基于抗原/抗体反应的生物检测技术及其应用研究
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摘要
抗原抗体反应在生物检测技术中具有非常广泛的应用，可以用于传染病、免疫异常性疾病、肿瘤等相关疾病的诊断、疗效评价及发病机理的研究。传统的检测方法（如免疫沉淀、ELISA等）已经不能满足现代生命科学和现代医学在快速、高通量检测和分析方面的需求。近年来新发展起来的蛋白质芯片技术，在高通量、高灵敏度等方面的独特性质受到国内外的重视。但是，蛋白质芯片的制备技术还不很完善，如固定化方法对蛋白质活性的影响较大，玻璃表面修饰后的荧光背景较高等，影响了蛋白质芯片的推广应用。本论文针对蛋白质芯片在制备过程中存在的问题，研究了玻璃表面的修饰方法及其对蛋白质的固定化和反应性的影响，对蛋白质芯片的制备工艺进行了优化，取得了明显的效果。在此基础上，研制了TORCH感染检测蛋白质芯片，可用于产妇的孕期检查。该芯片与目前应用的ELISA法检测技术相比，一次试验可完成TORCH感染4项指标的检测。此外，我们提出了一种基于蛋白质芯片的细胞检测方法，制备了血型分型抗体阵列芯片，并成功地用于人红细胞血型检测。
纳米粒子在生物检测中的应用越来越广泛，胶体金由于其特殊的性质而倍受关注。其显色性和导电性已经在免疫组织化学、DNA杂交检测等方面的研究中获得应用。但是，其优异的光学非线性特性在生物检测中的应用尚未引起人们的重视。本研究采用胶体金为介质，用超瑞利散射（Hyper-Rayleigh Scattering , HRS）技术检测其二阶非线性光学特性，获得了抗原抗体反应诱导的胶体金HRS信号的变化，并进行了机理分析。该方法为溶液中抗原抗体反应的高灵敏度检测提供了新途径。
本论文所获得的主要研究成果如下：
1. 以玻璃为基质，对四种蛋白质的固定方法进行了实验对比研究，优化蛋白质芯片的制备技术。
(1) 用四种不同的修饰方法（戊二醛修饰法、琼脂糖修饰法、巯基修饰法和聚赖氨酸修饰法）对用于制备蛋白质芯片的玻璃表面进行了修饰，并用接触角测定仪和原子力显微镜进行表征。研究表明：修饰后玻片的疏水性都有较大的提高，其中醛基修饰和聚赖氨酸修饰玻片的接触角较大，达到34o。原子力显微镜表征结果显示琼脂糖修饰玻片具有三维多孔结构，表面积大。
(2) 通过直接法和间接法，研究了不同修饰表面对蛋白质的固定效果：直接法是在玻片上固定Cy3荧光标记的蛋白质，间接法是先在玻片上固定抗原，然后与Cy3荧光标记的抗体的反应。实验结果指出：琼脂糖修饰玻片固定蛋白质的能力最大。与常用的戊二醛修饰方法相比，琼脂糖修饰玻片固定蛋白质的能力高30％。
(3) 对蛋白质在玻片的固定饱和度进行了评价。结果表明，琼脂糖修饰玻片与蛋白质的结合能力较强，蛋白质结合牢固。戊二醛修饰玻片上固定的蛋白质达到饱和所需蛋白质的量为琼脂糖修饰玻片所需的2.5倍。
2. 蛋白质芯片在TORCH检测中的应用研究
TORCH是指发生在孕期的各种微生物感染所致的一类临床综合症，包括弓形体（Toxoplasmagondii，Tox）、风疹病毒（Rubellavirus，RV）、巨细胞病毒（Cytomegalovirus，CMV）、单纯疱疹病毒（Herpessimples Virus，HSV）及其他感染（Others，包括微小病毒属、人免疫缺陷病毒、肝炎病毒、梅毒等）。目前临床多采用ELISA方法进行检测，各项检测指标需要逐项进行，检测时间长，样品需要量大。利用蛋白质芯片技术将TORCH检测集成化，研制了可一次完成四项指标（Tox，RV，CMV，HSVⅡ）检测的TORCH蛋白芯片，克服了以往需要逐项检查的缺点，缩短了检测时间，减少了样本用量，同时还提高了检测灵敏度。与ELISA方法相比，检测灵敏度提高了2倍。
3. 对蛋白质芯片在细胞检测中的应用进行了研究
首次提出了一种基于蛋白质芯片的细胞检测方法，可以同时鉴别被测样品中多种不同的细胞。以血型分型试剂抗A和抗B与不同血型的红细胞的结合反应作为研
究体系，采用显微镜和CCD进行观察和图象采集，对蛋白质芯片在细胞检测中的应用进行了研究。结果表明，蛋白质芯片对于细胞的检测灵敏度较高，细胞含量检测可以达到1000个细胞/ml，并且特异性高。
为了提高抗体与细胞的结合能力，提出了一种抗体化学修饰技术。在抗体固定前，先采用了三种不同的化学修饰方法（戊二醛、聚赖氨酸、和聚乙二醇（PEG））对抗体进行处理，再把修饰后的抗体固定在玻片表面。对细胞的检测结果表明，采用抗体化学修饰技术可明显地提高抗体与细胞结合能力。研究发现，对于不同的抗体化学修饰技术存在一个最佳修饰条件，如对于戊二醛抗体化学修饰法，戊二醛的最佳反应浓度约0.5％。该方法为大规模、高通量的细胞检测研究提供了新的途径。
4. 利用超瑞利散射（Hyper-Rayleigh Scattering, HRS）技术研究了溶液中抗原与抗体的反应。
HRS现象是一种二阶非线性光学现象。本论文通过研究首次发现，胶体金标记的抗原抗体反应将引起胶体金的二阶非线性光学特性变化；HRS信号变化是由于溶液中抗体抗原反应产生的胶体金聚集效应引起的。在此基础上，提出了通过测定胶体金的二阶非线性光学特性，分析和检测水溶液中蛋白质与蛋白质反应的新方法。研究结果表明，用HRS技术检测水溶液中抗原或抗体时，灵敏度
Immunological detection based on the interactions of antigen/antibody has been used widely. It can be used in disease diagnosis, curative effect evaluation, pathogenesis research. However, traditional methods, such as immuno-precipitation, ELISA, cannot meet the fast and high throughput analysis need in modern life science and medicine. Protein microarray is regarded as an important tool because of its advantages in high throughput, high sensitivity, and simple use.
