吖啶酯DMAE·NHS的合成及TSH和TT_4化学发光免疫分析的研究
摘要
在各种非放免疫分析方法中，化学发光免疫分析(CLIA)具有灵敏度高、检测范围宽、精密度好、标记物稳定、可实现全自动化、应用范围广等优点，CLIA是一种先进的免疫分析方法。以吖啶酯衍生物为发光标记物的CLIA的特点是：标记简单、标记物稳定、不需要催化剂、自然本底低、受外界干扰因素少、信噪比高、发光体系简单等。
目前国内尚未见有关DMAE·NHS的合成及其应用于CLIA的研究报导，本论文研究了吖啶酯DMAE·NHS的有机合成方法，DMAE·NHS的发光性能，DMAE·NHS及其与抗体偶联物的热稳定性，DMAE·NHS标记TSH单克隆抗体和链亲和素，TSH CLIA，TSH BAS-CLIA和TT_4 BAS-CLIA。
DMAE·NHS的合成是本论文的关键和难点，我们对文献方法进行改进：文献方法用氢氧化钠与3,5-二甲基-4-羟基苯甲酸反应制得3,5-甲基-4-羟基苯甲酸钠，再用3,5-二甲基-4-羟基苯甲酸钠与苄氯作用制备3,5-二甲基-4-羟基苯甲酸苄酯，我们用氢氧化钾代替氢氧化钠，使合成取得成功；在合成2',6'-二甲基-4'-(N-琥珀酰亚胺氧羰基)苯基-吖啶-9-甲酸酯时，文献方法对粗产品进行两次硅胶柱层析纯化，第一次柱层析用氯仿/乙酸乙酯(4：1，Ⅴ：Ⅴ)作溶剂和淋洗剂，第二次柱层析用己烷/丙酮(2：1，Ⅴ：Ⅴ)作溶剂和淋洗剂，按照文献方法得到的不是所需要的化合物，我们只进行第一次柱层析纯化，然后用己烷/丙酮(2：1，Ⅴ：Ⅴ)进行研磨，过滤，洗涤，除去溶于己烷/丙酮(2：1，Ⅴ：Ⅴ)的部分，得到所需要的产品。合成产物经IR、~1HNMR、MS和元素分析表征为所期望的化合物。
对DMAE·NHS发光性能的研究表明，DMAE·NHS的发光为闪光，0.4s时发光强度达到最大，发光半衰期约为0.9s，发光强度为6.11×10~(18)cps/mol。发光启动试剂的组成对其发光强度有影响，表面活性剂如CTAC，TritonX-100，Tween-20等能增强其发光。DMAE·NHS与抗体偶联物DMAE·NHS-Ab的发光性能与DMAE·NHS相似，DMAE·NHS与Ab的分子比为2.4时其发光强度为1.8×10~(19)cps/molAb。DMAE·NHS及DMAE·NHS-Ab具有良好的热稳定性，4℃或冰冻下，在pH7.0的PB缓冲液中，半年后它们的发光活性或免疫活性基本没有
中国原子能科学研究院博士学位论文
下降，室温或37’C下，由于水解而降低发光活性，它们的热稳定性随温度升高＿
而降低，随熔液pH值增大DMAE佣S的热稳定性降低，而DMAE棚S－Ah在
中性溶液中最稳定。
以 DMAE·NHS为发光标记物，研究建立了两种 TSH CLIA方法，均采用一
步夹心免疫分析法，在第一种方法中一株抗TSH单克隆抗体包被微孔板，另一
株标记DMAE·N’HS，方法的分析灵敏度为0．olmIUA，批内变异系数为
4．29巧．71％，批间变异系数为 4．36ed．14％，平均回收率为 98％，与 TSH IRMA法
的相关方程为 Y－0．20＋0．92X，相关系数为 0．993，与 Ciba Co讪ng TSH CLIA测
量值的相关方程为 Y—0．16＋0．92兄相关系数为 0．986。在第二种方法中，引入
生物素－链亲和素放大系统①AS），该方法用到的两株TSH单抗，一株包被微孔
板，另一株标记生物素，DMAE·NHS标记链亲和素，该方法亦具有良好的精密
性和准确性，与 TSH IRMA及 Ciba Co而flg TSH CLIA测量值呈明显相关，其分
析灵敏度较第一种方法高，为0刀07mIU／l，这两种方法之间亦相关良好。论文最
．后对TT CLIA方法进行了研究，并初步建立起T乙 BAS－CLIA方法，该方法
＿用T4单抗包被微孔板，T4ESA标记生物素，DMAE·NHS标记链亲和素，采用
8
。一步法竞争免疫分析。
，研究结果表明：DMAE·NHS具有良好的发光性能，易与抗体偶联，偶联物
稳定，发光体系简单，是一种理想的化学发光免疫分析标记物，以DMAE·NHS
做发光标记物建立的 TSH CLIA、TSH BAS－CLIA和 TT4 BAS－CLIA方法，标准
曲线线性好、信噪比高、检测范围宽、具有较高的灵敏度，良好的精密度、准确
性和健全性，与 IRMA方法及 Ciba Coming CLIA的临床测量值相关性良好。
Chemiluminescent immunoassay (CLIA) is one of the advanced immunoassay of non-radioisotopic immunoassay because of its high sensitivity,wide dynamic range,high accuracy,stable labeled protein,full automation and extensive application field. CLIA using acridinium ester derivatives as chemiluminescent label has advantages of low background,high signal-to-noise ratio,no need of catalyst and simple luminescence system.
