血清SP-D定量检测方法的建立及其作为肺相关疾病标志物的探讨
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摘要
目的：
1.采用多肽合成的方式制备SP-D抗原，以及制备SP-D多克隆抗体；
2.建立豚鼠HGPRT基因缺陷型瘤细胞系，并制备豚鼠SP-D单克隆抗体；
3.制备诊断用的纳米磁微粒；
4.建立SP-D纳米磁微粒化学发光免疫分析法（NM-CLIA）为基础的SP-D定量免疫学检测技术平台；
5. NM-CLIA定量检测人血清SP-D，并初步探讨血清SP-D在肺相关疾病中的临床意义。
方法：
1.应用DNAStar生物分析软件，采用Jameson-Wolf法和Emini法对SP-D蛋白的抗原指数和蛋白表面可及性进行预测，多肽合成SP-D抗原，经HPLC纯化，质谱鉴定，并采用重氮法制备SP-D-BSA，以此免疫原制备SP-D多克隆抗体；
2.豚鼠转化胚胎细胞104C1细胞经200rad60Coγ射线辐照诱导，并用8-AG培养筛选出HGPRT基因缺陷型瘤细胞，以此豚鼠瘤细胞制备豚鼠SP-D单克隆抗体；*本课题由国家自然科学基金资助（30872417）
3.采用共沉淀法制备葡聚糖包覆的纳米磁性微粒，经表面羧基化处理后得到诊断用的纳米磁微粒，并以EDC法制备成纳米磁微粒-SA；
4.建立血清SP-D纳米磁微粒化学发光免疫分析法，并对其进行方法学评价；
5.以建立的NM-CLIA试剂定量测定健康人、矽尘暴露人群、ILD患者、ALI患者和COPD患者血清中SP-D蛋白水平，并初步评价其作为肺相关疾病诊断生物标志物的临床意义。
结果：
1.应用DNAStar生物分析软件预测到三个SP-D线性抗原表位暴露机率相对较大氨基酸序列分别为：1（20-35aa），2（42.71aa），8（316-332aa），本次实验选择线性抗原表位暴露机率最大一段多肽：1（20-35aa），氨基酸序列为：eaemktyshrtmpsac（16aa；MW:1875Da）HPLC纯化后多肽纯度为95.5%，经质谱鉴定偶联比为：SP-D：BSA=4.9:1；多抗纯度>95%，效价>1：106；
2.建立了能稳定分泌豚鼠SP-D单克隆抗体的5株杂交瘤细胞株，分别命名为2B2、3F1、3H4、4C7和5A3，经类似物的交叉反应实验证实，其中2B2、3F1、4C7和5A3的特异性良好，与SP-D可进行特异性结合，对SP-A、SP-B、SP-C不发生交叉反应。经连续冻存40次后，3F1分泌抗体的能力最稳定；经Protein G亲和纯化系统大量制备SP-D单克隆抗体，纯度>97%，效价>1：106，并能识别特异性的SP-D蛋白的（20-35aa）肽段。
3.制备所得纳米磁性微粒粒径约300nm，均一性良好。并以EDC法制备成纳米磁微粒-SA，37℃烘箱破坏试验7天后，稳定性良好；
4.建立了SP-D NM-CLIA定量检测方法；该法最低检测限为0.22ng/ml，线性范围1.0-1000.0ng/ml，Hook效应在50000ng/ml才出现；批内与批间变异系数均<6.0%；稀释回收率为91.17%～106.42%，添加回收率为91.00%～109.63%；与SP-A（10000ng/ml）、SP-B（10000ng/ml）和SP-C（10000ng/ml）交叉反应率低于0.1%；0.2mg/ml胆红素、5mg/ml血红蛋白、10mg/ml甘油三酯、1500U/ml的类风湿因子（RF）、抗凝剂，研究样本冻融3次和冻存120天对测值的无明显影响；整个检测所需时间约22min；
5.与正常对照组（29.33±7.41ng/ml）比较，矽尘暴露组（48.67±17.79ng/ml）、0+组（95.23±24.83ng/ml）、I期矽肺组（160.28±39.05ng/ml）、间质性肺病(ILD)组（85.57±25.12ng/ml）、急性肺损伤(ALI)组（93.64±44.94ng/ml）、慢性阻塞性肺病(COPD)组血清SP-D含量均增高，差异均有统计学意义（P<0.01）；与矽尘暴露组比较， ILD组、ALI组、COPD组、0+组和I期矽肺组血清SP-D含量均增高，差异均有统计学意义（P<0.01）；矽肺I期组血清SP-D含量仍然高于0+组、ILD组、ALI组、COPD组，差异均有统计学意义（P<0.01）；血清SP-D含量与矽肺病情分级呈正相关性（rs=0.764,P<0.01）。COPD组血清SP-D含量高于ILD组、ALI组，差异均有统计学意义（P<0.01）；0+组、ILD组和ALI组三组间差异无统计学意义。
结论：
1.成功制备了SP-D多肽蛋白及其多抗；
2.成功建立了豚鼠HGPRT基因缺陷型豚鼠瘤细胞系，并制备了豚鼠SP-D单克隆抗体；
3.成功制备出了诊断用的纳米磁微粒；
4.成功建立了性能稳定的血清SP-D定量检测NM-CLIA法；
5.血清SP-D水平可作为肺相关疾病辅助诊断的生物标志物。
Objective:
1. To prepare the surfactant protein D (SP-D) by chemical synthesismethod; and prepare anti-SP-D polyclonal antibody (pAb).
2. Establishment and identification of hypoxanthine-guaninephosphoribosyl transferase (HGPRT) deficient guinea pig hybridoma cellline; and preparation of anti-SP-D monoclonal antibody (mAb) with thisguinea pig hybridoma cell line.
3. To prepare nano-magnetic particles for diagnositic reagents.
4. Development and methological evaluation of a nano-magneticparticles chemiluminescence immunoassay for quantifying serum SP-D.
5. Determination of SP-D in patiens with various lung-related diseasesand control subjects with established NM-CLIA, and investigating theclinical significance of serum SP-D as marker of lung-related diseases
Methods:
1. Prediction and synthesis of SP-D antigen epitopes usingJameson-Wolf and Emini methods, HPLC purification and massspectrometric identification. Preparation of SP-D-BSA conjμgation complex, and preparation of rabbit anti-SP-D pAb using SP-D-BSA asimmunogen.
2. The establishment and screening of HGPRT Deficient Cell Line byirradiating guinea pig transformation embryonic cell104C1with the60Coγ-Ray source, and preparation of anti-SP-D mAb.
3. Dextrans coating nano-magnetic particles were prepared byco-precipitation, and surface modification of it with carboxyl group fordiagnositic reagents, and prepareation of NM-SA with EDC method.
4. Development of a NM-CLIA method for quantifying serum SP-D,and the performance of NM-CLIA were evaluated
5. The level of serum SP-D in silica-exposed popμlation, patients withinterstitial lung disease (ILD), acute lung injury (ALI) and chronicobstructive pμlmonary disease (COPD) were determined by the establishedNM-CLIA; and preliminary evaluation of the clinical significance of serumSP-D as the biomarker of lung-related diseases was performed..
