【摘要】：化學(xué)發(fā)光免疫分析法(Chemiluminescence immunoassay, CLIA)是將化學(xué)發(fā)光反應(yīng)和免疫反應(yīng)相互結(jié)合,具有高靈敏度、高特異性,以放射免疫分析為理論基礎(chǔ)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(shè)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(lǐng)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(jié)合多標記模式構(gòu)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(xué)發(fā)光反應(yīng)動力學(xué)分辨策略為基礎(chǔ),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(xué)發(fā)光動力學(xué)特征具有明顯差異的兩個化學(xué)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(fù)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(dāng)加入共反應(yīng)劑時,具有閃光型和輝光型動力學(xué)特征的兩個反應(yīng)同時被觸發(fā),在反應(yīng)后0.2 s和500 s時,分別記錄這兩個反應(yīng)的化學(xué)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(gòu)建的方法沒有明顯的信號重疊,并能夠成功應(yīng)用于檢測小鼠血清樣品中MIgG和MIgM,檢測結(jié)果與商業(yè)化的ELISA方法檢測結(jié)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(lǐng)域有很好的應(yīng)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(xué)位授予單位】：西南大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：R917

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本文編號：2446455