Protein microarray is a powerful tool used to investagate the interactions of protein-protein. One of the key problems about protein microarray is how to retain the activity of protein when it is immobilized in the array. In this paper, four modification methods of glass slides used for preparing protein microarray were discussed: glutaraldehyde modification, agarose film coated, mercapto-group modofication, and poly-l-lysine modification. The effects of the modification methods on the immobilization efficiency and on the activity were discussed to optimalization the preparating condition of protein array. The application of protein microarray in TORCH (short for Toxoplasmagondii，Others， Rubellavirus， Cytomegalovirus，Herpessimples Virus) infection diagnosis was also investigated. Protein microarray is also used in cell detection to determine the cell surface antigens with human red cells and blood group antibodies as the experimental system. To improve the combination efficiency, antibodies used for cell detection were cross-linked with glutaraldehyde, poly-l-lysine, and polyethylene glycol (PEG). The experimental work provided a foundation for detecting different cells in one sample at the same time.
Nanoparticles have been used wildly in bio-detection. Gold nanoparticles attracted more and more interest because of their specific character. Hyper-Rayleigh Scattering (HRS) is a new research area in biomolecule detection. Gold nanoparticles were used in HRS system for analysis the interaction of antibody-antigen in aqueous solution. The special colour and the conductivity of gold nanoparticals have been used in immunity histochemistry and DNA hybridization. However, the non-linear optical properties of nanoparticles have been studied less, especially in bio-detection. Hyper-Rayleigh Scattering (HRS) is a new research area in biomolecule detection. Gold nanoparticles were used in HRS system for analyzing the interaction of antibody-antigen in aqueous solution.
The major contributions were described as follows:
1. Modification methods of glass slides in preparation of protein microarray
(1) The glass slides were modified with four different methods: glutaraldehyde modification, agarose film coated, mercapto-group modofication, and poly-l-lysine modification. The modified glass slide surfaces were physically analyzed with AFM and an optical bench-type contact angle gonimeter. The results shows that the slides modified with poly-L-lysin or glutaraldehyde have the contact angle of 34o,while the pure slide has the contactangle of 4o. The result of AFM shows that the slide modified with agarose has a 3-dimension configuration which has the biggest surface area.
