WO2007121668A1

WO2007121668A1 - Direct chemiluminescent reagent for magnetic separation and immunoassay method using thereof

Info

Publication number: WO2007121668A1
Application number: PCT/CN2007/001295
Authority: WO
Inventors: Wei Rao; Hongtao Song; Tinghua Li; Jia Shao; Ying Yan
Original assignee: Shenzhen New Industries Biomedical Engineering Co., Ltd.
Priority date: 2006-04-21
Filing date: 2007-04-20
Publication date: 2007-11-01
Also published as: CN100464189C; CN1888901A

Abstract

A direct chemiluminescent reagent for magnetic separation and immunoassay method using thereof, comprises an isoluminol derivative as a luminescent label, immuno-magnetic nano-beads coated with goat anti-FITC polyclonal antibodies as separating agents, NaOH and H2O2 as chemiluminescent substrates. The reagent is stable and undecomposable, which won't be influenced by environmental factors. The immunoassay method is rapid with high accuracy.

Description

磁分离直接化学发光试剂及其免疫测定方法技术领域本发明涉及一种磁分离直接化学发光试剂及采用该试剂的测试方法，特别是釆用异鲁米诺衍生物分作为标记物、纳米磁性微珠作为分离材料的试剂。背景技术现代临床医学技术的飞速发展，要求对越来越多人血或尿液中的微量活性物质进行精确定量测定，为临床医生诊断疾病，制定有效的治疗方案，治疗效果评价提供准确的依据，目前，实现人血清或尿液中微量活性物质精确定量分析的方法主要有放射免疫分析方法、酶免疫分析方法、酶促化学发光或吖啶酯标记的直接化学发光方法及三联吡啶钌标记的电化学发光方法。 BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a magnetic separation direct chemiluminescence reagent and a test method using the same, in particular, an isoluminol derivative is used as a label, nano magnetic micro Beads are used as reagents for separating materials. BACKGROUND OF THE INVENTION The rapid development of modern clinical medical technology requires accurate quantitative determination of a large number of human active blood or urine active substances, providing an effective treatment plan for clinicians to diagnose diseases, and providing an accurate basis for evaluation of therapeutic effects. At present, methods for accurate quantitative analysis of trace active substances in human serum or urine mainly include radioimmunoassay methods, enzyme immunoassay methods, direct chemiluminescence methods for enzymatic chemiluminescence or acridinium ester labeling, and pyridinium-labeled Electrochemiluminescence method.

放射免疫分析法是采用放射性同位素 I¹²⁵作为示踪剂，标记在抗原或相应抗体上，在 γ射线探测仪上，通过对 ⁵放射线强度的测定，来确定待测物质的含量，这种方法虽然较好地解决了微量活性物质的定量分析目的，但其存在的缺陷是很明显的：放射性同位素 I¹²⁵作为示踪技术，在试剂的制造、贮存、应用操作过程中，对环境带来污染，给人体造成了严重的伤害；同时，由于 ²⁵半衰期的限制，决定了放射免疫分析试剂的有效期只有一个月；放免试剂的有效期也只有一个月；分离过程的非特异性导致结果的准确率差，还不能做急诊标本。 Radioimmunoassay uses a radioisotope I ¹²⁵ as a tracer, labeled on an antigen or a corresponding antibody, and determines the content of the substance to be tested by measuring the intensity of ⁵ radiation on a gamma ray detector. The purpose of quantitative analysis of trace active substances is better solved, but the defects are obvious: The radioactive isotope I ^{125 is} used as a tracer technology to pollute the environment during the manufacture, storage and application of reagents. It causes serious harm to the human body; at the same time, due to the limitation of ²⁵ half-life, it is determined that the radioimmunoassay reagent is only valid for one month; the exemption reagent is only valid for one month; the non-specificity of the separation process leads to poor accuracy of the result, Can not do emergency specimens.

