1. 免疫磁性海藻酸钠复合微球的研制及在肿瘤早期诊断中的应用 2. BirA酶基因表达载体的构建、原核表达及表达产物的活性鉴定
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摘要
磁性高分子微球是通过适当的方法使高分子与无机物结合起来形成具有一定磁性及特殊结构的微球,其表面可赋予多种反应性功能基团,如-OH,-COOH和-NH2等,具有良好的生物相容性,通过与生物活性物质中的配基偶联,能够识别并结合相应的抗原、抗体或核酸等,然后可在外加磁场中进行分离富集。因此,磁性高分子微球在细胞分离、固定化酶、靶向药物和免疫检测等生物学领域有着广泛的应用前景。本课题旨在制备一种新型的纳米磁性复合微球,此种纳米级别的微球与普通的微球相比磁响应敏感度极高,同时具有超顺磁特性,在液相分散体中不易聚集、稳定性高,其比表面积非常大,负载蛋白质的能力强,将其制备免疫磁性微球可作为公共载体和分离工具。然后同免疫分析技术和化学发光技术相结合,设计开发出新一代磁性富集、检测系统和磁性化学发光免疫分析系统用于肿瘤早期诊断。此种检测方法的建立,将会带来巨大的社会效益和经济效益。
1.反相微乳-包埋方法制备磁性海藻酸钠复合微球
以正庚烷为油相,AOT为表面活性剂,海藻酸钠水溶液和碳包铁纳米颗粒混合作为水相,在高速搅拌和超声的条件下形成微乳液,加入氯化钙和环氧氯丙烷进行交联固化制备磁性海藻酸钠复合微球。系统地考察了反应过程中海藻酸钠的浓度、表面活性剂的浓度及搅拌速度对所制微球性能的影响,并对微球进行了初步的性能表征。实验证明此方法克服了目前磁性高分子微球单分散性差、磁响应性弱和粒径偏大的缺点,得到外观为球形、分散性好、平均粒径为279nm、具有强的磁响应性的磁性高分子微球。
2.在磁性微球表面交联抗体制备免疫磁性海藻酸钠复合微球
研究磁性复合微球表面化学基团的植入方法,在微球表面接入6-氨基正己酸“手臂”分子,将功能基团的手臂延伸6个碳原子长度,然后以两步法蛋白缩和的方法将羊抗鼠IgG二抗与微球联接,制备免疫磁性微球,并对抗体交联微球的反应条件进行优化。实验证明用此方法制备的免疫磁性微球抗体吸附量达111.2μg/mg,交联抗体后流式细胞术检测98.3%的磁性微球具有免疫活性。
3.建立肿瘤细胞磁性富集与检测系统,对肺癌患者外周血微转移癌细胞进行临床验证
制备羊抗鼠IgG二抗包被的免疫磁性微球,将免疫磁性分离技术与免疫细胞化学技术相结合建立肺癌细胞磁性富集与检测系统。鉴定该检测系统的特异性、灵敏性和稳定性,并进行肺癌患者外周血肿瘤细胞的临床验证。与单纯的免疫细胞化学检测技术及RT-PCR检测技术相比较,本论文建立的磁性富集与检测系统具有灵敏度高、特异性强和稳定性好的特点,进行肺癌临床验证结果显示本检测系统检测率与RT-PCR检测技术所检测的结果相当,无显著性差异(p>0.05),同时无假阳性结果出现,比单纯的免疫细胞化学检测技术检出率高,存在显著差异(p<0.05)。
4.建立磁性化学发光免疫分析系统,应用于肿瘤标志物AFP的测定。
制备羊抗兔IgG二抗包被的免疫磁性微球,将免疫磁性分离技术与化学发光技术相结合,建立磁性化学发光免疫分析系统,优化检测系统的条件,鉴定其特异性、灵敏性和稳定性,并应用于患者外周血AFP的测定。实验证明本论文建立的磁性化学发光免疫分析系统具有灵敏度高、特异性强和稳定性好的特点,进行患者外周血AFP的测定结果有很好的临床相符性。
本研究制备的新型纳米磁性复合微球,以碳包铁(Fe@C)纳米颗粒作为磁性内核,解决了氧化铁内核磁性能不够的难题,性能优良;在交联抗体时于微球与抗体之间连接一个手臂分子,避免了由于位阻效应导致的微球表面抗体与相应抗原结合不完全的弊端,更有利于微球功能的发挥。在此基础上,纳米磁性复合微球作为公共载体和分离工具应用于磁性富集与检测系统和磁性化学发光免疫分析系统,实验证明方便易行,具有广泛的应用前景。
目的:构建生物素-蛋白连接酶(BirA酶)基因的表达载体,并在大肠杆菌BL-21 (DE3)中表达具有生物学活性的BirA酶。
方法:用PCR法扩增BirA酶基因。将PCR产物克隆入pGEX-4T-2中构建BirA酶-GST融合蛋白基因的重组表达载体pGEX-BirA。经测序验证后,在大肠杆菌BL-21(DE3)中诱导表达,表达产物采用谷胱苷肽-琼脂糖层析柱进行纯化。以带有生物素酶底物肽(BirA substrate peptide, BSP)的HLA-A2-肽复合物为底物,用ELISA和Western blot鉴定表达产物的生物素化活性。
结果:成功地构建了pGEX-BirA原核表达质粒,并在大肠杆菌BL-21(DE3)中诱导表达Mr为61300的BirA酶-GST融合蛋白,表达产物经谷胱苷肽-琼脂糖层析柱纯化后,得到N端带有GST标签的BirA酶蛋白。ELISA和Western blot的结果显示,表达产物具有酶活性,能使HLA-A2-肽复合物生物素化。
结论:成功地制备了具有生物学活性的BirA酶,为研究蛋白质分子的相互作用提供了有效的制剂。
Magnetic polymer microspheres (MMS) are those microspheres synthesized by polymer and inorganic material on molecule scale, which have the ability of magnetism and special structure. Since varied functional groups linking on the surface of microspheres, such as -OH,-COOH, -NH2 and so on, MMS are biocompatible and can conjugate with some ligands of bioactive compound,these give them the ability to recognize and bind to corresponding antigen, antibody, nucleic acid etc. Then this compound can be separated and enriched through the magnetic field. So MMS are promising for the new tool on varial areas such as cell separation, enzyme immobilization, drug targeting, immunodetection and so on. In this thesis, a kind of neotype nano- MMS were prepared, these nano-MMS had some characteristics when compared with ordinary MMS, such as strongly magnetic response, superparamagnetism, highly stability, largely specific surface area and so on. MMs can be taked as publicly carrier and separate tool. Then associated with immune analytical technique and chemiluminescence reaction, new system of magnetic enrichment and detedtion and magnetic chemiluminescence immune assay system were designed and exploited, and applied in the early diagnose of tumor. These establishments of such detection can bring out strongly social effects and economic returns.