(2) The capacity of protein immobilization is studied by two methods: one is direct and the other is indirect. In direct methods, Cy3-labeled IgG was spotted on the modified glasses. In indirect methods, human-IgG was spotted on the glass and incubated with Cy3-IgG. The results were obtained by comparing the fluoresecent intensities. The results indicated that the glass modified with argrose has 30% higher capacity than that modified with glutaraldehyde.
(3) The saturation of protein immobilized on glass slides was evaluated by spotting human-IgG in different concerntrations and incubated with Cy3-IgG. The results indicated that the glass slide modified with agrose has the strongest ability to combine Protein.
2. Protein microarray in TORCH infection diagnosis
Protein microarrays for detection of TORCH antibodies in human sera were fabricated by pr
纳米粒子在生物检测中的应用越来越广泛，胶体金由于其特殊的性质而倍受关注。其显色性和导电性已经在免疫组织化学、DNA杂交检测等方面的研究中获得应用。但是，其优异的光学非线性特性在生物检测中的应用尚未引起人们的重视。本研究采用胶体金为介质，用超瑞利散射（Hyper-Rayleigh Scattering , HRS）技术检测其二阶非线性光学特性，获得了抗原抗体反应诱导的胶体金HRS信号的变化，并进行了机理分析。该方法为溶液中抗原抗体反应的高灵敏度检测提供了新途径。
本论文所获得的主要研究成果如下：
1. 以玻璃为基质，对四种蛋白质的固定方法进行了实验对比研究，优化蛋白质芯片的制备技术。
(1) 用四种不同的修饰方法（戊二醛修饰法、琼脂糖修饰法、巯基修饰法和聚赖氨酸修饰法）对用于制备蛋白质芯片的玻璃表面进行了修饰，并用接触角测定仪和原子力显微镜进行表征。研究表明：修饰后玻片的疏水性都有较大的提高，其中醛基修饰和聚赖氨酸修饰玻片的接触角较大，达到34o。原子力显微镜表征结果显示琼脂糖修饰玻片具有三维多孔结构，表面积大。
(2) 通过直接法和间接法，研究了不同修饰表面对蛋白质的固定效果：直接法是在玻片上固定Cy3荧光标记的蛋白质，间接法是先在玻片上固定抗原，然后与Cy3荧光标记的抗体的反应。实验结果指出：琼脂糖修饰玻片固定蛋白质的能力最大。与常用的戊二醛修饰方法相比，琼脂糖修饰玻片固定蛋白质的能力高30％。
(3) 对蛋白质在玻片的固定饱和度进行了评价。结果表明，琼脂糖修饰玻片与蛋白质的结合能力较强，蛋白质结合牢固。戊二醛修饰玻片上固定的蛋白质达到饱和所需蛋白质的量为琼脂糖修饰玻片所需的2.5倍。
2. 蛋白质芯片在TORCH检测中的应用研究
TORCH是指发生在孕期的各种微生物感染所致的一类临床综合症，包括弓形体（Toxoplasmagondii，Tox）、风疹病毒（Rubellavirus，RV）、巨细胞病毒（Cytomegalovirus，CMV）、单纯疱疹病毒（Herpessimples Virus，HSV）及其他感染（Others，包括微小病毒属、人免疫缺陷病毒、肝炎病毒、梅毒等）。目前临床多采用ELISA方法进行检测，各项检测指标需要逐项进行，检测时间长，样品需要量大。利用蛋白质芯片技术将TORCH检测集成化，研制了可一次完成四项指标（Tox，RV，CMV，HSVⅡ）检测的TORCH蛋白芯片，克服了以往需要逐项检查的缺点，缩短了检测时间，减少了样本用量，同时还提高了检测灵敏度。与ELISA方法相比，检测灵敏度提高了2倍。
3. 对蛋白质芯片在细胞检测中的应用进行了研究
首次提出了一种基于蛋白质芯片的细胞检测方法，可以同时鉴别被测样品中多种不同的细胞。以血型分型试剂抗A和抗B与不同血型的红细胞的结合反应作为研
究体系，采用显微镜和CCD进行观察和图象采集，对蛋白质芯片在细胞检测中的应用进行了研究。结果表明，蛋白质芯片对于细胞的检测灵敏度较高，细胞含量检测可以达到1000个细胞/ml，并且特异性高。
为了提高抗体与细胞的结合能力，提出了一种抗体化学修饰技术。在抗体固定前，先采用了三种不同的化学修饰方法（戊二醛、聚赖氨酸、和聚乙二醇（PEG））对抗体进行处理，再把修饰后的抗体固定在玻片表面。对细胞的检测结果表明，采用抗体化学修饰技术可明显地提高抗体与细胞结合能力。研究发现，对于不同的抗体化学修饰技术存在一个最佳修饰条件，如对于戊二醛抗体化学修饰法，戊二醛的最佳反应浓度约0.5％。该方法为大规模、高通量的细胞检测研究提供了新的途径。
4. 利用超瑞利散射（Hyper-Rayleigh Scattering, HRS）技术研究了溶液中抗原与抗体的反应。
HRS现象是一种二阶非线性光学现象。本论文通过研究首次发现，胶体金标记的抗原抗体反应将引起胶体金的二阶非线性光学特性变化；HRS信号变化是由于溶液中抗体抗原反应产生的胶体金聚集效应引起的。在此基础上，提出了通过测定胶体金的二阶非线性光学特性，分析和检测水溶液中蛋白质与蛋白质反应的新方法。研究结果表明，用HRS技术检测水溶液中抗原或抗体时，灵敏度
Immunological detection based on the interactions of antigen/antibody has been used widely. It can be used in disease diagnosis, curative effect evaluation, pathogenesis research. However, traditional methods, such as immuno-precipitation, ELISA, cannot meet the fast and high throughput analysis need in modern life science and medicine. Protein microarray is regarded as an important tool because of its advantages in high throughput, high sensitivity, and simple use.