Results：
1. SP-D antigen epitopes were predicted by DNAStar bioinformaticssoftware and synthetized. Monoclonal antibody was prepared withhybridoma technique, HPLC purification and mass spectrometricidentification.
2. The establishment and screening of five hybridoma cell lines were2B2,3F1,3H4,4C7and5A3, respectively.The cross-reaction assay of analogues shoed that2B2,3F1,4C7and5A3coμld specific binding withSP-D antigen rather than SP-A, SP-B and SP-C. The hybridoma antibodystable secretion was3F1after40times of freezing and thawing. Theanti-SP-D monoclonal antibodies were prepared. We obtained high affinityand titers by protein G purified, it coμld specificly recognize20-35aa ofSP-D protein sequence.
3. The prepared nano-magnetic particle size about was300nm, andgood uniformity. Prepareation of NM-SA with EDC method had goodstability by37℃oven destroyed for7days.
4. A novel quantitative method using NM-CLIA for detection of SP-Dwas developed. The linear range was from1.0to1000.0ng/ml; theminimum detection limit was0.22ng/ml; the intra-and inter-assay CVvalues were both <6.0%. The dilution and adjunction recovery rate were91.17%~106.42%and91.00%~109.63%, respectively. The Cross-reactivityrate with SP-A（10000ng/ml）、SP-B（10000ng/ml）and SP-C（10000ng/ml）were all <0.1%. After three times of freezing and thawing and storage120days at-20℃, or existence of0.2mg/ml bilirubin,5mg/ml hemoglobin,10mg/mltriglyceride,1500U/ml rheumatoid factor, and anticoagμlants, itsdetection performance was no significant change. The total time of theassay was about22minutes.
5. The concentration of SP-D in the serum was significantly elevatedin silica-exposed worker group, ILD group, ALI groups and COPD group compared with contro（lP<0.01）; Compared with silica-exposed group, thelevels of SP-D in the serum of silicosis suspect (0+),silicosis phase I group,ILD group, ALI group and COPD group were significantly elevated (P<0.01, respectively). The levels of SP-D in the serum of silicosis phase Igroup were remarkably increased (P<0.01, respectively) compared withsilicosis suspect (0+), ILD group, ALI group and COPD group. The levelsof serum SP-D increased with the development of silicosis stage（0.764,P<0.01）, COPD group was also higher than ILD group and ALIgroup.(P <0.01, respectively). But there were no significant differencesamong silicosis suspect (0+), ILD group and ALI group.
Conclusions:
1. The SP-D and its pAbs are successfμlly prepared.
2. The establishment and screening of guinea pig hybridoma cell lines,and the SP-D mAbs have successfμlly carried out.
3. Diagnostic nano-magnetic particles are successfμlly prepared.
4. A high-performance NM-CLIA for the quantitative measurement ofserum SP-D with the guinea pig anti-SP-D mAb and rabbit anti-SP-D pAbhas been developed.
5. Serum SP-D levels can be used as auxiliary diagnosis biomarker oflung-related diseases.
1.采用多肽合成的方式制备SP-D抗原，以及制备SP-D多克隆抗体；
2.建立豚鼠HGPRT基因缺陷型瘤细胞系，并制备豚鼠SP-D单克隆抗体；
3.制备诊断用的纳米磁微粒；
4.建立SP-D纳米磁微粒化学发光免疫分析法（NM-CLIA）为基础的SP-D定量免疫学检测技术平台；
5. NM-CLIA定量检测人血清SP-D，并初步探讨血清SP-D在肺相关疾病中的临床意义。
方法：
1.应用DNAStar生物分析软件，采用Jameson-Wolf法和Emini法对SP-D蛋白的抗原指数和蛋白表面可及性进行预测，多肽合成SP-D抗原，经HPLC纯化，质谱鉴定，并采用重氮法制备SP-D-BSA，以此免疫原制备SP-D多克隆抗体；
2.豚鼠转化胚胎细胞104C1细胞经200rad60Coγ射线辐照诱导，并用8-AG培养筛选出HGPRT基因缺陷型瘤细胞，以此豚鼠瘤细胞制备豚鼠SP-D单克隆抗体；*本课题由国家自然科学基金资助（30872417）
3.采用共沉淀法制备葡聚糖包覆的纳米磁性微粒，经表面羧基化处理后得到诊断用的纳米磁微粒，并以EDC法制备成纳米磁微粒-SA；
4.建立血清SP-D纳米磁微粒化学发光免疫分析法，并对其进行方法学评价；
5.以建立的NM-CLIA试剂定量测定健康人、矽尘暴露人群、ILD患者、ALI患者和COPD患者血清中SP-D蛋白水平，并初步评价其作为肺相关疾病诊断生物标志物的临床意义。
结果：
1.应用DNAStar生物分析软件预测到三个SP-D线性抗原表位暴露机率相对较大氨基酸序列分别为：1（20-35aa），2（42.71aa），8（316-332aa），本次实验选择线性抗原表位暴露机率最大一段多肽：1（20-35aa），氨基酸序列为：eaemktyshrtmpsac（16aa；MW:1875Da）HPLC纯化后多肽纯度为95.5%，经质谱鉴定偶联比为：SP-D：BSA=4.9:1；多抗纯度>95%，效价>1：106；
2.建立了能稳定分泌豚鼠SP-D单克隆抗体的5株杂交瘤细胞株，分别命名为2B2、3F1、3H4、4C7和5A3，经类似物的交叉反应实验证实，其中2B2、3F1、4C7和5A3的特异性良好，与SP-D可进行特异性结合，对SP-A、SP-B、SP-C不发生交叉反应。经连续冻存40次后，3F1分泌抗体的能力最稳定；经Protein G亲和纯化系统大量制备SP-D单克隆抗体，纯度>97%，效价>1：106，并能识别特异性的SP-D蛋白的（20-35aa）肽段。
3.制备所得纳米磁性微粒粒径约300nm，均一性良好。并以EDC法制备成纳米磁微粒-SA，37℃烘箱破坏试验7天后，稳定性良好；
4.建立了SP-D NM-CLIA定量检测方法；该法最低检测限为0.22ng/ml，线性范围1.0-1000.0ng/ml，Hook效应在50000ng/ml才出现；批内与批间变异系数均<6.0%；稀释回收率为91.17%～106.42%，添加回收率为91.00%～109.63%；与SP-A（10000ng/ml）、SP-B（10000ng/ml）和SP-C（10000ng/ml）交叉反应率低于0.1%；0.2mg/ml胆红素、5mg/ml血红蛋白、10mg/ml甘油三酯、1500U/ml的类风湿因子（RF）、抗凝剂，研究样本冻融3次和冻存120天对测值的无明显影响；整个检测所需时间约22min；
5.与正常对照组（29.33±7.41ng/ml）比较，矽尘暴露组（48.67±17.79ng/ml）、0+组（95.23±24.83ng/ml）、I期矽肺组（160.28±39.05ng/ml）、间质性肺病(ILD)组（85.57±25.12ng/ml）、急性肺损伤(ALI)组（93.64±44.94ng/ml）、慢性阻塞性肺病(COPD)组血清SP-D含量均增高，差异均有统计学意义（P<0.01）；与矽尘暴露组比较， ILD组、ALI组、COPD组、0+组和I期矽肺组血清SP-D含量均增高，差异均有统计学意义（P<0.01）；矽肺I期组血清SP-D含量仍然高于0+组、ILD组、ALI组、COPD组，差异均有统计学意义（P<0.01）；血清SP-D含量与矽肺病情分级呈正相关性（rs=0.764,P<0.01）。COPD组血清SP-D含量高于ILD组、ALI组，差异均有统计学意义（P<0.01）；0+组、ILD组和ALI组三组间差异无统计学意义。
结论：
1.成功制备了SP-D多肽蛋白及其多抗；
2.成功建立了豚鼠HGPRT基因缺陷型豚鼠瘤细胞系，并制备了豚鼠SP-D单克隆抗体；
3.成功制备出了诊断用的纳米磁微粒；
4.成功建立了性能稳定的血清SP-D定量检测NM-CLIA法；
5.血清SP-D水平可作为肺相关疾病辅助诊断的生物标志物。
Objective:
1. To prepare the surfactant protein D (SP-D) by chemical synthesismethod; and prepare anti-SP-D polyclonal antibody (pAb).