Different aspects associating the DMAE-NHS-based CLIA were studied in this thesis,including synthesis of acridinium ester (DMAE-NHS),chemiluminescent characteristics of DMAE-NHS,labelling antibody or streptavidin with DMAE-NHS,two-site sandwich chemiluminescent immunoassay (CLIA) for TSH,two-site sandwich chemiluminescent immunoassay involved biotin-streptavidin system(BAS-CLIA) for TSH,and competitive chemiluminescent immunoassay using biotin-streptavidin system(B AS-CLIA) for TLV
The desired acridinium ester,DMAE-NHS,was synthesized according to the reference method with some modifications. The products were identified by IR,NMR,MS and elemental analysis. In our method,K.OH was used in place of NaOH to synthesize benzyl ester of 3,5-dimethyl-4-hydroxybenzoic acid,2',6'-dimethyl-4'-(n-succinimidyloxycarbonyl) phenyl-acridinium-9-carboxylate was purified on a silica gel column with chloroform/ethylacetate(4:l,v/v) as eluent and further purified by triturating with hexane/acetone(2:l,v/v).
The luminescence produced by DMAE-NHS is a flash light with maximum
emission at 0.4s and decay half-time of 0.9s. The luminescence intensity is 6.11x10 cps/mol,which is affected by the composition of trigger and surfactant. Chemiluminescent characteristics of labeled antibody are similar to the DMAE-NHS. Specific luminescence activity of the labeled antibody is l.SxlO1 cps/mol with an average incorporation ratio of 2.4 mole of DMAE-NHS per mole of antibody. The stability of DMAE-NHS and labeled antibody was tested under various pH values and
temperatures. In aqueous solution,the stability of DMAE-NHS decreases with the increase of pH value and temperature due to its decomposition by hydrolysis. The labeled antibody stored in neutral media is more stable than that in acidic or basic media. There is no significant loss of chemiluminescence and immunological activity of DMAE-NHS and labeled antibody stored in pH 7.0 phosphate buffer at 4C for six months.
A two-site sandwich CLIA for TSH and a two-site sandwich BAS-CLIA for TSH were developed. In the two-site sandwich CLIA for TSH,two anti-TSH monoclonal antibodies were involved,one was coated on microwells and another was labeled with acridinium ester (DMAE-NHS). The assay sensitivity is 0.01mlU/L. Intrassay and interassay coefficients of variation are 4.29-6.71 and 4.36-6.14%,respectively. Average recovery is 98%. Correlation coefficients and correlation equations of the assay with TSH IRMA and Ciba Corning are 0.993,0.986,Y=0.20 + 0.92X and Y=-0.16+0.92X,respectively. The two-site sandwich BAS-CLIA for TSH employs the same two monoclonal antibodies. Differently,one of the monoclonal antibodies was biotinylated instead of being labeled directly by DMAE-NHS,and DMAE-NHS labeled streptavidin was used as tracer. The assay sensitivity of the two-site sandwich BAS-CLIA for TSH was higher than the former.
A competitive CLIA for serum TTj was also developed. In this assay,the biotinylated T4-BSA conjugate competes with the T4 in the serum to bind the limited amount of anti-T4 McAb. The amount of the McAb-Ti-BSA-biotin complex was determined after a reaction with DMAE-NHS labeled streptavidin.