Protein microarray is a powerful tool used to investagate the interactions of protein-protein. One of the key problems about protein microarray is how to retain the activity of protein when it is immobilized in the array. In this paper, four modification methods of glass slides used for preparing protein microarray were discussed: glutaraldehyde modification, agarose film coated, mercapto-group modofication, and poly-l-lysine modification. The effects of the modification methods on the immobilization efficiency and on the activity were discussed to optimalization the preparating condition of protein array. The application of protein microarray in TORCH (short for Toxoplasmagondii，Others， Rubellavirus， Cytomegalovirus，Herpessimples Virus) infection diagnosis was also investigated. Protein microarray is also used in cell detection to determine the cell surface antigens with human red cells and blood group antibodies as the experimental system. To improve the combination efficiency, antibodies used for cell detection were cross-linked with glutaraldehyde, poly-l-lysine, and polyethylene glycol (PEG). The experimental work provided a foundation for detecting different cells in one sample at the same time.
Nanoparticles have been used wildly in bio-detection. Gold nanoparticles attracted more and more interest because of their specific character. Hyper-Rayleigh Scattering (HRS) is a new research area in biomolecule detection. Gold nanoparticles were used in HRS system for analysis the interaction of antibody-antigen in aqueous solution. The special colour and the conductivity of gold nanoparticals have been used in immunity histochemistry and DNA hybridization. However, the non-linear optical properties of nanoparticles have been studied less, especially in bio-detection. Hyper-Rayleigh Scattering (HRS) is a new research area in biomolecule detection. Gold nanoparticles were used in HRS system for analyzing the interaction of antibody-antigen in aqueous solution.
The major contributions were described as follows:
1. Modification methods of glass slides in preparation of protein microarray
(1) The glass slides were modified with four different methods: glutaraldehyde modification, agarose film coated, mercapto-group modofication, and poly-l-lysine modification. The modified glass slide surfaces were physically analyzed with AFM and an optical bench-type contact angle gonimeter. The results shows that the slides modified with poly-L-lysin or glutaraldehyde have the contact angle of 34o,while the pure slide has the contactangle of 4o. The result of AFM shows that the slide modified with agarose has a 3-dimension configuration which has the biggest surface area.
(2) The capacity of protein immobilization is studied by two methods: one is direct and the other is indirect. In direct methods, Cy3-labeled IgG was spotted on the modified glasses. In indirect methods, human-IgG was spotted on the glass and incubated with Cy3-IgG. The results were obtained by comparing the fluoresecent intensities. The results indicated that the glass modified with argrose has 30% higher capacity than that modified with glutaraldehyde.
(3) The saturation of protein immobilized on glass slides was evaluated by spotting human-IgG in different concerntrations and incubated with Cy3-IgG. The results indicated that the glass slide modified with agrose has the strongest ability to combine Protein.
2. Protein microarray in TORCH infection diagnosis
Protein microarrays for detection of TORCH antibodies in human sera were fabricated by pr
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65. 王福勇，刘克义，于振华. 体外检测抗原抗体反应的新制剂——胶体金探针，中国实验临床免疫学杂志，1993,5(4).