2. Establishment and identification of hypoxanthine-guaninephosphoribosyl transferase (HGPRT) deficient guinea pig hybridoma cellline; and preparation of anti-SP-D monoclonal antibody (mAb) with thisguinea pig hybridoma cell line.
3. To prepare nano-magnetic particles for diagnositic reagents.
4. Development and methological evaluation of a nano-magneticparticles chemiluminescence immunoassay for quantifying serum SP-D.
5. Determination of SP-D in patiens with various lung-related diseasesand control subjects with established NM-CLIA, and investigating theclinical significance of serum SP-D as marker of lung-related diseases
Methods:
1. Prediction and synthesis of SP-D antigen epitopes usingJameson-Wolf and Emini methods, HPLC purification and massspectrometric identification. Preparation of SP-D-BSA conjμgation complex, and preparation of rabbit anti-SP-D pAb using SP-D-BSA asimmunogen.
2. The establishment and screening of HGPRT Deficient Cell Line byirradiating guinea pig transformation embryonic cell104C1with the60Coγ-Ray source, and preparation of anti-SP-D mAb.
3. Dextrans coating nano-magnetic particles were prepared byco-precipitation, and surface modification of it with carboxyl group fordiagnositic reagents, and prepareation of NM-SA with EDC method.
4. Development of a NM-CLIA method for quantifying serum SP-D,and the performance of NM-CLIA were evaluated
5. The level of serum SP-D in silica-exposed popμlation, patients withinterstitial lung disease (ILD), acute lung injury (ALI) and chronicobstructive pμlmonary disease (COPD) were determined by the establishedNM-CLIA; and preliminary evaluation of the clinical significance of serumSP-D as the biomarker of lung-related diseases was performed..
Results：
1. SP-D antigen epitopes were predicted by DNAStar bioinformaticssoftware and synthetized. Monoclonal antibody was prepared withhybridoma technique, HPLC purification and mass spectrometricidentification.
2. The establishment and screening of five hybridoma cell lines were2B2,3F1,3H4,4C7and5A3, respectively.The cross-reaction assay of analogues shoed that2B2,3F1,4C7and5A3coμld specific binding withSP-D antigen rather than SP-A, SP-B and SP-C. The hybridoma antibodystable secretion was3F1after40times of freezing and thawing. Theanti-SP-D monoclonal antibodies were prepared. We obtained high affinityand titers by protein G purified, it coμld specificly recognize20-35aa ofSP-D protein sequence.
3. The prepared nano-magnetic particle size about was300nm, andgood uniformity. Prepareation of NM-SA with EDC method had goodstability by37℃oven destroyed for7days.
4. A novel quantitative method using NM-CLIA for detection of SP-Dwas developed. The linear range was from1.0to1000.0ng/ml; theminimum detection limit was0.22ng/ml; the intra-and inter-assay CVvalues were both <6.0%. The dilution and adjunction recovery rate were91.17%~106.42%and91.00%~109.63%, respectively. The Cross-reactivityrate with SP-A（10000ng/ml）、SP-B（10000ng/ml）and SP-C（10000ng/ml）were all <0.1%. After three times of freezing and thawing and storage120days at-20℃, or existence of0.2mg/ml bilirubin,5mg/ml hemoglobin,10mg/mltriglyceride,1500U/ml rheumatoid factor, and anticoagμlants, itsdetection performance was no significant change. The total time of theassay was about22minutes.
5. The concentration of SP-D in the serum was significantly elevatedin silica-exposed worker group, ILD group, ALI groups and COPD group compared with contro（lP<0.01）; Compared with silica-exposed group, thelevels of SP-D in the serum of silicosis suspect (0+),silicosis phase I group,ILD group, ALI group and COPD group were significantly elevated (P<0.01, respectively). The levels of SP-D in the serum of silicosis phase Igroup were remarkably increased (P<0.01, respectively) compared withsilicosis suspect (0+), ILD group, ALI group and COPD group. The levelsof serum SP-D increased with the development of silicosis stage（0.764,P<0.01）, COPD group was also higher than ILD group and ALIgroup.(P <0.01, respectively). But there were no significant differencesamong silicosis suspect (0+), ILD group and ALI group.
Conclusions:
1. The SP-D and its pAbs are successfμlly prepared.
2. The establishment and screening of guinea pig hybridoma cell lines,and the SP-D mAbs have successfμlly carried out.
3. Diagnostic nano-magnetic particles are successfμlly prepared.
4. A high-performance NM-CLIA for the quantitative measurement ofserum SP-D with the guinea pig anti-SP-D mAb and rabbit anti-SP-D pAbhas been developed.
5. Serum SP-D levels can be used as auxiliary diagnosis biomarker oflung-related diseases.
引文
[1] Mason R J, Greene K, Dennis R V. Surfactant protein A and surfactant protein D inhealth and disease[J]. Am. J.Physiol,1998,275:L1–L13.
[2] Francis X M, Jeffrey A W. The pμlmonary collectins, SP-A and SP-D, orchestrateinnate immunity in the lung[J]. J. Clin. Invest,2002,109:707-712.
[3]. Akella A, Deshpande SB.Pμlmonary surfactants and their role in pathophysiologyof lung disorders. Indian J Exp Biol.2013,51(1):5-22.
[4] Uday Kishore, Andrés López Bernal, Mohammed F. Kamran，et al. Surfactantproteins SP-A and SP-D in human health and disease[J]. Arch Immunol Ther Exp,2005,53:399–417.
[5] Hermans C, Bernard A. Lung Epithelium-specific Proteins [J]. Am J Respir CritCare Med,1999,159:646–678.