In conclusion,the DMAE-NHS has many advantageous performances in CLIA. The assay methods established for TSH and TT4 are efficient and reliable.
目前国内尚未见有关DMAE·NHS的合成及其应用于CLIA的研究报导，本论文研究了吖啶酯DMAE·NHS的有机合成方法，DMAE·NHS的发光性能，DMAE·NHS及其与抗体偶联物的热稳定性，DMAE·NHS标记TSH单克隆抗体和链亲和素，TSH CLIA，TSH BAS-CLIA和TT_4 BAS-CLIA。
DMAE·NHS的合成是本论文的关键和难点，我们对文献方法进行改进：文献方法用氢氧化钠与3,5-二甲基-4-羟基苯甲酸反应制得3,5-甲基-4-羟基苯甲酸钠，再用3,5-二甲基-4-羟基苯甲酸钠与苄氯作用制备3,5-二甲基-4-羟基苯甲酸苄酯，我们用氢氧化钾代替氢氧化钠，使合成取得成功；在合成2',6'-二甲基-4'-(N-琥珀酰亚胺氧羰基)苯基-吖啶-9-甲酸酯时，文献方法对粗产品进行两次硅胶柱层析纯化，第一次柱层析用氯仿/乙酸乙酯(4：1，Ⅴ：Ⅴ)作溶剂和淋洗剂，第二次柱层析用己烷/丙酮(2：1，Ⅴ：Ⅴ)作溶剂和淋洗剂，按照文献方法得到的不是所需要的化合物，我们只进行第一次柱层析纯化，然后用己烷/丙酮(2：1，Ⅴ：Ⅴ)进行研磨，过滤，洗涤，除去溶于己烷/丙酮(2：1，Ⅴ：Ⅴ)的部分，得到所需要的产品。合成产物经IR、~1HNMR、MS和元素分析表征为所期望的化合物。
对DMAE·NHS发光性能的研究表明，DMAE·NHS的发光为闪光，0.4s时发光强度达到最大，发光半衰期约为0.9s，发光强度为6.11×10~(18)cps/mol。发光启动试剂的组成对其发光强度有影响，表面活性剂如CTAC，TritonX-100，Tween-20等能增强其发光。DMAE·NHS与抗体偶联物DMAE·NHS-Ab的发光性能与DMAE·NHS相似，DMAE·NHS与Ab的分子比为2.4时其发光强度为1.8×10~(19)cps/molAb。DMAE·NHS及DMAE·NHS-Ab具有良好的热稳定性，4℃或冰冻下，在pH7.0的PB缓冲液中，半年后它们的发光活性或免疫活性基本没有
中国原子能科学研究院博士学位论文
下降，室温或37’C下，由于水解而降低发光活性，它们的热稳定性随温度升高＿
而降低，随熔液pH值增大DMAE佣S的热稳定性降低，而DMAE棚S－Ah在
中性溶液中最稳定。
以 DMAE·NHS为发光标记物，研究建立了两种 TSH CLIA方法，均采用一
步夹心免疫分析法，在第一种方法中一株抗TSH单克隆抗体包被微孔板，另一
株标记DMAE·N’HS，方法的分析灵敏度为0．olmIUA，批内变异系数为
4．29巧．71％，批间变异系数为 4．36ed．14％，平均回收率为 98％，与 TSH IRMA法
的相关方程为 Y－0．20＋0．92X，相关系数为 0．993，与 Ciba Co讪ng TSH CLIA测
量值的相关方程为 Y—0．16＋0．92兄相关系数为 0．986。在第二种方法中，引入
生物素－链亲和素放大系统①AS），该方法用到的两株TSH单抗，一株包被微孔
板，另一株标记生物素，DMAE·NHS标记链亲和素，该方法亦具有良好的精密
性和准确性，与 TSH IRMA及 Ciba Co而flg TSH CLIA测量值呈明显相关，其分
析灵敏度较第一种方法高，为0刀07mIU／l，这两种方法之间亦相关良好。论文最
．后对TT CLIA方法进行了研究，并初步建立起T乙 BAS－CLIA方法，该方法
＿用T4单抗包被微孔板，T4ESA标记生物素，DMAE·NHS标记链亲和素，采用
8
。一步法竞争免疫分析。
，研究结果表明：DMAE·NHS具有良好的发光性能，易与抗体偶联，偶联物
稳定，发光体系简单，是一种理想的化学发光免疫分析标记物，以DMAE·NHS
做发光标记物建立的 TSH CLIA、TSH BAS－CLIA和 TT4 BAS－CLIA方法，标准
曲线线性好、信噪比高、检测范围宽、具有较高的灵敏度，良好的精密度、准确
性和健全性，与 IRMA方法及 Ciba Coming CLIA的临床测量值相关性良好。
Chemiluminescent immunoassay (CLIA) is one of the advanced immunoassay of non-radioisotopic immunoassay because of its high sensitivity,wide dynamic range,high accuracy,stable labeled protein,full automation and extensive application field. CLIA using acridinium ester derivatives as chemiluminescent label has advantages of low background,high signal-to-noise ratio,no need of catalyst and simple luminescence system.
