渭 g / mL, IC (50) 1.131 渭 g / mL.
【学位授予单位】：华中科技大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R392.1

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