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68. Elghanian, R., Storhoff, JJ., Mucic, RC., Letsinger, RL., and Mirkin, CA. Selective Colorimetric Detection of Polynucleotides Based on the Distance-Dependent Optical Properties of Gold Nanoparticles. Science, 1997, 277: 1078-1081.
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72. Huang R.P. Detection of multiple proteins in an antibody-based microarray system[J]. Journal of Immunological Methods, 2001,255: 1-13
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37. Lueking A, Horn M, Eickhoff H,etal Protein microarrays for gene expression and antibody screening[J].Anal Biochem，1999,270(1):103-111.
38. Arenkov P, Kukhtin A, Gemmell A, et al. Protein microchips: use for immunoassay and enzymatic reactions. Anal Biochem, 2000, 278:123~131
39. Huang R P. Detection of multiple proteins in an antibody-based protein microarray system. Journal of Immunological Methods, 2001,255: 1~13
40. de Wildt R M T, Mundy C R, Gorick B D, et al. Antibody arrays for high-throughput screening of antibody-antigen intractions. Nature Biotechnol, 2000,18:989~994
41. Uetz P, Hughes R E. Systematic and large-scale two-hybrid screens. Current Opinion in Microbiology, 2000, 3:303~308
42. Flores A, Briand J F, Gadal O, et al. A protein-protein interaction map of yeast RNA polymerase III. PNAS, 1999, 96: 7815~7820
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44. Zhu H, Klemic J F, Chang S, et al. Analysis of yeast protein kinase using protein chips. Nature Genetics, 2000, 26:283~289
45. Cohen CB, Chin DE, Jeong S. A microchip-based enzyme assay for protein kinase A. Analytical Biochemistry. 1999, 273(1): 89-97
46. Ito E, Iwahashi Y, Yanagisawaa Y, et al. Two short sequences have positive effects on the human p27 Kip1 gene transcription. Gene, 1999, 228: 93~100
47. Bulyk M L, Huang X, Choo Y, et al. Exploring the DNA-binding specificities of zinc fingers with DNA microarrays. PNAS, 2001, 98:7158~7163
48. Bulyk ML, Gentalen E, Lockhart DJ, et al. Quantifying DNA-protein interactions by double-stranded DNA arrays. Nature Biotechnol, 1999,17: 573~578
Ge H. UPA, a universal protein array system for quantitative detection of protein-protein,
49. protein-DNA, protein-RNA and protein-ligand interactions. Nucleic Acids Res, 2000, 28:e3, I~VII
50. Senior K. Fingerprinting disease with protein chip array. Molecular Medicine Today, 1999, 5:326~32
51. Cahill, Dolores J. Protein and antibody arrays and their medical application. Journal of Immunological Methods, 2001, 250: 81-91.
52. Huang RP, Huang RC, Fan Y, et al. Simultaneous detection of multiple cytokines from conditioned media and patient's sera by an antibody-based protein array system. Analytical Biochemistry. 2001, 7: 55-62.
53. Fung ET, Thulasiraman V, et al. Protein biochip for differential profiling. Current Opinion Biotechnology. 2001, 12(1): 65-69.
54. 汪昕，崔一平，研究分子非线性光学特性的新技术——超瑞利散射技术[J]. 中国激光，1999， 26：15-20.
55. Clays K., Persoons A. Hyper-Rayleigh scattering in solution. Phys. Rev. Lett., 1991, 66(23):2980～2983.
56. Vance, F.W., Hupp, J.T. Probing the symmetry of the nonlinear optic chromophore: insight from polarized Hyper-Rayleigh scattering and electroabsorption spectroscopy. J. Am. Chem. Soc. 1999，121, 4047-4053.
57. Clays, K., Hendrickx, E., Triest, M., et al. Nonliear optical properties of proteins measured by Hyper-Rayleigh scattering in solution[J]. Science, 1993, 262: 1419-1421。
58. Wang, X., Zhang, Y., Fu, D., Lu, Z., Cui, Y. Hyper-Rayleigh scattering and multi-photon fluorescence of nanoscale CdS colloids in water. Proceedings of SPIE, 1999, 384, 3899-3903.