Different aspects associating the DMAE-NHS-based CLIA were studied in this thesis,including synthesis of acridinium ester (DMAE-NHS),chemiluminescent characteristics of DMAE-NHS,labelling antibody or streptavidin with DMAE-NHS,two-site sandwich chemiluminescent immunoassay (CLIA) for TSH,two-site sandwich chemiluminescent immunoassay involved biotin-streptavidin system(BAS-CLIA) for TSH,and competitive chemiluminescent immunoassay using biotin-streptavidin system(B AS-CLIA) for TLV
The desired acridinium ester,DMAE-NHS,was synthesized according to the reference method with some modifications. The products were identified by IR,NMR,MS and elemental analysis. In our method,K.OH was used in place of NaOH to synthesize benzyl ester of 3,5-dimethyl-4-hydroxybenzoic acid,2',6'-dimethyl-4'-(n-succinimidyloxycarbonyl) phenyl-acridinium-9-carboxylate was purified on a silica gel column with chloroform/ethylacetate(4:l,v/v) as eluent and further purified by triturating with hexane/acetone(2:l,v/v).
The luminescence produced by DMAE-NHS is a flash light with maximum
emission at 0.4s and decay half-time of 0.9s. The luminescence intensity is 6.11x10 cps/mol,which is affected by the composition of trigger and surfactant. Chemiluminescent characteristics of labeled antibody are similar to the DMAE-NHS. Specific luminescence activity of the labeled antibody is l.SxlO1 cps/mol with an average incorporation ratio of 2.4 mole of DMAE-NHS per mole of antibody. The stability of DMAE-NHS and labeled antibody was tested under various pH values and
temperatures. In aqueous solution,the stability of DMAE-NHS decreases with the increase of pH value and temperature due to its decomposition by hydrolysis. The labeled antibody stored in neutral media is more stable than that in acidic or basic media. There is no significant loss of chemiluminescence and immunological activity of DMAE-NHS and labeled antibody stored in pH 7.0 phosphate buffer at 4C for six months.
A two-site sandwich CLIA for TSH and a two-site sandwich BAS-CLIA for TSH were developed. In the two-site sandwich CLIA for TSH,two anti-TSH monoclonal antibodies were involved,one was coated on microwells and another was labeled with acridinium ester (DMAE-NHS). The assay sensitivity is 0.01mlU/L. Intrassay and interassay coefficients of variation are 4.29-6.71 and 4.36-6.14%,respectively. Average recovery is 98%. Correlation coefficients and correlation equations of the assay with TSH IRMA and Ciba Corning are 0.993,0.986,Y=0.20 + 0.92X and Y=-0.16+0.92X,respectively. The two-site sandwich BAS-CLIA for TSH employs the same two monoclonal antibodies. Differently,one of the monoclonal antibodies was biotinylated instead of being labeled directly by DMAE-NHS,and DMAE-NHS labeled streptavidin was used as tracer. The assay sensitivity of the two-site sandwich BAS-CLIA for TSH was higher than the former.
A competitive CLIA for serum TTj was also developed. In this assay,the biotinylated T4-BSA conjugate competes with the T4 in the serum to bind the limited amount of anti-T4 McAb. The amount of the McAb-Ti-BSA-biotin complex was determined after a reaction with DMAE-NHS labeled streptavidin.
In conclusion,the DMAE-NHS has many advantageous performances in CLIA. The assay methods established for TSH and TT4 are efficient and reliable.
引文
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45. Ian W, Anthony K C J. Stuaet W. Two-site immunochemiluminometric assay for Human α_1-Fetoprotein[J]. Clin Chem, 1983,29(8):1480-1483.
46. George G K, Carol M P, Patricia G S. Multesite immunochemiluminometric assay for simultaneously measuring whole-molecule and amino-terminal fragments of human parathyrin[J]. Clin Chem, 1992,38(5):628-635.
