化学发光免疫分析在游离甲状腺素测定中的应用研究
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摘要
化学发光免疫分析方法灵敏、特异、简便,在生命、环境科学领域得到了广泛的应用。本论文主要围绕游离甲状腺素的化学发光免疫分析方法的建立和优化开展了一些研究。通过结合抗体-抗原的高特异免疫分析技术、高效的酶标记技术和化学发光检测体系,建立了测定体液内甲状腺激素的化学发光免疫分析方法,对大批量临床样品筛选、检测和体外诊断试剂的研发有一定临床应用价值和学术意义。
首先,简要介绍了化学发光免疫分析的原理和甲状腺激素的种类、来源和作用功能,并对游离甲状腺激素的检测技术和方法进行了综述。
其次,建立了一种简单鉴定小分子的酶标记物质量方法,通过实验证明,本方法可以在选用原料时保证酶标记物质量的合格,从而保证了后续实验结果的准确性。
再次,建立了一种基于磁性微粒子化学发光免疫分析定量测定人血清中的游离甲状腺素的方法,该方法具有灵敏度高、高特异、快速、稳定性高、重复性好等特点。本方法使用辣根过氧化物酶标记的甲状腺素类似物与血清中的游离甲状腺素共同竞争结合羊抗甲状腺素抗体；表面包被有抗异硫氰酸荧光素(fluorescein isothiocyanate, FITC)的磁性微粒子用作免疫反应的固相载体和分离剂,以辣根过氧化物酶(horseradish peroxidase, HRP)催化H2O2-luminol化学发光体系作为检测体系；对免疫反应的试验条件及各项物理化学参数如：反应温度、温育时间、稀释度、磁性微粒子和发光底物的用量等进行了测定和优化。在最佳条件下,该方法的线性范围是0-122 pmol L-1,检测限为0.25 pmol L-1;批内变异和批间变异均小于14.6%；通过本法对实际血清样本检测,并将结果与商品化试剂盒的测定结果进行相关分析,其相关系数r2=0.9592,证实本法可用于人血清中游离甲状腺素的临床测定。
最后,使用通用二抗固相法制备固相抗甲状腺素抗体,使用鲁米诺-双氧水-辣根过氧化物酶化学发光体系作为检测体系,发展了一种通用二抗固相化学发光酶免疫分析方法并将其应用于临床游离甲状腺素的测定。驴抗羊IgG(即二抗)通过物理吸附包被在96孔聚苯乙烯微孔板上,对其进行封闭后,作为一个通用的固相应用于各种物质的免疫分析。向该固相中加入甲状腺素的多克隆羊源抗体,样品抗原和酶标记物后进行温育反应,羊抗甲状腺素抗体通过与二抗之间的免疫反应被间接固定化在聚苯乙烯微孔板上,样品抗原和酶标记物竞争结合羊抗甲状腺素抗体,形成免疫复合物,通过测定酶促化学发光体系的发光强度来达到定量检测的目的。对一些相关参数和实验条件诸如免疫试剂的稀释度、温育时间、化学发光反应时间以及底物液加样体积进行了考查和优化,并对建立的方法进行了评价。本方法和国外商品化的CLIA试剂盒同时测定119例人血清样品,二者的临床测值的符合率r2达到了0.8121,证实本方法可应用于实际的临床诊断。
Chemiluminescence immunoassay applicated widely in life and environmental science fields for its sensitivity, specific, and simple. It combines the advantages of the immunoassay and chemiluminescent analysis. Our work carried out a series study of chemiluminescence immunoassay for the determination of free in human serum. It is momentous practice and academic significance for the screening and detection of numbers clinic samples and the development of the in-vitro diagnostic reagents.
First, The principle of chemiluminescent immunoassay were reviewed. The categories, sources and functions of thyroid hormone were introduced. Then, various detection methods for free thyroxine were summarized.
Secondly, a simple method to the quality identification of the small molecule substance enzyme markers was proposed. The experiment showed that the proposed method could be used in selecting the experimental raw material to ensure the accuracy of the future experiment research.