1. Preparation of magnetic Alginate Sodium composite Microsphere by means of reverse phase microemulsion and imbed process
Take heptane as organic dispersed medium, AOT as surfactant, sodium alginate solution and Fe@C nanoparticle as aqueous phase, the system of reverse phase microemulsion was formed, and calcium chlorination and epichlorohydrin were been taked as crossing agents. Preparation conditions such as the concentration of sodium alginate, the concentration of AOT and stirring speed were described, and then the microsphere properties also were described in this paper. Results showed that the microspheres had a spherical appearance, with narrow size distribution, and the average diameter is 279nm, while the microspheres also showed strongly magnetic response.
2. Immunomagnetic Alginate Sodium composite microspheres (IMM) were prepared in this part.
The imbedding method of chemical group on the surface of MMS are discussed, and 6-Aminocaproic Acid with six carbon chain length were attached onto MMS as“spacer arm”, then goat anti-mouse IgG antibodies were binded with“spacer arm”through cross-linking reaction by preparing immunomagnetic alginate microspheres. Preparation conditions and MMS properties were described. Results showed that 98.3% immunomagnetic microsphere showed successfully cross-linked actived antibody and there were 111.2μg antibodies immobilized onto 1mg immunomagnetic microspheres.
3. Tumor cell magnetic enrichment and detection method were established and applied in the detection of tumor cells in peripheral blood of lung adenocarcinoma patients. IMM coated with goat anti-mouse IgG antibodies were prepared, system of magnetic enrichment and detedtion were established by combining immunomagnetic separation with immunocytochemistry technique. The specificity, sensitivity and stability of the system were identified. In the detection of tumor cells in peripheral blood of lung adenocarcinoma patients, three kinds of method (enrichment and detection method that established in this thesis, immunocytochemistry and RT-PCR method) were used to compare their variability.
The result showed that Tumor cell magnetic enrichment and detection system had the abilities of high sensitivity, specificity and stability. In the detection of clinic patients, the new detection system had no significant difference compared with RT-PCR methods (p>0.05), and had significant difference compared with immunocytochemistry methods (p<0.05).
4. Magnetic chemiluminescence immune assay system were established and applied in the detection of tumor marker AFP in peripheral blood.
IMM coated with goat anti-rabbit IgG antibodies were prepared, magnetic chemiluminescence immune assay system was established by combined immunomagnetic separation with chemiluminescence reaction. The specificity, sensitivity and stability of the system were identified. In the detection of tumor marker AFP in peripheral blood, the result showed that system of Magnetic chemiluminescence immune assay had the abilities of high sensitivity, specificity and stability, and the result of AFP in the blood have matched with clinic diagnosis.
In this thesis, neotype Immunomagnetic composite microspheres were prepared taking Fe@C nanoparticle as magnetic materials. This resolved the problem of poor magnetism when taking ferric oxide as magnetic materials. Considering stereo conformation between antibody and antigen reaction, antibodies were binding on microspheres through“arm”molecule. In this basis, nano- MMS are promising for the new tool in more areas besides of system of magnetic enrichment and detedtion and magnetic chemiluminescence immune assay system.
Objective: To construct the expression vector of biotin-protein ligase (BirA enzyme) gene and express the BirA enzyme with bioactivity in E.coli BL-21 (DE3).
Methods: The BirA gene was amplified from E.coli genome by PCR and it was cloned into pGEX-4T-2 to construct the recombinant plasmid pGEX-BirA. After being verified by DNA sequencing, the fusion protein was expressed under IPTG induction in the E.coli BL-21 (DE3). The expressed product was purified through Glutathione-agarose chromatography column. The enzyme activity of the expressed product was identified by ELISA and Western blot.
Results: The recombinant prokaryotic expression vector pGEX-BirA was constructed and the fusion protein GST-BirA with Mr being 61300 and bioactivity was expressed successfully. After the expressed product was purified through Glutathione-agarose chromatography column, the results of ELISA and Western blot showed that the expressed product could make HLA-A2 peptide complex biotinylation.
Conclusion: BirA enzyme with bioactivity is prepared successfully which provide an effective reagent for studying the interaction between protein and protein.