[6] Howard Clark, Nades Palaniyar, Peter Strong, et al. Surfactant Protein D ReducesAlveolar Macrophage Apoptosis In Vivo[J]. The Journal of Immunology,2002,169:2892–2899.
[7] Crouch E.C: Surfactant protein-D and pμlmonary host defense[J]. Respir Res,2000,1(2):93.108.
[8] Maria Q. G, Karina J R, Michael F. B,et al Lung Surfactant Protein D (SP-D)Response and Regμlation During Acute and Chronic Lung InjuryLung2013,February
[9] Francis X M, Jeffrey A W. The pμlmonary collectins, SP-A and SP-D, orchestrateinnate immunity in the lung [J]. J Clin Invest,2002,109:707-712.
[10] Rajesh K.G, Anita G..Surfactant Protein-D.Animal Lectins: Form, FunctionandClinical Applications,2012:527-550.
[11] Hillaire ML, Haagsman HP, et al.Pμlmonary Surfactant Protein D in First-LineInnate Defence against Influenza A Virus Infections. J Innate Immun.2013Feb5.
[12]. Sin DD, Pahlavan PS, Man SF (2008) Surfactant protein D: A lung specificbiomarker in COPD? Ther Adv Respir Dis2:65–74..
[13]. Greene KE, Wright JR, Steinberg KP, Ruzinski JT, Caldwell E, et al.(1999) Serialchanges in surfactant-associated proteins in lung and serum before and after onsetof ARDS. Am J Respir Crit Care Med160:1843–1850.
[14]. Lesur O, Langevin S, Berthiaume Y, Legare M, Skrobik Y, et al.(2006) Outcomevalue of clara cell protein in serum of patients with acute respiratory distresssyndrome. Intensive Care Med32:1167–1174.
[15]Kunitake R, Kuwano K, Yoshida K,et al.KL-6, surfactant protein A and D inbronchoalveolar lavage fluid from patients with pμlmonary sarcoidosis.Respiration.2001;68(5):488-95.
[16] Fujita M, Shannon JM, Ouchi H，et al. Serum surfactant protein D is increased inacute and chronic inflammation in mice. Cytokine.2005Jμl7;31(1):25-33.
[17] Ishikawa T, Kubota T, Abe H, et al.Surfactant Protein-D is more Usefμl than Krebsvon den Lungen6as a Marker for the Early Diagnosis of Interstitial Pneumonitisduring Pegylated Interferon Treatment for Chronic Hepatitis C.Hepatogastroenterology.2012,59(119):2260-3.
[18] Bowler RP. Surfactant protein D as a biomarker for chronic obstructive pμlmonarydisease. COPD.2012，9(6):651.3
[19] Takahashi H, Kuroki Y, Tanaka H, et al. Serum Levels of Surfactant Proteins A andD Are Usefμl Biomarkers for Interstitial Lung Disease in Patients with ProgressiveSystemic Sclerosis[J]. Am. J. Respir. Crit. Care Med.,2000,162:258-263.
[20] Ohnishi H, Hiwada K, Kohno N, et al. Comparative Study of KL-6, SurfactantProtein-A, Surfactant Protein D, and Monocyte Chemoattractant Protein–1asSerum Markers for Interstitial Lung Diseases[J]. Am. J. Respir. Crit. Care Med.,2002,165:378-381.
[21] Takahashi H, Kuroki Y, Abe S, et al. Serum Surfactant Proteins A and D asPrognostic Factors in Idiopathic Pμlmonary Fibrosis and Their Relationship toDisease Extent[J]. Am. J. Respir. Crit. Care Med.,2000,162:1109-1114.
[22] Kunitake R, Kuwano K, Yoshida K,et al.KL-6, surfactant protein A and D inbronchoalveolar lavage fluid from patients with pμlmonary sarcoidosis.Respiration.2001;68(5):488-95.
[23]刘萍,王世鑫,陈蕾,等.矽肺患者血清克拉拉细胞蛋白和表面活性蛋白D的改变.中华劳动卫生职业病杂志，2007,25(1):18-21;
[24] Bjerk SM, Baker JV, Emery S, et al.(2013) Biomarkers and Bacterial PneumoniaRisk in Patients with Treated HIV Infection: A Case-Control Study. PLoS ONE8(2): e56249
[25] Xie F, Wang X, Ding Z,et al.Serum surfactant protein D is associated with theprognosis in patients with chronic kidney disease. J Cardiovasc Med (Hagerstown).2013Jan9.
[26] Hiroyuki Y, Hiroshi S, Yoichi N, et al.Serum levels of surfactant protein D predictthe anti-tumor activity of gefitinib in patients with advanced non-small cell lungcancer。Cancer Chemotherapy and Pharmacology,2011,67(2):331.338.
[27] Hill J, Heslop C, Man SF, Frohlich J,et al.Circμlating surfactant protein-D and therisk of cardiovascμlar morbidity and mortality. Eur Heart J.2011,32(15):1918-25
[28]熊忠良，汪宏才，赵海忠,等。浅谈几种实验动物在兽用生物制品试验研究中的应用。湖北畜牧兽医，2010，8：10-12
[29] Imre Kacskovics，Judit Cervenak，Anna Erdei，et al．Recent advances using FcRnoverexpression in transgenic animals to overcome impediments of standardantibody technologies to improve the generation of specific antibodies[J]．mAbs，2011，3(5)：431.439．
[30] Yongchen Guo，et al．Immunoglobμlin Genomics in the Guinea Pig (Caviaporcellus)[J]．PLoS ONE，2012，7(6)：1.18．
[31].He, S., et al., beta-cyclodextrins-based inclusion complexes of CoFe(2)O(4)magnetic nanoparticles as catalyst for the luminol chemiluminescence system andtheir applications in hydrogen peroxide detection. Talanta,2010.82(1):377-83.
[32].Wang, X., et al., A molecμlarly imprinted polymer-coated nanocomposite ofmagnetic nanoparticles for estrone recognition. Talanta,2009.78(2):327-32.
[33] JAMESON B A, WOLF H. The antigenic index: a novel algorithm for predicingantigenic determinants[J]. Comput Appl Biosci,1988,4(1):181.186.
[34] EMMINI E A, HUGHES J V, PERLOW D S, et al. Induction of hepatitis avirus-neutralizing antibody by a virus-specific synthetic peptide [J]. J Viorol,1985,55(3):836-839.
[35] Jiang Hu，Xiong Yong-hua，Xu Yang，et al．Preparation of Aflatoxin B1ArtificialAntigen by EDC Method[J]．Food Science，2005，26（7）：125-128．
[36] Quarrie SA,Galfre G.Use of different hapten-protein conjμgates immobilized onnitrocellμlose to screen monoclonal antibodies to abscisic acid[J].AnalyticalBiochem,1985,151:389
[37] Determann RM, Millo JL, Waddy S, Lutter R, Garrard CS, et al.(2009) PlasmaCC16levels are associated with development of ALI/ARDS in patients withventilator-associated pneumonia: A retrospective observational study. BMC PμlmMed9:49. doi:10.1186/1471.2466-9-49.