47. Pal C K, Lawrenece R, Patricia G S. Development and evaluation of an osteocalcin chemiluminoimmunoassay[J]. Clin Chem, 1993,39(7): 1369-1374.
48. Roelant C, Vande V. Chemiluminescencent compositions,chemiluminescent process and their uses in ananlytical assays. European Patent Application, 1991,408 463 A1.
49. Zomer G, Stavenuter J F. Acridinium compounds as chemiluminogenic label. European Patent Application ,1989, 324 202.
50. Mattingly P G, Bennett L G. Chemiluminescencent acridinium and phenantridinium salts. European Patent Application, 1988, 273 115.
51. Godwin O, Patrick S C, Peter C, Roger JT. A competitive Chemiluminescencent immunoassay for the sensitive measurement of apolipoprotein B100[J]. Clinical Biochemistry, 1995,28(2):117-122.
52. Josefina M B, Neus G R. Evaluation of Ciba Corning ACS:180~(TM) Automated Immunoassay System[J]. Clin Chem, 1994,40(3):407-410.
53. Hiroshi S, Hiroshi M, Yuki T, Toshinori K. Enhancement of the sensitivity of a chemiluminescencent immunoassay for estradiol based on hapten heterology[J].Clinical Biochemistry, 1996,29:509-513.
54. Law S J, Chang S C S, Palmacci S A. Polysubstituted aryl acredinium esters. US Patent, 1988,745,181.
55. Krouwer J S, Rabinowitz R. How to improveestimates of imprecision[J]. Clin Chem, 1984,30:290-292.
56. Russell C H, Laurence R T. The use of acridinium ester-labelled streptavidin in immunoassays[J]. J. Immunological Methods, 1987,101:91-96.
57. Littig J S, Nieman T A. Quantitation of acridinium esters using electrogenerated chemiluminescence and flow injection[J]. Anal.Chem,1992,64:1140-1144.
58. Littig J S, Nieman T A. Flow injection chemiluminescence study of acridinium ester stability and kinetics of decomposition[J]. J. Biolumin Chemilumin,1993,8:25-31.
59. Zia R, Frank M. Stable and versatile active acridinium ester I[J]. Luminescence, 2000,15:239-244.
60. Steijger O M, Kamminga D A, Lingeman H, Brinkman U A T. An acridinium sulphonylamide as a new chemiluminescent label for the determination of carboxylic acids in liquid chromatography[J]. J. Biolumin Chemilumin, 1998,13:31-40.
61. Javier F, Luis J. Effect of surfactants on the intensity of chemiluminescence emission from acridinium ester labeled proteins[J]. J. Biolumin Chemilumin,1988,2:121-128.
62. Bronstein JCV, Thorpe GHG, Kricka L J, Armstrong G. Chemiluminescencent assay of alkaline phosphatase applied in an ultrasensitive enzyme immunoassay of thyrotropin[J]. Clin Chem,1989,35:1441-1446.
63. Irena Y B, Newton M. Method of detecting a substance using enzymatically-induced decomposition of dixoetanes. United States Patent, 1990, 4,978,614.
64. Urdea M S, Warner B D. Chemiluminescencent doubletriggered 1,2-dioxetanes. European Patent Application,1990, 401 001.
65. Irena B, Brooks E, John C V. 1,2-Dioxeanes:novel chemiluminescencent enzyme substrates.Applications to immunoassays[J]. J Biolumin Chemilumin, 1989,4:99-111.
66. Mitsuko K, Masako M, Akio T. Anew highly sensitive chemiluminescencent of alkaline phosphatase using lucigenin and its application to enzyme immunoassays[J]. J Biolumin Chemilumin, 1995,10:1-7.
67. Thorpe G H G; Bronstein I., Kricka L J, Edwards B. Chemiluminescencent enzyme immunoassays of α-fetoprotein based on an adamantyl dioxetane phenyl phosphate substrate[J]. Clin Chem, 1989,35:2319-2321.
68. Paul S, Hashem A, Louis J. Chemiluminescencent substrates for alkaline phosphatase:application to ultrasensitive enzyme-linked immunoassays and DNA probes[J]. Clin Chem, 1989,35:1863-1864.
69. Hidehido S, Masako M, Akio T. Chemiluminescencent assay of alkaline phosphatase using phenacyl phosphate[J]. Analytica Chimica Acta,1995,306:161-166.