1.反相微乳-包埋方法制备磁性海藻酸钠复合微球
以正庚烷为油相,AOT为表面活性剂,海藻酸钠水溶液和碳包铁纳米颗粒混合作为水相,在高速搅拌和超声的条件下形成微乳液,加入氯化钙和环氧氯丙烷进行交联固化制备磁性海藻酸钠复合微球。系统地考察了反应过程中海藻酸钠的浓度、表面活性剂的浓度及搅拌速度对所制微球性能的影响,并对微球进行了初步的性能表征。实验证明此方法克服了目前磁性高分子微球单分散性差、磁响应性弱和粒径偏大的缺点,得到外观为球形、分散性好、平均粒径为279nm、具有强的磁响应性的磁性高分子微球。
2.在磁性微球表面交联抗体制备免疫磁性海藻酸钠复合微球
研究磁性复合微球表面化学基团的植入方法,在微球表面接入6-氨基正己酸“手臂”分子,将功能基团的手臂延伸6个碳原子长度,然后以两步法蛋白缩和的方法将羊抗鼠IgG二抗与微球联接,制备免疫磁性微球,并对抗体交联微球的反应条件进行优化。实验证明用此方法制备的免疫磁性微球抗体吸附量达111.2μg/mg,交联抗体后流式细胞术检测98.3%的磁性微球具有免疫活性。
3.建立肿瘤细胞磁性富集与检测系统,对肺癌患者外周血微转移癌细胞进行临床验证
制备羊抗鼠IgG二抗包被的免疫磁性微球,将免疫磁性分离技术与免疫细胞化学技术相结合建立肺癌细胞磁性富集与检测系统。鉴定该检测系统的特异性、灵敏性和稳定性,并进行肺癌患者外周血肿瘤细胞的临床验证。与单纯的免疫细胞化学检测技术及RT-PCR检测技术相比较,本论文建立的磁性富集与检测系统具有灵敏度高、特异性强和稳定性好的特点,进行肺癌临床验证结果显示本检测系统检测率与RT-PCR检测技术所检测的结果相当,无显著性差异(p>0.05),同时无假阳性结果出现,比单纯的免疫细胞化学检测技术检出率高,存在显著差异(p<0.05)。
4.建立磁性化学发光免疫分析系统,应用于肿瘤标志物AFP的测定。
制备羊抗兔IgG二抗包被的免疫磁性微球,将免疫磁性分离技术与化学发光技术相结合,建立磁性化学发光免疫分析系统,优化检测系统的条件,鉴定其特异性、灵敏性和稳定性,并应用于患者外周血AFP的测定。实验证明本论文建立的磁性化学发光免疫分析系统具有灵敏度高、特异性强和稳定性好的特点,进行患者外周血AFP的测定结果有很好的临床相符性。
本研究制备的新型纳米磁性复合微球,以碳包铁(Fe@C)纳米颗粒作为磁性内核,解决了氧化铁内核磁性能不够的难题,性能优良;在交联抗体时于微球与抗体之间连接一个手臂分子,避免了由于位阻效应导致的微球表面抗体与相应抗原结合不完全的弊端,更有利于微球功能的发挥。在此基础上,纳米磁性复合微球作为公共载体和分离工具应用于磁性富集与检测系统和磁性化学发光免疫分析系统,实验证明方便易行,具有广泛的应用前景。
目的:构建生物素-蛋白连接酶(BirA酶)基因的表达载体,并在大肠杆菌BL-21 (DE3)中表达具有生物学活性的BirA酶。
方法:用PCR法扩增BirA酶基因。将PCR产物克隆入pGEX-4T-2中构建BirA酶-GST融合蛋白基因的重组表达载体pGEX-BirA。经测序验证后,在大肠杆菌BL-21(DE3)中诱导表达,表达产物采用谷胱苷肽-琼脂糖层析柱进行纯化。以带有生物素酶底物肽(BirA substrate peptide, BSP)的HLA-A2-肽复合物为底物,用ELISA和Western blot鉴定表达产物的生物素化活性。
结果:成功地构建了pGEX-BirA原核表达质粒,并在大肠杆菌BL-21(DE3)中诱导表达Mr为61300的BirA酶-GST融合蛋白,表达产物经谷胱苷肽-琼脂糖层析柱纯化后,得到N端带有GST标签的BirA酶蛋白。ELISA和Western blot的结果显示,表达产物具有酶活性,能使HLA-A2-肽复合物生物素化。
结论:成功地制备了具有生物学活性的BirA酶,为研究蛋白质分子的相互作用提供了有效的制剂。
Magnetic polymer microspheres (MMS) are those microspheres synthesized by polymer and inorganic material on molecule scale, which have the ability of magnetism and special structure. Since varied functional groups linking on the surface of microspheres, such as -OH,-COOH, -NH2 and so on, MMS are biocompatible and can conjugate with some ligands of bioactive compound,these give them the ability to recognize and bind to corresponding antigen, antibody, nucleic acid etc. Then this compound can be separated and enriched through the magnetic field. So MMS are promising for the new tool on varial areas such as cell separation, enzyme immobilization, drug targeting, immunodetection and so on. In this thesis, a kind of neotype nano- MMS were prepared, these nano-MMS had some characteristics when compared with ordinary MMS, such as strongly magnetic response, superparamagnetism, highly stability, largely specific surface area and so on. MMs can be taked as publicly carrier and separate tool. Then associated with immune analytical technique and chemiluminescence reaction, new system of magnetic enrichment and detedtion and magnetic chemiluminescence immune assay system were designed and exploited, and applied in the early diagnose of tumor. These establishments of such detection can bring out strongly social effects and economic returns.
1. Preparation of magnetic Alginate Sodium composite Microsphere by means of reverse phase microemulsion and imbed process
Take heptane as organic dispersed medium, AOT as surfactant, sodium alginate solution and Fe@C nanoparticle as aqueous phase, the system of reverse phase microemulsion was formed, and calcium chlorination and epichlorohydrin were been taked as crossing agents. Preparation conditions such as the concentration of sodium alginate, the concentration of AOT and stirring speed were described, and then the microsphere properties also were described in this paper. Results showed that the microspheres had a spherical appearance, with narrow size distribution, and the average diameter is 279nm, while the microspheres also showed strongly magnetic response.