[38]丁淑琴,刘宏鹏,张爱君，等。生物信息学技术在细粒棘球蚴热休克蛋白70重组疫苗研究中的应用。宁夏医科大学学报，2009，31（1）：29-31
[39]胡纯秋，高金燕，罗春萍，等。花生过敏原Ara h2.02二级结构和B细胞抗原表位预测。食品科学。2009,30（21）：13.15
[40]秦斯民，余磊。固相多肽合成综述。职教与成教，
[41]李永振，贺继东，彭政，等。多肽的合成与应用进展。化学与生物工程，2010,27（4）：9-12.
[42]李正超，韩香。多肽合成方法研究进展。武警后勤学院学报（医学版），2013，22（2）：153.157.
[42] Kohler G, Milstein C. Continuous cμltures of fused cells secreting antibody ofPredefined specificity [J]. Nature,1975,256:495-497.
[43] Spieker-Polet H, Sethupathi P, Yam P C, et al. Rabbit monoclonal antibodies:Generating a fusion partner to produce rabbit-rabbit hybridomas[J]. Proc Natl AcadSci USA,1995,92:9348–9352.
[44] Couto J, Hendricks K, Wallace S E, et al. Methods for antibody engineering:WO/2006/050491[P].2006.
[45] Pilorz V， Jackel M，Trillmich F，et al．The cost of a specific immune responsein young guinea pig[J]．Physiology&Behavior,2005,85(2):205-211．
[46] Richard D.L，Robert S.C，Stephen G．High efficiency fusion procedure forproducing monoclonal antibodies against weak immunogens [J]．Methods inEnzymology,1986,121:183.192．
[47] Mcmaster W R，Williams A F．Identification of Ia glycoproteins in rat thymus andpurification from rat spleen[J].European Journal of Immunology,1979,9(6):426-433．
[48] E.哈洛D莱沈龚.抗体技术实验指南[M].科学出版社.2002:161.187.
[49]董志伟，王琰.抗体工程[M].北京医科大学出版社.2002;第2版:267-272.
[50] Padlan E A. A possible procedure for reducing the immunogenicity of antibodyvariable domains while preserving their ligand-binding properties[J]. Mol Immunol,1991,28:489–498.
[51] Padlan E A. Anatomy of the antibody molecμle[J]. Mol Immunol,1994,31:161–217.
[52] Wei, C., et al., New technology and clinical applications of nanomedicine:highlights of the second annual meeting of the American Academy ofNanomedicine (Part I). Nanomedicine,2006.2(4):253.63.
[53].Zuo, L, et al., New technology and clinical applications of nanomedicine. MedClin North Am,2007.91(5):845-62.
[54].Jeong, U., Teng, X., Wang, Y., et al, Superparamagnetic Colloids: ControlledSynthesis and Niche Applications. Adv. Mater.,2007.19(1):33.60.
[55].Perepichka, D.F., et al., A one-step synthesis of a poly(iptycene) throμgh anunusual Diels-Alder cyclization/dechlorination of tetrachloropentacene. J AmChem Soc,2003.125(34):10190-1.
[56].Gupta A.K,M. Gupta, Synthesis and surface engineering of iron oxidenanoparticles for biomedical applications. Biomaterials,2005.26(18):3995-4021.
[57].Li Z., W.L., Gao M. Y. and Lei H., One-Pot Reaction to SynthesizeBiocompatible Magnetite Nanoparticles.. Adv. Mater.,2005.17(8):1001.1005.
[58].Moroz, P,S.K. Jones, and B.N. Gray, Magnetically mediated hyperthermia: currentstatus and future directions. Int J Hyperthermia,2002.18(4):267-84.
[59].肖勤，林金明,化学发光免疫分析新进展.分析试验室,2011(01):111.122.
[60].Lu, A.H., E.L. Salabas, and F. Schuth, Magnetic nanoparticles: synthesis,protection,functionalization, and application. Angew Chem Int Ed Engl,2007,46(8):1222.44.
[61].Wang, X., et al., A molecμlarly imprinted polymer-coated nanocomposite ofmagnetic nanoparticles for estrone recognition. Talanta,2009.78(2):
[1]. Wei, C., et al., New technology and clinical applications of nanomedicine:highlights of the second annual meeting of the American Academy ofNanomedicine (Part I). Nanomedicine,2006.2(4): p.253.63.
[2]. Zuo, L., et al., New technology and clinical applications of nanomedicine. MedClin North Am,2007.91(5): p.845-62.
[3]. Jeong, U., Teng, X., Wang, Y., et al, Superparamagnetic Colloids: ControlledSynthesis and Niche Applications. Adv. Mater.,2007.19(1): p.33.60.
[4]. Gupta, A.K. and M. Gupta, Synthesis and surface engineering of iron oxidenanoparticles for biomedical applications. Biomaterials,2005.26(18): p.3995-4021.
[5]. Perepichka, D.F., et al., A one-step synthesis of a poly(iptycene) through anunusual Diels-Alder cyclization/dechlorination of tetrachloropentacene. J AmChem Soc,2003.125(34): p.10190-1.
[6]. Moroz, P., S.K. Jones, and B.N. Gray, Magnetically mediated hyperthermia:current status and future directions. Int J Hyperthermia,2002.18(4): p.267-84.
[7]. Li Z., W.L., Gao M.Y. and Lei H., One-Pot Reaction to SynthesizeBiocompatible Magnetite Nanoparticles.. Adv. Mater.,2005.17(8): p.1001.1005.
[8].肖勤等,化学发光免疫分析新进展.分析试验室,2011(01): p.111.122.
[9]. Lu, A.H., E.L. Salabas, and F. Schuth, Magnetic nanoparticles: synthesis,protection, functionalization, and application. Angew Chem Int Ed Engl,2007.46(8): p.1222.44.
[10].王士婷等, Fe3O4@m-SiO2磁性纳米颗粒的制备及其药物缓释行为.硅酸盐学报,2013(03): p.281.287.
[11].赵东方等.,聚丙烯酰胺凝胶法制备NiFe_2O_4纳米颗粒及性能.人工晶体学报,2013(02): p.316-321+350.
[12].唐宏志等., MgFe2O4纳米颗粒制备及其电磁性能研究.功能材料,2013(06).
[13].赵鹏等,蛋白改性纳米磁性颗粒的制备与性能研究.生物医学工程学杂志,2009(05): p.1005-1009.
[14].詹凤等,水热法制备纳米Fe_3O_4磁性颗粒.金属功能材料,2012(01): p.20-25.
[15].黄双等.,微乳液法制备纳米Fe_3O_4磁性颗粒的研究.武汉工程职业技术学院学报,2006(03): p.39-44.
[16]. Wang, Z., et al., In situ amplified chemiluminescent detection of DNA andimmunoassay of IgG using special-shaped gold nanoparticles as label. ClinChem,2006.52(10): p.1958-61.
[17]. Zhang, Z.F., et al., Gold nanoparticle-catalyzed luminol chemiluminescence andits analytical applications. Anal Chem,2005.77(10): p.3324-9.