Thirdly, a magnetic particles-based chemiluminescence enzyme immunoassay with high sensitivity, specificity, rapidity, and reproducibility was proposed for the determination of free thyroxine in human serum. A competitive assay has been proposed with horseradish peroxidase labeled thyroxine analog. The immunomagnetic particles coated with anti-fluorescein isothiocyanate antibody was used as dispersed solid phase and separation means for the immunoassay. Experimental conditions, such as temperature, the volume of magnetic particles and substrate, incubation time, dilution ratio and other relevant variables upon the immunoassay have been examined and optimized. The proposed method exhibited high performance which the linear range was 0-122 pmol L-1 and the detection limit was 0.25 pmol L-1. A coefficient of variance of less than 14.6% was obtained for both intra-assay and inter-assay precision. The present method has been successfully applied to the analysis of free thyroxine in human serum. The diagnostic accordance rate of the method for normal serum, hyperthyroidism and hypothyroidism are satisfactory. Good correlations were obtained between the results by the proposed method and the commercial radioimmunoassay kit.The proposed method exhibits good potential in the fabrication of FT4 diagnostic kits which could be used in the clinical analysis and facilitated the development of automated operation systems in the clinical practice.
Finally, a solid phase CLEIA for the determination of free thyroxine in human serum was established, with the highly sensitive HRP-luminol-H2O2 system as chemiluminescence detection system, donkey anti-goat IgG as secondary antibody. The donkey anti-goat IgG was indirectly immobilized on the 96-wells polystyrene microplate, the gaot anti-thyroxine through the immunoreaction with secondary antibody to prepare the solid phase antibody, a competitive assay has been proposed with horseradish peroxidase labeled thyroxine analog. After the immune complex formed, added the the substrate solution, detected the chemiluminescence intensity for the quantitative detection. The effect of several experimental conditions, such as temperature, the volume of magnetic particles and substrate, incubation time, dilution ratio and other relevant variables upon the immunoassay were examined and optimized. The assay was evaluated and every methodological index can meet the demand of quantitative analysis. This method has been successfully applied to the evaluation of free thyroxine in 119 human serum and the results showed a good correlation (r2=0.8121) with the commercially available RIA kit, which shown that it can be used in the clinical practice.
首先,简要介绍了化学发光免疫分析的原理和甲状腺激素的种类、来源和作用功能,并对游离甲状腺激素的检测技术和方法进行了综述。
其次,建立了一种简单鉴定小分子的酶标记物质量方法,通过实验证明,本方法可以在选用原料时保证酶标记物质量的合格,从而保证了后续实验结果的准确性。
再次,建立了一种基于磁性微粒子化学发光免疫分析定量测定人血清中的游离甲状腺素的方法,该方法具有灵敏度高、高特异、快速、稳定性高、重复性好等特点。本方法使用辣根过氧化物酶标记的甲状腺素类似物与血清中的游离甲状腺素共同竞争结合羊抗甲状腺素抗体；表面包被有抗异硫氰酸荧光素(fluorescein isothiocyanate, FITC)的磁性微粒子用作免疫反应的固相载体和分离剂,以辣根过氧化物酶(horseradish peroxidase, HRP)催化H2O2-luminol化学发光体系作为检测体系；对免疫反应的试验条件及各项物理化学参数如：反应温度、温育时间、稀释度、磁性微粒子和发光底物的用量等进行了测定和优化。在最佳条件下,该方法的线性范围是0-122 pmol L-1,检测限为0.25 pmol L-1;批内变异和批间变异均小于14.6%；通过本法对实际血清样本检测,并将结果与商品化试剂盒的测定结果进行相关分析,其相关系数r2=0.9592,证实本法可用于人血清中游离甲状腺素的临床测定。
最后,使用通用二抗固相法制备固相抗甲状腺素抗体,使用鲁米诺-双氧水-辣根过氧化物酶化学发光体系作为检测体系,发展了一种通用二抗固相化学发光酶免疫分析方法并将其应用于临床游离甲状腺素的测定。驴抗羊IgG(即二抗)通过物理吸附包被在96孔聚苯乙烯微孔板上,对其进行封闭后,作为一个通用的固相应用于各种物质的免疫分析。向该固相中加入甲状腺素的多克隆羊源抗体,样品抗原和酶标记物后进行温育反应,羊抗甲状腺素抗体通过与二抗之间的免疫反应被间接固定化在聚苯乙烯微孔板上,样品抗原和酶标记物竞争结合羊抗甲状腺素抗体,形成免疫复合物,通过测定酶促化学发光体系的发光强度来达到定量检测的目的。对一些相关参数和实验条件诸如免疫试剂的稀释度、温育时间、化学发光反应时间以及底物液加样体积进行了考查和优化,并对建立的方法进行了评价。本方法和国外商品化的CLIA试剂盒同时测定119例人血清样品,二者的临床测值的符合率r2达到了0.8121,证实本方法可应用于实际的临床诊断。