2. Immunomagnetic Alginate Sodium composite microspheres (IMM) were prepared in this part.
The imbedding method of chemical group on the surface of MMS are discussed, and 6-Aminocaproic Acid with six carbon chain length were attached onto MMS as“spacer arm”, then goat anti-mouse IgG antibodies were binded with“spacer arm”through cross-linking reaction by preparing immunomagnetic alginate microspheres. Preparation conditions and MMS properties were described. Results showed that 98.3% immunomagnetic microsphere showed successfully cross-linked actived antibody and there were 111.2μg antibodies immobilized onto 1mg immunomagnetic microspheres.
3. Tumor cell magnetic enrichment and detection method were established and applied in the detection of tumor cells in peripheral blood of lung adenocarcinoma patients. IMM coated with goat anti-mouse IgG antibodies were prepared, system of magnetic enrichment and detedtion were established by combining immunomagnetic separation with immunocytochemistry technique. The specificity, sensitivity and stability of the system were identified. In the detection of tumor cells in peripheral blood of lung adenocarcinoma patients, three kinds of method (enrichment and detection method that established in this thesis, immunocytochemistry and RT-PCR method) were used to compare their variability.
The result showed that Tumor cell magnetic enrichment and detection system had the abilities of high sensitivity, specificity and stability. In the detection of clinic patients, the new detection system had no significant difference compared with RT-PCR methods (p>0.05), and had significant difference compared with immunocytochemistry methods (p<0.05).
4. Magnetic chemiluminescence immune assay system were established and applied in the detection of tumor marker AFP in peripheral blood.
IMM coated with goat anti-rabbit IgG antibodies were prepared, magnetic chemiluminescence immune assay system was established by combined immunomagnetic separation with chemiluminescence reaction. The specificity, sensitivity and stability of the system were identified. In the detection of tumor marker AFP in peripheral blood, the result showed that system of Magnetic chemiluminescence immune assay had the abilities of high sensitivity, specificity and stability, and the result of AFP in the blood have matched with clinic diagnosis.
In this thesis, neotype Immunomagnetic composite microspheres were prepared taking Fe@C nanoparticle as magnetic materials. This resolved the problem of poor magnetism when taking ferric oxide as magnetic materials. Considering stereo conformation between antibody and antigen reaction, antibodies were binding on microspheres through“arm”molecule. In this basis, nano- MMS are promising for the new tool in more areas besides of system of magnetic enrichment and detedtion and magnetic chemiluminescence immune assay system.
Objective: To construct the expression vector of biotin-protein ligase (BirA enzyme) gene and express the BirA enzyme with bioactivity in E.coli BL-21 (DE3).
Methods: The BirA gene was amplified from E.coli genome by PCR and it was cloned into pGEX-4T-2 to construct the recombinant plasmid pGEX-BirA. After being verified by DNA sequencing, the fusion protein was expressed under IPTG induction in the E.coli BL-21 (DE3). The expressed product was purified through Glutathione-agarose chromatography column. The enzyme activity of the expressed product was identified by ELISA and Western blot.
Results: The recombinant prokaryotic expression vector pGEX-BirA was constructed and the fusion protein GST-BirA with Mr being 61300 and bioactivity was expressed successfully. After the expressed product was purified through Glutathione-agarose chromatography column, the results of ELISA and Western blot showed that the expressed product could make HLA-A2 peptide complex biotinylation.
Conclusion: BirA enzyme with bioactivity is prepared successfully which provide an effective reagent for studying the interaction between protein and protein.
引文
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13. Guo LA, Zhu BQ, Chen DJ. [Purification of vancomycin by using magnetic affinity technique]. Sheng Wu Gong Cheng Xue Bao. 2001;17:584-586.
14. Horton JK, Davies M, Woodhead JS, Weeks I. A rapid and sensitive method for estimating low concentrations of albumin in human urine. Clin Chim Acta. 1989;186:45-51.
15. Iwata A, Satoh K, Murata M, Hikata M, Hayakawa T, Yamaguchi T. Virus concentration using sulfonated magnetic beads to improve sensitivity in nucleic acid amplification tests. Biol Pharm Bull. 2003;26:1065-1069.
16. Jiang XY, Bai S, Sun Y. Fabrication and characterization of rigid magnetic monodisperse microspheres for protein adsorption. J Chromatogr B Analyt Technol Biomed Life Sci. 2007;10:10.
17. Basso D, Guariso G, Fasolo M, et al. A new indirect chemiluminescent immunoassay to measure anti-tissue transglutaminase antibodies. J Pediatr Gastroenterol Nutr. 2006;43:613-618.
18. Dungchai W, Siangproh W, Lin JM, Chailapakul O, Lin S, Ying X. Development of a sensitive micro-magnetic chemiluminescence enzyme immunoassay for the determination of carcinoembryonic antigen. Anal Bioanal Chem. 2007;387:1965-1971.
19. Alpeeva IS, Yu Sakharov I. Luminol-hydrogen peroxide chemiluminescence produced by sweet potato peroxidase. Luminescence. 2007;22:92-96.
20. Dasgupta A, Kang E, Datta P. A new enzyme-linked chemiluminescentimmunosorbent digoxin assay is virtually free from interference of spironolactone, potassium canrenoate, and their common metabolite canrenone. J Clin Lab Anal. 2006;20:204-208.