70. Deboever J, Mares A, Stans G, et al. Comparison of chemiluminescencent and chromogenic substrates of alkaline phosphatase in a direct immunoassays for plasma estradiol[J]. Analytica Chimica Acta 1995;303:143-148.
71. Wood W G Spiroadamantane dioxetane substrates stable label for luminescence-enhaced enzyme immunoassays[J]. J.Clin Chem,Clin Biochem 1990, 28:481-482.
72. Nicbolas R H, Eckert B, Kraiss S. Electrochimiluminescence: leading-edge technology for automated immunoassays analyte detection[J]. Clin Chem, 1996,42:1575-1578.
73. Marta S C, Antonia E, Virtudes C. New Electrochimiluminescence immunoassays for the determination of cyfra 21-1:analytical evaluation and clinical diagnostic performance in urin samples of patients with bladder cancer[J]. Clin Chem, 1999,45:1944-1953.
74. Xi C, Masanori S, Yongjing L. Study of the Electrochimiluminescence based on tri(2,2'-bipyridine)ruthenium(Ⅱ)and alcohols in a flow injection system[J]. J Microchemical, 1998,58:13-20.
75. Loranelle LS, Timothy A N. Stopped-flow analysis of Ru(bpy)_3~(3+) chimiluminescence reations[J]. J Biolumin Chemilumin,1998,13;85-90.
76. Gary F B, Haresh P S, John H K, et al. Electrochemiluminescence Detection for Development of Immunoassays and DNA Probe Assays for Clinical Diagnostics[J]. Clin. Chem, 1991,37(1534-1539.
77. Jonathan K L, Michael J P. Electrogenerated Chemiluminescence: An Oxidative-Reduction Type ECL Reaction Sequence Using Tripropyl Amine[J]. J. Electrchem. Soc,1990,137:3127-3131.
78. Daniel R D. A new non-isotopic detection system for immunoassays[J]. Nature, 1995, 377:758-760.
79. Kenten J H, Gudibande S, Link J, Willey J J, et al. Improved electrochemiluminescent label for DNA probe assays: rapid quantitative assays of HIV- 1 polymerase chain reaction products[J]. Clim. Chem,1992,38:873-879.
80. Peter W, Darcy L, Dennis M, Katby H. Immunoanalyzer with concurrent radom access to chemiluminescence and nephelometric assays[J]. Clim.Chem, 1996,42:1575-1578.
81. Takashi L, Masatoshi Y, Koichi S,Yoshihiro S, Yukil S. The developmint of luminomaster~(TM),a fully automated chimiluminescen enzyme immunoassays system[J]. J Biolumin Chemilumin, 1995,10:219-27.
82. Alain B,Vincent F, Joel A. Application of a long-term enhanced xanthine oxidase induced luminescence in solid-phase immunoassays[J]. Analytical Biochemistry, 1990,187:20-26.
83. Baret V F. T_4 and ultrasensitive TSH immunoassays using luminescent enhanced xanthine oxidase assay[J]. J Biolumin Chemilumin, 1989,4:149-153.
84．贺佑丰，王玉肖，王衍真等译．放射免疫分析与相关技术进展[A]．北京：原子能出版社，1991．397-400．
85. Hua C, Shifeng L, Feng L, Yugang S, Xiangqin L. A novel chemiluminescent method for the determination of salicylic acid in bactericidal solution[J]. Anal Bioanal Chem, 2002,372:601-604.
86. Zheng-Hua S, Shuang H. Chemiluminescence assay for uric acid in human serum and urine using flow-injection with immunobilized reagents technology[J]. Anal Bioanal Chem, 2002, 372:327-332.
87. Ian W, Iraj B, Frank M C. Acridinium ester as high-specficactivity labels in immunoassay[J]. Clin Chem, 1983,29:1474-1479.
88. Knwaguchi M, Suzuki N. Stabilized phenyl acridinium esters for chemiluminescent immunoassay. Bioluiminescence and Chemiluminescence [M]. Simonoseki.Japan:1983.482-484.
89. Javier F, Luis J. Effect of surfactants on the intensity of chemiluminescence emission from acridinium ester labeled proteins[J]. J. Biolum Chemilum, 1988,2:121-128.
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44. Ian W, Paul M, Anthony K C. Measurement of C9 concentrations using an immunochemiluminometric assay[J]. J Immunological Methods, 1985,80:33-38.