[18]. Huang, X., et al., A resonance energy transfer between chemiluminescent donorsand luminescent quantum-dots as acceptors (CRET). Angew Chem Int Ed Engl,2006.45(31): p.5140-3.
[19]. Shi, H., et al., Determination of cortisol in human blood sera by a new Ag(III)complex-luminol chemiluminescent system. Anal Biochem,2009.387(2): p.178-83.
[20]. Bi, S., et al., Gold nanolabels for new enhanced chemiluminescenceimmunoassay of alpha-fetoprotein based on magnetic beads. Chemistry,2009.15(18): p.4704-9.
[21]. Previte, M.J., K. Aslan, and C.D. Geddes, Spatial and temporal control ofmicrowave triggered chemiluminescence: a protein detection platform. AnalChem,2007.79(18): p.7042.52.
[22]. He, S., et al., beta-cyclodextrins-based inclusion complexes of CoFe(2)O(4)magnetic nanoparticles as catalyst for the luminol chemiluminescence systemand their applications in hydrogen peroxide detection. Talanta,2010.82(1): p.377-83.
[23]. Chen, H., et al., Chemiluminescence of luminol catalyzed by silver nanoparticles.J Colloid Interface Sci,2007.315(1): p.158-63.
[24]. Xu, S.L. and H. Cui, Luminol chemiluminescence catalysed by colloidalplatinum nanoparticles. Luminescence,2007.22(2): p.77-87.
[25]. Zhan, W. and A.J. Bard, Electrogenerated chemiluminescence.83. Immunoassayof human C-reactive protein by using Ru(bpy)3(2+)-encapsulated liposomes aslabels. Anal Chem,2007.79(2): p.459-63.
[26]. Wang, X., et al., A molecularly imprinted polymer-coated nanocomposite ofmagnetic nanoparticles for estrone recognition. Talanta,2009.78(2): p.327-32.
[27]. Fu, Z., et al., A channel-resolved approach coupled with magnet-capturedtechnique for multianalyte chemiluminescent immunoassay. Biosens Bioelectron,2008.23(10): p.1422.8.
[28]. Fu, Z., et al., Channel-resolved multianalyte immunosensing system forflow-through chemiluminescent detection of alpha-fetoprotein andcarcinoembryonic antigen. Biosens Bioelectron,2008.23(7): p.1063.9.
[29]. Liu, H., et al., Sampling-resolution strategy for one-way multiplexedimmunoassay with sequential chemiluminescent detection. Anal Chem,2008.80(14): p.5654-9.
[30].郑国金等,管式磁性微粒子化学发光免疫分析法测定人尿液中的雌三醇.分析化学,2011(01): p.62.66.
[31].李智勇等,板式磁颗粒化学发光免疫分析在癌胚抗原测定中的应用.中国科学:化学,2010(05): p.594.
[32].李智勇等,板式磁颗粒化学发光免疫分析测定人血清中糖类抗原125.化学学报,2010(02): p.162.168.
[33].李智洋等,一种实用的基于化学发光和磁性纳米颗粒的E.coli O157:H7免疫鉴定方法.化学学报,2010(03): p.251.256.
[2] Francis X M, Jeffrey A W. The pμlmonary collectins, SP-A and SP-D, orchestrateinnate immunity in the lung[J]. J. Clin. Invest,2002,109:707-712.
[3]. Akella A, Deshpande SB.Pμlmonary surfactants and their role in pathophysiologyof lung disorders. Indian J Exp Biol.2013,51(1):5-22.
[4] Uday Kishore, Andrés López Bernal, Mohammed F. Kamran，et al. Surfactantproteins SP-A and SP-D in human health and disease[J]. Arch Immunol Ther Exp,2005,53:399–417.
[5] Hermans C, Bernard A. Lung Epithelium-specific Proteins [J]. Am J Respir CritCare Med,1999,159:646–678.
[6] Howard Clark, Nades Palaniyar, Peter Strong, et al. Surfactant Protein D ReducesAlveolar Macrophage Apoptosis In Vivo[J]. The Journal of Immunology,2002,169:2892–2899.
[7] Crouch E.C: Surfactant protein-D and pμlmonary host defense[J]. Respir Res,2000,1(2):93.108.
[8] Maria Q. G, Karina J R, Michael F. B,et al Lung Surfactant Protein D (SP-D)Response and Regμlation During Acute and Chronic Lung InjuryLung2013,February
[9] Francis X M, Jeffrey A W. The pμlmonary collectins, SP-A and SP-D, orchestrateinnate immunity in the lung [J]. J Clin Invest,2002,109:707-712.
[10] Rajesh K.G, Anita G..Surfactant Protein-D.Animal Lectins: Form, FunctionandClinical Applications,2012:527-550.
[11] Hillaire ML, Haagsman HP, et al.Pμlmonary Surfactant Protein D in First-LineInnate Defence against Influenza A Virus Infections. J Innate Immun.2013Feb5.
[12]. Sin DD, Pahlavan PS, Man SF (2008) Surfactant protein D: A lung specificbiomarker in COPD? Ther Adv Respir Dis2:65–74..
[13]. Greene KE, Wright JR, Steinberg KP, Ruzinski JT, Caldwell E, et al.(1999) Serialchanges in surfactant-associated proteins in lung and serum before and after onsetof ARDS. Am J Respir Crit Care Med160:1843–1850.
[14]. Lesur O, Langevin S, Berthiaume Y, Legare M, Skrobik Y, et al.(2006) Outcomevalue of clara cell protein in serum of patients with acute respiratory distresssyndrome. Intensive Care Med32:1167–1174.
[15]Kunitake R, Kuwano K, Yoshida K,et al.KL-6, surfactant protein A and D inbronchoalveolar lavage fluid from patients with pμlmonary sarcoidosis.Respiration.2001;68(5):488-95.
[16] Fujita M, Shannon JM, Ouchi H，et al. Serum surfactant protein D is increased inacute and chronic inflammation in mice. Cytokine.2005Jμl7;31(1):25-33.
[17] Ishikawa T, Kubota T, Abe H, et al.Surfactant Protein-D is more Usefμl than Krebsvon den Lungen6as a Marker for the Early Diagnosis of Interstitial Pneumonitisduring Pegylated Interferon Treatment for Chronic Hepatitis C.Hepatogastroenterology.2012,59(119):2260-3.
[18] Bowler RP. Surfactant protein D as a biomarker for chronic obstructive pμlmonarydisease. COPD.2012，9(6):651.3
[19] Takahashi H, Kuroki Y, Tanaka H, et al. Serum Levels of Surfactant Proteins A andD Are Usefμl Biomarkers for Interstitial Lung Disease in Patients with ProgressiveSystemic Sclerosis[J]. Am. J. Respir. Crit. Care Med.,2000,162:258-263.
[20] Ohnishi H, Hiwada K, Kohno N, et al. Comparative Study of KL-6, SurfactantProtein-A, Surfactant Protein D, and Monocyte Chemoattractant Protein–1asSerum Markers for Interstitial Lung Diseases[J]. Am. J. Respir. Crit. Care Med.,2002,165:378-381.