21. Duarte AM, de Andrade HM, do Monte SJ, de Toledo Vde P, Guimaraes TM. Assessment of chemiluminescence and PCR effectiveness in relation to conventional serological tests for the diagnosis of Chagas' disease. Rev Soc Bras Med Trop. 2006;39:385-387.
22. Fan A, Lau C, Lu J. Magnetic bead-based chemiluminescent metal immunoassay with a colloidal gold label. Anal Chem. 2005;77:3238-3242.
23. Sun Y, Wang B, Wang H, Jiang J. Controllable preparation of magnetic polymer microspheres with different morphologies by miniemulsion polymerization. J Colloid Interface Sci. 2007;308:332-336.
24. Dobakhti F, Rahimi F, Dehpour AR, et al. Stabilizing effects of calcium alginate microspheres on mycobacterium bovis BCG intended for oral vaccination. J Microencapsul. 2006;23:844-854.
25. Gomez d'Ayala G, De Rosa A, Laurienzo P, Malinconico M. Development of a new calcium sulphate-based composite using alginate and chemically modified chitosan for bone regeneration. J Biomed Mater Res A. 2007;81:811-820.
26. Scocca S, Faustini M, Villani S, et al. Alginate/polymethacrylate copolymer microparticles for the intestinal delivery of enzymes. Curr Drug Deliv. 2007;4:103-108.
27. de Kerchove AJ, Elimelech M. Formation of polysaccharide gel layers in the presence of Ca2+ and K+ ions: measurements and mechanisms. Biomacromolecules. 2007;8:113-121.
28. Safarikova M, Roy I, Gupta MN, Safarik I. Magnetic alginate microparticles for purification of alpha-amylases. J Biotechnol. 2003;105:255-260.
29. Chang JY, Lin JH, Yao CH, Chen JH, Lai TY, Chen YS. In vivo evaluation of a biodegradable EDC/NHS-cross-linked gelatin peripheral nerve guide conduit material.Macromol Biosci. 2007;7:500-507.
30. Zhao C, Sun YL, Zobitz ME, An KN, Amadio PC. Enhancing the strength of the tendon-suture interface using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride and cyanoacrylate. J Hand Surg [Am]. 2007;32:606-611.
31. Pall GS, Codony-Servat C, Byrne J, Ritchie L, Hamilton A. Carbodiimide-mediated cross-linking of RNA to nylon membranes improves the detection of siRNA, miRNA and piRNA by northern blot. Nucleic Acids Res. 2007;35:e60.
32. Englert C, Blunk TB, Muller R, et al. Bonding of articular cartilage utilising a combination of biochemical degradation and surface cross-linking. Arthritis Res Ther. 2007;9:R47.
33. Sauermann M, Hahne F, Schmidt C, et al. High-Throughput Flow Cytometry-Based Assay to Identify Apoptosis-Inducing Proteins. J Biomol Screen. 2007;3:3.
34. Pattanapanyasat K, Phuang-Ngern Y, Lerdwana S, et al. Evaluation of a single-platform microcapillary flow cytometer for enumeration of absolute CD4+ T-lymphocyte counts in HIV-1 infected Thai patients. Cytometry B Clin Cytom. 2007;1:1.
35. Weiss DJ. An indirect flow cytometric test for detection of anti-neutrophil antibodies in dogs. Am J Vet Res. 2007;68:464-467.
36. Kim SJ, Ikeda N, Shiba E, Takamura Y, Noguchi S. Detection of breast cancer micrometastases in peripheral blood using immunomagnetic separation and immunocytochemistry. Breast Cancer. 2001;8:63-69.
37. Wirtschafter A, Benninger MS, Moss TJ, Umiel T, Blazoff K, Worsham MJ. Micrometastatic tumor detection in patients with head and neck cancer: a preliminary report. Arch Otolaryngol Head Neck Surg. 2002;128:40-43.
38. Yamamoto K, Kitayama W, Denda A, Sasahira T, Kuniyasu H, Kirita T. Expression of receptor for advanced glycation end products during rat tongue carcinogenesis by 4-nitroquinoline 1-oxide and effect of a selective cyclooxygenase-2 inhibitor, etodolac. Pathobiology. 2006;73:317-324.
39. Uen YH, Lin SR, Wu CH, et al. Clinical significance of MUC1 and c-Met RT-PCR detection of circulating tumor cells in patients with gastric carcinoma. Clin Chim Acta. 2006;367:55-61.
40. Aquino A, Prete SP, Balduzzi A, et al. A novel method for monitoring response to chemotherapy based on the detection of circulating cancer cells: a case report. J Chemother. 2002;14:412-416.
41. Guney K, Yoldas B, Ozbilim G, Derin AT, Sarihan S, Balkan E. Detection of micrometastatic tumor cells in head and neck squamous cell carcinoma. A possible predictor of recurrences? Saudi Med J. 2007;28:216-220.
42. Mu XL, Li LY. [Detection and clinic implication of circulating tumor cell in patients with lung cancer]. Zhongguo Yi Xue Ke Xue Yuan Xue Bao. 2004;26:313-318.
43. Nakamura T, Yasumura T, Hayashi K, et al. Immunocytochemical detection of circulating esophageal carcinoma cells by immunomagnetic separation. Anticancer Res. 2000;20:4739-4744.
44. Meng S, Tripathy D, Frenkel EP, et al. Circulating tumor cells in patients with breast cancer dormancy. Clin Cancer Res. 2004;10:8152-8162.
45. Cristofanilli M. Circulating tumor cells, disease progression, and survival in metastatic breast cancer. Semin Oncol. 2006;33:S9-14.