Chemiluminescence immunoassay applicated widely in life and environmental science fields for its sensitivity, specific, and simple. It combines the advantages of the immunoassay and chemiluminescent analysis. Our work carried out a series study of chemiluminescence immunoassay for the determination of free in human serum. It is momentous practice and academic significance for the screening and detection of numbers clinic samples and the development of the in-vitro diagnostic reagents.
First, The principle of chemiluminescent immunoassay were reviewed. The categories, sources and functions of thyroid hormone were introduced. Then, various detection methods for free thyroxine were summarized.
Secondly, a simple method to the quality identification of the small molecule substance enzyme markers was proposed. The experiment showed that the proposed method could be used in selecting the experimental raw material to ensure the accuracy of the future experiment research.
Thirdly, a magnetic particles-based chemiluminescence enzyme immunoassay with high sensitivity, specificity, rapidity, and reproducibility was proposed for the determination of free thyroxine in human serum. A competitive assay has been proposed with horseradish peroxidase labeled thyroxine analog. The immunomagnetic particles coated with anti-fluorescein isothiocyanate antibody was used as dispersed solid phase and separation means for the immunoassay. Experimental conditions, such as temperature, the volume of magnetic particles and substrate, incubation time, dilution ratio and other relevant variables upon the immunoassay have been examined and optimized. The proposed method exhibited high performance which the linear range was 0-122 pmol L-1 and the detection limit was 0.25 pmol L-1. A coefficient of variance of less than 14.6% was obtained for both intra-assay and inter-assay precision. The present method has been successfully applied to the analysis of free thyroxine in human serum. The diagnostic accordance rate of the method for normal serum, hyperthyroidism and hypothyroidism are satisfactory. Good correlations were obtained between the results by the proposed method and the commercial radioimmunoassay kit.The proposed method exhibits good potential in the fabrication of FT4 diagnostic kits which could be used in the clinical analysis and facilitated the development of automated operation systems in the clinical practice.
Finally, a solid phase CLEIA for the determination of free thyroxine in human serum was established, with the highly sensitive HRP-luminol-H2O2 system as chemiluminescence detection system, donkey anti-goat IgG as secondary antibody. The donkey anti-goat IgG was indirectly immobilized on the 96-wells polystyrene microplate, the gaot anti-thyroxine through the immunoreaction with secondary antibody to prepare the solid phase antibody, a competitive assay has been proposed with horseradish peroxidase labeled thyroxine analog. After the immune complex formed, added the the substrate solution, detected the chemiluminescence intensity for the quantitative detection. The effect of several experimental conditions, such as temperature, the volume of magnetic particles and substrate, incubation time, dilution ratio and other relevant variables upon the immunoassay were examined and optimized. The assay was evaluated and every methodological index can meet the demand of quantitative analysis. This method has been successfully applied to the evaluation of free thyroxine in 119 human serum and the results showed a good correlation (r2=0.8121) with the commercially available RIA kit, which shown that it can be used in the clinical practice.
引文
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