45. Ian W, Anthony K C J. Stuaet W. Two-site immunochemiluminometric assay for Human α_1-Fetoprotein[J]. Clin Chem, 1983,29(8):1480-1483.
46. George G K, Carol M P, Patricia G S. Multesite immunochemiluminometric assay for simultaneously measuring whole-molecule and amino-terminal fragments of human parathyrin[J]. Clin Chem, 1992,38(5):628-635.
47. Pal C K, Lawrenece R, Patricia G S. Development and evaluation of an osteocalcin chemiluminoimmunoassay[J]. Clin Chem, 1993,39(7): 1369-1374.
48. Roelant C, Vande V. Chemiluminescencent compositions,chemiluminescent process and their uses in ananlytical assays. European Patent Application, 1991,408 463 A1.
49. Zomer G, Stavenuter J F. Acridinium compounds as chemiluminogenic label. European Patent Application ,1989, 324 202.
50. Mattingly P G, Bennett L G. Chemiluminescencent acridinium and phenantridinium salts. European Patent Application, 1988, 273 115.
51. Godwin O, Patrick S C, Peter C, Roger JT. A competitive Chemiluminescencent immunoassay for the sensitive measurement of apolipoprotein B100[J]. Clinical Biochemistry, 1995,28(2):117-122.
52. Josefina M B, Neus G R. Evaluation of Ciba Corning ACS:180~(TM) Automated Immunoassay System[J]. Clin Chem, 1994,40(3):407-410.
53. Hiroshi S, Hiroshi M, Yuki T, Toshinori K. Enhancement of the sensitivity of a chemiluminescencent immunoassay for estradiol based on hapten heterology[J].Clinical Biochemistry, 1996,29:509-513.
54. Law S J, Chang S C S, Palmacci S A. Polysubstituted aryl acredinium esters. US Patent, 1988,745,181.
55. Krouwer J S, Rabinowitz R. How to improveestimates of imprecision[J]. Clin Chem, 1984,30:290-292.
56. Russell C H, Laurence R T. The use of acridinium ester-labelled streptavidin in immunoassays[J]. J. Immunological Methods, 1987,101:91-96.
57. Littig J S, Nieman T A. Quantitation of acridinium esters using electrogenerated chemiluminescence and flow injection[J]. Anal.Chem,1992,64:1140-1144.
58. Littig J S, Nieman T A. Flow injection chemiluminescence study of acridinium ester stability and kinetics of decomposition[J]. J. Biolumin Chemilumin,1993,8:25-31.
59. Zia R, Frank M. Stable and versatile active acridinium ester I[J]. Luminescence, 2000,15:239-244.
60. Steijger O M, Kamminga D A, Lingeman H, Brinkman U A T. An acridinium sulphonylamide as a new chemiluminescent label for the determination of carboxylic acids in liquid chromatography[J]. J. Biolumin Chemilumin, 1998,13:31-40.
61. Javier F, Luis J. Effect of surfactants on the intensity of chemiluminescence emission from acridinium ester labeled proteins[J]. J. Biolumin Chemilumin,1988,2:121-128.
62. Bronstein JCV, Thorpe GHG, Kricka L J, Armstrong G. Chemiluminescencent assay of alkaline phosphatase applied in an ultrasensitive enzyme immunoassay of thyrotropin[J]. Clin Chem,1989,35:1441-1446.
63. Irena Y B, Newton M. Method of detecting a substance using enzymatically-induced decomposition of dixoetanes. United States Patent, 1990, 4,978,614.
64. Urdea M S, Warner B D. Chemiluminescencent doubletriggered 1,2-dioxetanes. European Patent Application,1990, 401 001.
65. Irena B, Brooks E, John C V. 1,2-Dioxeanes:novel chemiluminescencent enzyme substrates.Applications to immunoassays[J]. J Biolumin Chemilumin, 1989,4:99-111.
66. Mitsuko K, Masako M, Akio T. Anew highly sensitive chemiluminescencent of alkaline phosphatase using lucigenin and its application to enzyme immunoassays[J]. J Biolumin Chemilumin, 1995,10:1-7.
67. Thorpe G H G; Bronstein I., Kricka L J, Edwards B. Chemiluminescencent enzyme immunoassays of α-fetoprotein based on an adamantyl dioxetane phenyl phosphate substrate[J]. Clin Chem, 1989,35:2319-2321.
68. Paul S, Hashem A, Louis J. Chemiluminescencent substrates for alkaline phosphatase:application to ultrasensitive enzyme-linked immunoassays and DNA probes[J]. Clin Chem, 1989,35:1863-1864.