46. Chen XM, Chen GY, Wang ZR, Zhu FS, Wang XL, Zhang X. Detection of micrometastasis of gastric carcinoma in peripheral blood circulation. World J Gastroenterol. 2004;10:804-808.
47. Fu Z, Liu H, Ju H. Flow-through multianalyte chemiluminescent immunosensing system with designed substrate zone-resolved technique for sequential detection of tumor markers. Anal Chem. 2006;78:6999-7005.
48. Leary TP, Gutierrez RA, Muerhoff AS, Birkenmeyer LG, Desai SM, Dawson GJ. A chemiluminescent, magnetic particle-based immunoassay for the detection of hepatitis C virus core antigen in human serum or plasma. J Med Virol. 2006;78:1436-1440.
49. Dasgupta A, Kang E, Olsen M, Actor JK, Datta P. New enzyme-linked chemiluminescent immunosorbent digoxin assay is free from interference of Chinese medicine DanShen. Ther Drug Monit. 2006;28:775-778.
50. Li ZP, Liu CH, Fan YS, Wang YC, Duan XR. A chemiluminescent metalloimmunoassay based on silver deposition on colloidal gold labels. Anal Biochem. 2006;359:247-252.
51. Kohen F, De Boever J, Kim JB. Recent advances in chemiluminescence-based immunoassays for steroid hormones. J Steroid Biochem. 1987;27:71-79.
52. Barker DF, Campbell AM. The birA gene of Escherichia coli encodes a biotin holoenzyme synthetase [J]. J Mol Biol, 1981,146(4): 451-467.
53. Barker DF, Campbell AM. Genetic and biochemical characterization of the birA gene and its product: evidence for a direct role of biotin holoenzyme synthetase in repression of the biotin operon in Escherichia coli [J]. J Mol Biol, 1981, 146(4): 469-492.
54. Gillespie G, Mutis T, Schrama E, et al. HLA class I-minor histocompatibility antigen tetramers select cytotoxic T cells with high avidity to the natural ligand [J]. Hematol J, 2000, 1(6): 403-410
55. Constantin CM, Bonney EE, Altman JD, et al. Major histocompatibility complex(MHC) tetramer technology: an evaluation. Biol Res Nur,. 2002 , 4(2):115-27
56. McMichael AJ, Kelleher A. The arrival of HLA class II tetramers[J]. J Clin Invest, 1999, 104(12):1669-70.
57. Kotzin BL, Falta MT, Crawford F, et al. Use of soluble peptide-DR4 tetramers to detect synovial T cells specific for cartilage antigens in patiens with rheumatoid arthritis[J]. Proc Natl Acad Sci USA, 2000, 97(1):291-296
58. Schatz PJ. Use of peptide libraries to map the substrate specificity of a peptide-modifying enzyme: a 13 residue consensus peptide specifies biotinylation in Escherichia coli [J]. Biotechnology (N Y), 1993, 11(10): 1138-1143.
59. Allan DS, Lepin EJ, Braud VM, et al. Tetrameric complexes of HLA-E, HLA-F, andHLA-G[J]. J Immunol Methods. 2002 , 268(1):43-50.
60. Bousso P. Generation of MHC-peptide tetramers: a new opportunity for dissection T cell immune responses[J]. Microbes Infect, 2000, 2(4):425-429
61. Altman JD, Moss PAH, Goulder PJR, et al. Phenotypic analysis of antigen-specific T lymphocytes [J]. Science, 1996, 274 (5284): 94-96.
62. Skinner PJ, Daniels MA, Schmidt CS, et al. Cutting edge: In situ tetramer staining of antigen-specific T cells in tissues[J]. J Immunol., 2000,165(2):613-617
63. Xu XN, Screaton GR. MHC/peptide tetramer-based studies of T cell function[J]. J Immunol Methods, 2002, 268(1):21-28
64. Maile R, Wang B, schooler W, et al. antigen-specific modulation of an immune response by in vivo administration of soluble MHC class I tetramers[J]. J.Immunol, 2001, 167(7):3708-3714
65. Robert B, Guilaume P, Luescher I, et al.. Antibody-conjugated MHC class I tetramers can target tumor cells for specific lysis by T lymphocytes[J]. Eur J Immunol, 2000, 30(11):3165-70
66. Ladasky JJ, Shum BP, Canavez F, et al. Residue 3 of beta2-microglobulin affects binding of class I MHC molecules by the W6/32 antibody [J]. Immunogenetics, 1999, 49(4): 312-320
67.翁秀芳,陈浩,吴雄文,等.可溶性HLA-A2-肽复合物的体外折叠与生物素化[J].细胞与分子免疫学杂志,2004,3:259-263.
68.萨姆布鲁克J,费里奇EF,曼里阿缔斯T,著.分子克隆实验指南[M].第2版,北京:科学出版社,1992.
69. O’callaghan CA, Byford MF,Wyer JR, et al. BirA enzyme: production and application in the study of membrane receptor-ligand interactions by site-specific biotinylation[J].Anal Biochem,1999,266(1):9-15.
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12. Chen MJ, Chen KN, Kuo YT. Optimal thermotolerance of bifidobacterium bifidum in gellan-alginate microparticles. Biotechnol Bioeng. 2007;9:9.
13. Guo LA, Zhu BQ, Chen DJ. [Purification of vancomycin by using magnetic affinity technique]. Sheng Wu Gong Cheng Xue Bao. 2001;17:584-586.
14. Horton JK, Davies M, Woodhead JS, Weeks I. A rapid and sensitive method for estimating low concentrations of albumin in human urine. Clin Chim Acta. 1989;186:45-51.
15. Iwata A, Satoh K, Murata M, Hikata M, Hayakawa T, Yamaguchi T. Virus concentration using sulfonated magnetic beads to improve sensitivity in nucleic acid amplification tests. Biol Pharm Bull. 2003;26:1065-1069.