[21] Takahashi H, Kuroki Y, Abe S, et al. Serum Surfactant Proteins A and D asPrognostic Factors in Idiopathic Pμlmonary Fibrosis and Their Relationship toDisease Extent[J]. Am. J. Respir. Crit. Care Med.,2000,162:1109-1114.
[22] Kunitake R, Kuwano K, Yoshida K,et al.KL-6, surfactant protein A and D inbronchoalveolar lavage fluid from patients with pμlmonary sarcoidosis.Respiration.2001;68(5):488-95.
[23]刘萍,王世鑫,陈蕾,等.矽肺患者血清克拉拉细胞蛋白和表面活性蛋白D的改变.中华劳动卫生职业病杂志，2007,25(1):18-21;
[24] Bjerk SM, Baker JV, Emery S, et al.(2013) Biomarkers and Bacterial PneumoniaRisk in Patients with Treated HIV Infection: A Case-Control Study. PLoS ONE8(2): e56249
[25] Xie F, Wang X, Ding Z,et al.Serum surfactant protein D is associated with theprognosis in patients with chronic kidney disease. J Cardiovasc Med (Hagerstown).2013Jan9.
[26] Hiroyuki Y, Hiroshi S, Yoichi N, et al.Serum levels of surfactant protein D predictthe anti-tumor activity of gefitinib in patients with advanced non-small cell lungcancer。Cancer Chemotherapy and Pharmacology,2011,67(2):331.338.
[27] Hill J, Heslop C, Man SF, Frohlich J,et al.Circμlating surfactant protein-D and therisk of cardiovascμlar morbidity and mortality. Eur Heart J.2011,32(15):1918-25
[28]熊忠良，汪宏才，赵海忠,等。浅谈几种实验动物在兽用生物制品试验研究中的应用。湖北畜牧兽医，2010，8：10-12
[29] Imre Kacskovics，Judit Cervenak，Anna Erdei，et al．Recent advances using FcRnoverexpression in transgenic animals to overcome impediments of standardantibody technologies to improve the generation of specific antibodies[J]．mAbs，2011，3(5)：431.439．
[30] Yongchen Guo，et al．Immunoglobμlin Genomics in the Guinea Pig (Caviaporcellus)[J]．PLoS ONE，2012，7(6)：1.18．
[31].He, S., et al., beta-cyclodextrins-based inclusion complexes of CoFe(2)O(4)magnetic nanoparticles as catalyst for the luminol chemiluminescence system andtheir applications in hydrogen peroxide detection. Talanta,2010.82(1):377-83.
[32].Wang, X., et al., A molecμlarly imprinted polymer-coated nanocomposite ofmagnetic nanoparticles for estrone recognition. Talanta,2009.78(2):327-32.
[33] JAMESON B A, WOLF H. The antigenic index: a novel algorithm for predicingantigenic determinants[J]. Comput Appl Biosci,1988,4(1):181.186.
[34] EMMINI E A, HUGHES J V, PERLOW D S, et al. Induction of hepatitis avirus-neutralizing antibody by a virus-specific synthetic peptide [J]. J Viorol,1985,55(3):836-839.
[35] Jiang Hu，Xiong Yong-hua，Xu Yang，et al．Preparation of Aflatoxin B1ArtificialAntigen by EDC Method[J]．Food Science，2005，26（7）：125-128．
[36] Quarrie SA,Galfre G.Use of different hapten-protein conjμgates immobilized onnitrocellμlose to screen monoclonal antibodies to abscisic acid[J].AnalyticalBiochem,1985,151:389
[37] Determann RM, Millo JL, Waddy S, Lutter R, Garrard CS, et al.(2009) PlasmaCC16levels are associated with development of ALI/ARDS in patients withventilator-associated pneumonia: A retrospective observational study. BMC PμlmMed9:49. doi:10.1186/1471.2466-9-49.
[38]丁淑琴,刘宏鹏,张爱君，等。生物信息学技术在细粒棘球蚴热休克蛋白70重组疫苗研究中的应用。宁夏医科大学学报，2009，31（1）：29-31
[39]胡纯秋，高金燕，罗春萍，等。花生过敏原Ara h2.02二级结构和B细胞抗原表位预测。食品科学。2009,30（21）：13.15
[40]秦斯民，余磊。固相多肽合成综述。职教与成教，
[41]李永振，贺继东，彭政，等。多肽的合成与应用进展。化学与生物工程，2010,27（4）：9-12.
[42]李正超，韩香。多肽合成方法研究进展。武警后勤学院学报（医学版），2013，22（2）：153.157.
[42] Kohler G, Milstein C. Continuous cμltures of fused cells secreting antibody ofPredefined specificity [J]. Nature,1975,256:495-497.
[43] Spieker-Polet H, Sethupathi P, Yam P C, et al. Rabbit monoclonal antibodies:Generating a fusion partner to produce rabbit-rabbit hybridomas[J]. Proc Natl AcadSci USA,1995,92:9348–9352.
[44] Couto J, Hendricks K, Wallace S E, et al. Methods for antibody engineering:WO/2006/050491[P].2006.
[45] Pilorz V， Jackel M，Trillmich F，et al．The cost of a specific immune responsein young guinea pig[J]．Physiology&Behavior,2005,85(2):205-211．
[46] Richard D.L，Robert S.C，Stephen G．High efficiency fusion procedure forproducing monoclonal antibodies against weak immunogens [J]．Methods inEnzymology,1986,121:183.192．
[47] Mcmaster W R，Williams A F．Identification of Ia glycoproteins in rat thymus andpurification from rat spleen[J].European Journal of Immunology,1979,9(6):426-433．
[48] E.哈洛D莱沈龚.抗体技术实验指南[M].科学出版社.2002:161.187.
[49]董志伟，王琰.抗体工程[M].北京医科大学出版社.2002;第2版:267-272.
[50] Padlan E A. A possible procedure for reducing the immunogenicity of antibodyvariable domains while preserving their ligand-binding properties[J]. Mol Immunol,1991,28:489–498.
[51] Padlan E A. Anatomy of the antibody molecμle[J]. Mol Immunol,1994,31:161–217.
[52] Wei, C., et al., New technology and clinical applications of nanomedicine:highlights of the second annual meeting of the American Academy ofNanomedicine (Part I). Nanomedicine,2006.2(4):253.63.
[53].Zuo, L, et al., New technology and clinical applications of nanomedicine. MedClin North Am,2007.91(5):845-62.
[54].Jeong, U., Teng, X., Wang, Y., et al, Superparamagnetic Colloids: ControlledSynthesis and Niche Applications. Adv. Mater.,2007.19(1):33.60.
[55].Perepichka, D.F., et al., A one-step synthesis of a poly(iptycene) throμgh anunusual Diels-Alder cyclization/dechlorination of tetrachloropentacene. J AmChem Soc,2003.125(34):10190-1.