69. Hidehido S, Masako M, Akio T. Chemiluminescencent assay of alkaline phosphatase using phenacyl phosphate[J]. Analytica Chimica Acta,1995,306:161-166.
70. Deboever J, Mares A, Stans G, et al. Comparison of chemiluminescencent and chromogenic substrates of alkaline phosphatase in a direct immunoassays for plasma estradiol[J]. Analytica Chimica Acta 1995;303:143-148.
71. Wood W G Spiroadamantane dioxetane substrates stable label for luminescence-enhaced enzyme immunoassays[J]. J.Clin Chem,Clin Biochem 1990, 28:481-482.
72. Nicbolas R H, Eckert B, Kraiss S. Electrochimiluminescence: leading-edge technology for automated immunoassays analyte detection[J]. Clin Chem, 1996,42:1575-1578.
73. Marta S C, Antonia E, Virtudes C. New Electrochimiluminescence immunoassays for the determination of cyfra 21-1:analytical evaluation and clinical diagnostic performance in urin samples of patients with bladder cancer[J]. Clin Chem, 1999,45:1944-1953.
74. Xi C, Masanori S, Yongjing L. Study of the Electrochimiluminescence based on tri(2,2'-bipyridine)ruthenium(Ⅱ)and alcohols in a flow injection system[J]. J Microchemical, 1998,58:13-20.
75. Loranelle LS, Timothy A N. Stopped-flow analysis of Ru(bpy)_3~(3+) chimiluminescence reations[J]. J Biolumin Chemilumin,1998,13;85-90.
76. Gary F B, Haresh P S, John H K, et al. Electrochemiluminescence Detection for Development of Immunoassays and DNA Probe Assays for Clinical Diagnostics[J]. Clin. Chem, 1991,37(1534-1539.
77. Jonathan K L, Michael J P. Electrogenerated Chemiluminescence: An Oxidative-Reduction Type ECL Reaction Sequence Using Tripropyl Amine[J]. J. Electrchem. Soc,1990,137:3127-3131.
78. Daniel R D. A new non-isotopic detection system for immunoassays[J]. Nature, 1995, 377:758-760.
79. Kenten J H, Gudibande S, Link J, Willey J J, et al. Improved electrochemiluminescent label for DNA probe assays: rapid quantitative assays of HIV- 1 polymerase chain reaction products[J]. Clim. Chem,1992,38:873-879.
80. Peter W, Darcy L, Dennis M, Katby H. Immunoanalyzer with concurrent radom access to chemiluminescence and nephelometric assays[J]. Clim.Chem, 1996,42:1575-1578.
81. Takashi L, Masatoshi Y, Koichi S,Yoshihiro S, Yukil S. The developmint of luminomaster~(TM),a fully automated chimiluminescen enzyme immunoassays system[J]. J Biolumin Chemilumin, 1995,10:219-27.
82. Alain B,Vincent F, Joel A. Application of a long-term enhanced xanthine oxidase induced luminescence in solid-phase immunoassays[J]. Analytical Biochemistry, 1990,187:20-26.
83. Baret V F. T_4 and ultrasensitive TSH immunoassays using luminescent enhanced xanthine oxidase assay[J]. J Biolumin Chemilumin, 1989,4:149-153.
84．贺佑丰，王玉肖，王衍真等译．放射免疫分析与相关技术进展[A]．北京：原子能出版社，1991．397-400．
85. Hua C, Shifeng L, Feng L, Yugang S, Xiangqin L. A novel chemiluminescent method for the determination of salicylic acid in bactericidal solution[J]. Anal Bioanal Chem, 2002,372:601-604.
86. Zheng-Hua S, Shuang H. Chemiluminescence assay for uric acid in human serum and urine using flow-injection with immunobilized reagents technology[J]. Anal Bioanal Chem, 2002, 372:327-332.
87. Ian W, Iraj B, Frank M C. Acridinium ester as high-specficactivity labels in immunoassay[J]. Clin Chem, 1983,29:1474-1479.
88. Knwaguchi M, Suzuki N. Stabilized phenyl acridinium esters for chemiluminescent immunoassay. Bioluiminescence and Chemiluminescence [M]. Simonoseki.Japan:1983.482-484.
89. Javier F, Luis J. Effect of surfactants on the intensity of chemiluminescence emission from acridinium ester labeled proteins[J]. J. Biolum Chemilum, 1988,2:121-128.