16. Jiang XY, Bai S, Sun Y. Fabrication and characterization of rigid magnetic monodisperse microspheres for protein adsorption. J Chromatogr B Analyt Technol Biomed Life Sci. 2007;10:10.
17. Basso D, Guariso G, Fasolo M, et al. A new indirect chemiluminescent immunoassay to measure anti-tissue transglutaminase antibodies. J Pediatr Gastroenterol Nutr. 2006;43:613-618.
18. Dungchai W, Siangproh W, Lin JM, Chailapakul O, Lin S, Ying X. Development of a sensitive micro-magnetic chemiluminescence enzyme immunoassay for the determination of carcinoembryonic antigen. Anal Bioanal Chem. 2007;387:1965-1971.
19. Alpeeva IS, Yu Sakharov I. Luminol-hydrogen peroxide chemiluminescence produced by sweet potato peroxidase. Luminescence. 2007;22:92-96.
20. Dasgupta A, Kang E, Datta P. A new enzyme-linked chemiluminescentimmunosorbent digoxin assay is virtually free from interference of spironolactone, potassium canrenoate, and their common metabolite canrenone. J Clin Lab Anal. 2006;20:204-208.
21. Duarte AM, de Andrade HM, do Monte SJ, de Toledo Vde P, Guimaraes TM. Assessment of chemiluminescence and PCR effectiveness in relation to conventional serological tests for the diagnosis of Chagas' disease. Rev Soc Bras Med Trop. 2006;39:385-387.
22. Fan A, Lau C, Lu J. Magnetic bead-based chemiluminescent metal immunoassay with a colloidal gold label. Anal Chem. 2005;77:3238-3242.
23. Sun Y, Wang B, Wang H, Jiang J. Controllable preparation of magnetic polymer microspheres with different morphologies by miniemulsion polymerization. J Colloid Interface Sci. 2007;308:332-336.
24. Dobakhti F, Rahimi F, Dehpour AR, et al. Stabilizing effects of calcium alginate microspheres on mycobacterium bovis BCG intended for oral vaccination. J Microencapsul. 2006;23:844-854.
25. Gomez d'Ayala G, De Rosa A, Laurienzo P, Malinconico M. Development of a new calcium sulphate-based composite using alginate and chemically modified chitosan for bone regeneration. J Biomed Mater Res A. 2007;81:811-820.
26. Scocca S, Faustini M, Villani S, et al. Alginate/polymethacrylate copolymer microparticles for the intestinal delivery of enzymes. Curr Drug Deliv. 2007;4:103-108.
27. de Kerchove AJ, Elimelech M. Formation of polysaccharide gel layers in the presence of Ca2+ and K+ ions: measurements and mechanisms. Biomacromolecules. 2007;8:113-121.
28. Safarikova M, Roy I, Gupta MN, Safarik I. Magnetic alginate microparticles for purification of alpha-amylases. J Biotechnol. 2003;105:255-260.
29. Chang JY, Lin JH, Yao CH, Chen JH, Lai TY, Chen YS. In vivo evaluation of a biodegradable EDC/NHS-cross-linked gelatin peripheral nerve guide conduit material.Macromol Biosci. 2007;7:500-507.
30. Zhao C, Sun YL, Zobitz ME, An KN, Amadio PC. Enhancing the strength of the tendon-suture interface using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride and cyanoacrylate. J Hand Surg [Am]. 2007;32:606-611.
31. Pall GS, Codony-Servat C, Byrne J, Ritchie L, Hamilton A. Carbodiimide-mediated cross-linking of RNA to nylon membranes improves the detection of siRNA, miRNA and piRNA by northern blot. Nucleic Acids Res. 2007;35:e60.
32. Englert C, Blunk TB, Muller R, et al. Bonding of articular cartilage utilising a combination of biochemical degradation and surface cross-linking. Arthritis Res Ther. 2007;9:R47.
33. Sauermann M, Hahne F, Schmidt C, et al. High-Throughput Flow Cytometry-Based Assay to Identify Apoptosis-Inducing Proteins. J Biomol Screen. 2007;3:3.
34. Pattanapanyasat K, Phuang-Ngern Y, Lerdwana S, et al. Evaluation of a single-platform microcapillary flow cytometer for enumeration of absolute CD4+ T-lymphocyte counts in HIV-1 infected Thai patients. Cytometry B Clin Cytom. 2007;1:1.
35. Weiss DJ. An indirect flow cytometric test for detection of anti-neutrophil antibodies in dogs. Am J Vet Res. 2007;68:464-467.
36. Kim SJ, Ikeda N, Shiba E, Takamura Y, Noguchi S. Detection of breast cancer micrometastases in peripheral blood using immunomagnetic separation and immunocytochemistry. Breast Cancer. 2001;8:63-69.
37. Wirtschafter A, Benninger MS, Moss TJ, Umiel T, Blazoff K, Worsham MJ. Micrometastatic tumor detection in patients with head and neck cancer: a preliminary report. Arch Otolaryngol Head Neck Surg. 2002;128:40-43.
38. Yamamoto K, Kitayama W, Denda A, Sasahira T, Kuniyasu H, Kirita T. Expression of receptor for advanced glycation end products during rat tongue carcinogenesis by 4-nitroquinoline 1-oxide and effect of a selective cyclooxygenase-2 inhibitor, etodolac. Pathobiology. 2006;73:317-324.
39. Uen YH, Lin SR, Wu CH, et al. Clinical significance of MUC1 and c-Met RT-PCR detection of circulating tumor cells in patients with gastric carcinoma. Clin Chim Acta. 2006;367:55-61.