59. Zhang, Y., Wang, X., Fu, D., Cheng, J., Shen, Y., Liu, J., Lu, Z. Second-order optical nonlinearity study of CdS nanoparticles via Hyper-Rayleigh scattering. J. Phys. and Chem. Solids, 2001, 62, 903-906.
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60. Supramolecular Science. 1998, 5, 495-498.
61. Vance, F.W., Lemon, B.I., Hupp, J.T. Enormous hyper-rayleigh scattering from nanocrystalline gold particle suspensions[J]. J. Phys. Chem. B, 1998, 102: 10091-10093.
62. Shen, Y., Tang, Z., Gui，M, et al. Nonliear optical response of colloidal gold nanoparticles studied by Hyper Rayleigh Scattering technique[J]. Chem. Lett., 2000, 46: 1140-1141.
63. Johnson, R.C., Li, J., Hupp, J.T., Schatz, G.C. Hyper-Rayleigh scattering studies of silver, copper, and platinum nanoparticle suspensions. Chem. Phys. Lett. , 2002, 356, 534-540.
64. 王美琪，王伟君，张正福. 免疫电镜定位酶的探针——蛋白A胶金体复合物的制备和检验，细胞生物学杂志，1993,15(1).
65. 王福勇，刘克义，于振华. 体外检测抗原抗体反应的新制剂——胶体金探针，中国实验临床免疫学杂志，1993,5(4).
66. Horisberger, M., and Clerc, M.F. Labelling of colloidal gold with protein A. A quantitative study. Histochem, 1985,82: 219-223.
67. Bendayan, M. A review of the potential and versatility of colloidal gold cytochemical labeling for molecular morphology. Biotech. Histochem, 2000,75: 203-242.
68. Elghanian, R., Storhoff, JJ., Mucic, RC., Letsinger, RL., and Mirkin, CA. Selective Colorimetric Detection of Polynucleotides Based on the Distance-Dependent Optical Properties of Gold Nanoparticles. Science, 1997, 277: 1078-1081.
69. Fritzsche, W. DNA-gold conjugates for the detection of specific molecular interactions. Reviews in Molecular Biotechnology, 2001, 82: 37-46.
70. Thanh, N.T.K., Rosenzweig, Z. Development of an aggregation-based immunoassay for anti-protein A using gold nanoparticles. Anal. Chem.2002, 74: 1624-1628.
71. Park, SJ., Taton, TA., Mirkin, CA. Array-based electrical detection of DNA with nanaoparticle probes. Science, 2002, 295: 1503-1506.
72. Huang R.P. Detection of multiple proteins in an antibody-based microarray system[J]. Journal of Immunological Methods, 2001,255: 1-13
73. Heng Zhu, Snyder M. Protein arrays and microarrays[J]. Current Opinion in Chemical Biology, 2001, 5: 40-45.
74. Mendoza L.G, Mcquary P, Mongan A, et al. High-throughput microarraybased enzyme-linked immunosorbent array (ELISA). Biotechnique, 1999, 27: 778-788.
75. Matthias Orschel, Andreas Katerkamp, Markus Meusel,et al. Evaluation of several methods to quantify immobilized proteins on gold and silica surfaces[J]. Colloids and Surfaces B: Biointerfaces, 1998,10: 273-279
76. Vivian G. Cheung, Michael Morley, Francisco Aguilar,et al. Making and reading microarrays[J]. Nature genetics supplement, 1999, 21:15-19
77. 陈亚利. 检测型生物芯片的研制及其应用研究[D]：[博士学位论文]. 南京：东南大学, 1999.
78. Brian B. Haab, Maitreya J. Dunham，Patrick O. Brown. Protein microarrays for highly parallel detection and quantitation of specific proteins and antibodies in complex solutions[J]. Genome Biology, 2001, 2: research 0004.1-0004.13
79. Victor Afanassiev, Vera Hanemann，Stefan Wolfl. Preparation of DNA and protein microarrays on glass slides coated with an agarose film[J]. Nucleic Acids Res. 2000, 28: e66
80. Edward R, Newton M D. Diagnosis of perinatal TORCH infections[J]. Clin Obst Gyne, 1999, 42(1): 59-70
81. Montoya J G, Liesenfeld O, Kinney S, et al. VIDAS test for avidity of Toxoplasma-specific immunoglobulin G for confirmatory testing of pregnant women[J]. J Clin Microbiol, 2002, 40(7): 2504-2508
82. 司徒振强，吴军正. 细胞培养[M]，西安：世界图书出版公司西安公司，1996：289-312