[56].Gupta A.K,M. Gupta, Synthesis and surface engineering of iron oxidenanoparticles for biomedical applications. Biomaterials,2005.26(18):3995-4021.
[57].Li Z., W.L., Gao M. Y. and Lei H., One-Pot Reaction to SynthesizeBiocompatible Magnetite Nanoparticles.. Adv. Mater.,2005.17(8):1001.1005.
[58].Moroz, P,S.K. Jones, and B.N. Gray, Magnetically mediated hyperthermia: currentstatus and future directions. Int J Hyperthermia,2002.18(4):267-84.
[59].肖勤，林金明,化学发光免疫分析新进展.分析试验室,2011(01):111.122.
[60].Lu, A.H., E.L. Salabas, and F. Schuth, Magnetic nanoparticles: synthesis,protection,functionalization, and application. Angew Chem Int Ed Engl,2007,46(8):1222.44.
[61].Wang, X., et al., A molecμlarly imprinted polymer-coated nanocomposite ofmagnetic nanoparticles for estrone recognition. Talanta,2009.78(2):
[1]. Wei, C., et al., New technology and clinical applications of nanomedicine:highlights of the second annual meeting of the American Academy ofNanomedicine (Part I). Nanomedicine,2006.2(4): p.253.63.
[2]. Zuo, L., et al., New technology and clinical applications of nanomedicine. MedClin North Am,2007.91(5): p.845-62.
[3]. Jeong, U., Teng, X., Wang, Y., et al, Superparamagnetic Colloids: ControlledSynthesis and Niche Applications. Adv. Mater.,2007.19(1): p.33.60.
[4]. Gupta, A.K. and M. Gupta, Synthesis and surface engineering of iron oxidenanoparticles for biomedical applications. Biomaterials,2005.26(18): p.3995-4021.
[5]. Perepichka, D.F., et al., A one-step synthesis of a poly(iptycene) through anunusual Diels-Alder cyclization/dechlorination of tetrachloropentacene. J AmChem Soc,2003.125(34): p.10190-1.
[6]. Moroz, P., S.K. Jones, and B.N. Gray, Magnetically mediated hyperthermia:current status and future directions. Int J Hyperthermia,2002.18(4): p.267-84.
[7]. Li Z., W.L., Gao M.Y. and Lei H., One-Pot Reaction to SynthesizeBiocompatible Magnetite Nanoparticles.. Adv. Mater.,2005.17(8): p.1001.1005.
[8].肖勤等,化学发光免疫分析新进展.分析试验室,2011(01): p.111.122.
[9]. Lu, A.H., E.L. Salabas, and F. Schuth, Magnetic nanoparticles: synthesis,protection, functionalization, and application. Angew Chem Int Ed Engl,2007.46(8): p.1222.44.
[10].王士婷等, Fe3O4@m-SiO2磁性纳米颗粒的制备及其药物缓释行为.硅酸盐学报,2013(03): p.281.287.
[11].赵东方等.,聚丙烯酰胺凝胶法制备NiFe_2O_4纳米颗粒及性能.人工晶体学报,2013(02): p.316-321+350.
[12].唐宏志等., MgFe2O4纳米颗粒制备及其电磁性能研究.功能材料,2013(06).
[13].赵鹏等,蛋白改性纳米磁性颗粒的制备与性能研究.生物医学工程学杂志,2009(05): p.1005-1009.
[14].詹凤等,水热法制备纳米Fe_3O_4磁性颗粒.金属功能材料,2012(01): p.20-25.
[15].黄双等.,微乳液法制备纳米Fe_3O_4磁性颗粒的研究.武汉工程职业技术学院学报,2006(03): p.39-44.
[16]. Wang, Z., et al., In situ amplified chemiluminescent detection of DNA andimmunoassay of IgG using special-shaped gold nanoparticles as label. ClinChem,2006.52(10): p.1958-61.
[17]. Zhang, Z.F., et al., Gold nanoparticle-catalyzed luminol chemiluminescence andits analytical applications. Anal Chem,2005.77(10): p.3324-9.
[18]. Huang, X., et al., A resonance energy transfer between chemiluminescent donorsand luminescent quantum-dots as acceptors (CRET). Angew Chem Int Ed Engl,2006.45(31): p.5140-3.
[19]. Shi, H., et al., Determination of cortisol in human blood sera by a new Ag(III)complex-luminol chemiluminescent system. Anal Biochem,2009.387(2): p.178-83.
[20]. Bi, S., et al., Gold nanolabels for new enhanced chemiluminescenceimmunoassay of alpha-fetoprotein based on magnetic beads. Chemistry,2009.15(18): p.4704-9.
[21]. Previte, M.J., K. Aslan, and C.D. Geddes, Spatial and temporal control ofmicrowave triggered chemiluminescence: a protein detection platform. AnalChem,2007.79(18): p.7042.52.
[22]. He, S., et al., beta-cyclodextrins-based inclusion complexes of CoFe(2)O(4)magnetic nanoparticles as catalyst for the luminol chemiluminescence systemand their applications in hydrogen peroxide detection. Talanta,2010.82(1): p.377-83.
[23]. Chen, H., et al., Chemiluminescence of luminol catalyzed by silver nanoparticles.J Colloid Interface Sci,2007.315(1): p.158-63.
[24]. Xu, S.L. and H. Cui, Luminol chemiluminescence catalysed by colloidalplatinum nanoparticles. Luminescence,2007.22(2): p.77-87.
[25]. Zhan, W. and A.J. Bard, Electrogenerated chemiluminescence.83. Immunoassayof human C-reactive protein by using Ru(bpy)3(2+)-encapsulated liposomes aslabels. Anal Chem,2007.79(2): p.459-63.
[26]. Wang, X., et al., A molecularly imprinted polymer-coated nanocomposite ofmagnetic nanoparticles for estrone recognition. Talanta,2009.78(2): p.327-32.
[27]. Fu, Z., et al., A channel-resolved approach coupled with magnet-capturedtechnique for multianalyte chemiluminescent immunoassay. Biosens Bioelectron,2008.23(10): p.1422.8.
[28]. Fu, Z., et al., Channel-resolved multianalyte immunosensing system forflow-through chemiluminescent detection of alpha-fetoprotein andcarcinoembryonic antigen. Biosens Bioelectron,2008.23(7): p.1063.9.
[29]. Liu, H., et al., Sampling-resolution strategy for one-way multiplexedimmunoassay with sequential chemiluminescent detection. Anal Chem,2008.80(14): p.5654-9.
[30].郑国金等,管式磁性微粒子化学发光免疫分析法测定人尿液中的雌三醇.分析化学,2011(01): p.62.66.
[31].李智勇等,板式磁颗粒化学发光免疫分析在癌胚抗原测定中的应用.中国科学:化学,2010(05): p.594.
[32].李智勇等,板式磁颗粒化学发光免疫分析测定人血清中糖类抗原125.化学学报,2010(02): p.162.168.
[33].李智洋等,一种实用的基于化学发光和磁性纳米颗粒的E.coli O157:H7免疫鉴定方法.化学学报,2010(03): p.251.256.
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