40. Aquino A, Prete SP, Balduzzi A, et al. A novel method for monitoring response to chemotherapy based on the detection of circulating cancer cells: a case report. J Chemother. 2002;14:412-416.
41. Guney K, Yoldas B, Ozbilim G, Derin AT, Sarihan S, Balkan E. Detection of micrometastatic tumor cells in head and neck squamous cell carcinoma. A possible predictor of recurrences? Saudi Med J. 2007;28:216-220.
42. Mu XL, Li LY. [Detection and clinic implication of circulating tumor cell in patients with lung cancer]. Zhongguo Yi Xue Ke Xue Yuan Xue Bao. 2004;26:313-318.
43. Nakamura T, Yasumura T, Hayashi K, et al. Immunocytochemical detection of circulating esophageal carcinoma cells by immunomagnetic separation. Anticancer Res. 2000;20:4739-4744.
44. Meng S, Tripathy D, Frenkel EP, et al. Circulating tumor cells in patients with breast cancer dormancy. Clin Cancer Res. 2004;10:8152-8162.
45. Cristofanilli M. Circulating tumor cells, disease progression, and survival in metastatic breast cancer. Semin Oncol. 2006;33:S9-14.
46. Chen XM, Chen GY, Wang ZR, Zhu FS, Wang XL, Zhang X. Detection of micrometastasis of gastric carcinoma in peripheral blood circulation. World J Gastroenterol. 2004;10:804-808.
47. Fu Z, Liu H, Ju H. Flow-through multianalyte chemiluminescent immunosensing system with designed substrate zone-resolved technique for sequential detection of tumor markers. Anal Chem. 2006;78:6999-7005.
48. Leary TP, Gutierrez RA, Muerhoff AS, Birkenmeyer LG, Desai SM, Dawson GJ. A chemiluminescent, magnetic particle-based immunoassay for the detection of hepatitis C virus core antigen in human serum or plasma. J Med Virol. 2006;78:1436-1440.
49. Dasgupta A, Kang E, Olsen M, Actor JK, Datta P. New enzyme-linked chemiluminescent immunosorbent digoxin assay is free from interference of Chinese medicine DanShen. Ther Drug Monit. 2006;28:775-778.
50. Li ZP, Liu CH, Fan YS, Wang YC, Duan XR. A chemiluminescent metalloimmunoassay based on silver deposition on colloidal gold labels. Anal Biochem. 2006;359:247-252.
51. Kohen F, De Boever J, Kim JB. Recent advances in chemiluminescence-based immunoassays for steroid hormones. J Steroid Biochem. 1987;27:71-79.
52. Barker DF, Campbell AM. The birA gene of Escherichia coli encodes a biotin holoenzyme synthetase [J]. J Mol Biol, 1981,146(4): 451-467.
53. Barker DF, Campbell AM. Genetic and biochemical characterization of the birA gene and its product: evidence for a direct role of biotin holoenzyme synthetase in repression of the biotin operon in Escherichia coli [J]. J Mol Biol, 1981, 146(4): 469-492.
54. Gillespie G, Mutis T, Schrama E, et al. HLA class I-minor histocompatibility antigen tetramers select cytotoxic T cells with high avidity to the natural ligand [J]. Hematol J, 2000, 1(6): 403-410
55. Constantin CM, Bonney EE, Altman JD, et al. Major histocompatibility complex(MHC) tetramer technology: an evaluation. Biol Res Nur,. 2002 , 4(2):115-27
56. McMichael AJ, Kelleher A. The arrival of HLA class II tetramers[J]. J Clin Invest, 1999, 104(12):1669-70.
57. Kotzin BL, Falta MT, Crawford F, et al. Use of soluble peptide-DR4 tetramers to detect synovial T cells specific for cartilage antigens in patiens with rheumatoid arthritis[J]. Proc Natl Acad Sci USA, 2000, 97(1):291-296
58. Schatz PJ. Use of peptide libraries to map the substrate specificity of a peptide-modifying enzyme: a 13 residue consensus peptide specifies biotinylation in Escherichia coli [J]. Biotechnology (N Y), 1993, 11(10): 1138-1143.
59. Allan DS, Lepin EJ, Braud VM, et al. Tetrameric complexes of HLA-E, HLA-F, andHLA-G[J]. J Immunol Methods. 2002 , 268(1):43-50.
60. Bousso P. Generation of MHC-peptide tetramers: a new opportunity for dissection T cell immune responses[J]. Microbes Infect, 2000, 2(4):425-429
61. Altman JD, Moss PAH, Goulder PJR, et al. Phenotypic analysis of antigen-specific T lymphocytes [J]. Science, 1996, 274 (5284): 94-96.
62. Skinner PJ, Daniels MA, Schmidt CS, et al. Cutting edge: In situ tetramer staining of antigen-specific T cells in tissues[J]. J Immunol., 2000,165(2):613-617
63. Xu XN, Screaton GR. MHC/peptide tetramer-based studies of T cell function[J]. J Immunol Methods, 2002, 268(1):21-28
64. Maile R, Wang B, schooler W, et al. antigen-specific modulation of an immune response by in vivo administration of soluble MHC class I tetramers[J]. J.Immunol, 2001, 167(7):3708-3714
65. Robert B, Guilaume P, Luescher I, et al.. Antibody-conjugated MHC class I tetramers can target tumor cells for specific lysis by T lymphocytes[J]. Eur J Immunol, 2000, 30(11):3165-70
66. Ladasky JJ, Shum BP, Canavez F, et al. Residue 3 of beta2-microglobulin affects binding of class I MHC molecules by the W6/32 antibody [J]. Immunogenetics, 1999, 49(4): 312-320
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