【摘要】：化學發(fā)光免疫分析法(Chemiluminescence immunoassay, CLIA)是將化學發(fā)光反應和免疫反應相互結合,具有高靈敏度、高特異性,以放射免疫分析為理論基礎建立起來的,用來檢測抗原或者抗體的一種非放射標記免疫分析法。這種方法具有無需光源、儀器設備簡單、靈敏度高、特異性強、線性范圍寬、無放射性污染、可實現(xiàn)自動化等優(yōu)點。多組分免疫分析(Multiplexed immunoassay, MIA)是目前臨床診斷、環(huán)境監(jiān)測、生物分析等領域的研究熱點,具有分析成本低、分析通量高、所需時間短、樣品消耗少等突出優(yōu)點。近年來,大多數(shù)報道的MIA方法通常是基于空間分辨或多標記模式。本文基于CLIA,結合多標記模式構建了檢測兩種免疫球蛋白的新方法,具體開展了以下工作：本文以新的化學發(fā)光反應動力學分辨策略為基礎,提出了一種近同時檢測兩種免疫球蛋白的多組分免疫分析方法。該方法利用化學發(fā)光動力學特征具有明顯差異的兩個化學發(fā)光標記物—吖啶酯和堿性磷酸酶作為信號探針,分別標記羊抗鼠IgG和兔抗鼠IgM,形成兩個免疫復合物,通過競爭免疫分析模式,對兩個分析物小鼠IgG (Mouse IgG, MIgG)和小鼠IgM(Mouse IgM, MIgM)進行檢測。當加入共反應劑時,具有閃光型和輝光型動力學特征的兩個反應同時被觸發(fā),在反應后0.2 s和500 s時,分別記錄這兩個反應的化學發(fā)光信號。該多組分免疫分析方法對于MIgG和MIgM的檢測范圍均為0.50-200 ng mL-1,檢測限為0.16 ngmL-1 (S/N=3)。所構建的方法沒有明顯的信號重疊,并能夠成功應用于檢測小鼠血清樣品中MIgG和MIgM,檢測結果與商業(yè)化的ELISA方法檢測結果相一致。該方法具有靈敏度、特異性較高和操作簡便等優(yōu)勢,在一些如藥物篩選,食品安全,環(huán)境監(jiān)測和臨床診斷等領域有很好的應用潛力。
[Abstract]:Chemiluminescent immunoassay (Chemiluminescence immunoassay, CLIA), which combines chemiluminescence reaction with immune reaction, has high sensitivity and specificity, and is based on the theory of RIA. A non-radiolabeled immunoassay used to detect antigens or antibodies. This method has the advantages of no light source, simple instrument and equipment, high sensitivity, strong specificity, wide linear range, no radioactive pollution and automation. Multi-component immunoassay (Multiplexed immunoassay, MIA) is a hot spot in the fields of clinical diagnosis, environmental monitoring, biological analysis and so on. It has the advantages of low analysis cost, high analysis flux, short time and low sample consumption. In recent years, most reported MIA methods are based on spatial resolution or multi-marker patterns. In this paper, a new method for detecting two kinds of immunoglobulin based on CLIA, combined with multi-labeled pattern is proposed. The following works are carried out in detail: based on the new chemiluminescence kinetic resolution strategy, A multi-component immunoassay method for near-simultaneous detection of two kinds of immunoglobulin was proposed. Using acridine ester and alkaline phosphatase as signal probes, two chemiluminescence markers, acridine ester and alkaline phosphatase, were used to label goat anti-mouse IgG and rabbit anti-mouse IgM, to form two immune complexes. Two analytes, mouse IgG (Mouse IgG, MIgG) and mouse IgM (Mouse IgM, MIgM), were detected by competitive immunoassays. When the co-reactant was added, the two reactions with the kinetic characteristics of flash and glow type were triggered at the same time. The chemiluminescence signals of the two reactions were recorded at 0.2 s and 500 s after the reaction, respectively. The detection range of both MIgG and MIgM is 0.50,200 ngmL-1 and the detection limit is 0.16 ngmL-1 (S/N=3). The constructed method has no obvious signal overlap and can be successfully applied to the detection of MIgG and MIgM, in mouse serum samples. The results are consistent with those of the commercial ELISA method. The method has the advantages of sensitivity, high specificity and simple operation. It has good application potential in some fields such as drug screening, food safety, environmental monitoring and clinical diagnosis.
【學位授予單位】：西南大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：R917

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本文編號：2446455