酶免疫分析方法是采用 HRP辣根过氧化物酶作为发光标记物，塑料微孔板作为载体和分离技术， 0PD邻苯二胺等作为显色底物， 1M¾S0₄作为酶显色反应的终止溶液，通过测定酶反应产物波长在 492rai处的吸光度值来分析待测物质的浓度，这种方法主要用来进行大批量人血清标本的定性筛査，其缺陷是显色产物的颜色强度随时间的变化而变化，不能固定不变，显色底物 0PD等是强致癌物，同时对相当一部分微量激章的灵敏度不够，反应时间太长，操作过程中难确认本以规范化。 Enzyme immunoassay method uses HRP horseradish peroxidase as luminescent marker, plastic microplate as carrier and separation technology, 0PD o-phenylenediamine as chromogenic substrate, 1M3⁄4S0 ₄ as stop solution for enzyme color reaction The concentration of the substance to be tested is analyzed by measuring the absorbance value of the enzyme reaction product at a wavelength of 492 rai. This method is mainly used for qualitative screening of large-scale human serum samples, and the defect is that the color intensity of the color-developing product is with time. Change and change, can not be fixed, chromogenic substrate 0PD, etc. are strong carcinogens, and the sensitivity of a considerable part of the micro-skills is not enough, the reaction time is too long, it is difficult to confirm the operation To standardize.

酶促化学发光免疫分析方法是采用碱性磷酸酶 AKP或辣根酶 HRP作为标记物，用 AMPPD (金刚烷）或鲁米诺、 ¾0₂作为发光底物；其优点是待测物转变成可测定的光信号，显著提高了分析的灵敏度，其缺点是影响酶活性的诸多因素，如环境温度、温育温度和时间、保存条件等有将直接影响测定结果，试剂的稳定性不好；同时，该方法发光反应启动缓慢，需要在 37°C温育一定的时间才能达到发光平台，会更强地影响发光测定的结果。 The enzymatic chemiluminescence immunoassay method uses alkaline phosphatase AKP or horseradish HRP as a label, and AMPPD (adamantane) or luminol, 3⁄40 ₂ as a luminescent substrate; the advantage is that the analyte is converted into a The measured optical signal significantly improves the sensitivity of the analysis. The disadvantage is that many factors affecting the enzyme activity, such as ambient temperature, incubation temperature and time, storage conditions, etc., will directly affect the measurement results, and the stability of the reagent is not good; The luminescence reaction of the method starts slowly, and it takes a certain time to incubate at 37 ° C to reach the illuminating platform, which will more strongly affect the luminescence measurement results.

另外，吖啶酯标记的直接化学发光方法、三联吡啶钌标记的电化学发光方法等在分析过程中带来试剂不稳定、分析结果准确率不高和测试速度不够快等缺陷，给准确分析微量活性物质的量带来了阻碍。发明内容本发明的目的在于针对上述现有技术中的不足之处，提供一种磁分离直接化学发光的试剂，特别是采用异鲁米诺衍生物作标记物、纳米磁珠作为分离材料的磁化学直接发光试剂。本发明另一目的是用上述磁分离直接化学发光试剂的测试方法。为实现上述目的，本发明是采用以下技术方案实现的- 所述的磁分离直接化学发光试剂包括发光标记物、分离试剂、 FITC-抗体联系物和标准抗原或抗体，其中，发光标记物是异鲁米诺衍生物，所述的异鲁米诺衍生物结构式是：

其中： Rl是 C₂¾、 C₃H₇或 C₄ , R2是 NH₂- (CH₂) ₄、 ¾- (CH₂) ₆、 N¾- ( CH₂) 8或腿 ₂- ( C¾ ) ₁₀, 优选 ABEI和 AHEI，其结构式中 Rl是 C₂H₅, R2是 NH₂- (CH₂) ₄ 或 NH₂- (CH₂) ₆; In addition, the acridine ester-labeled direct chemiluminescence method, the pyridinium-labeled electrochemiluminescence method, etc., bring defects such as unstable reagents, low accuracy of analysis results, and fast test speed, etc., for accurate analysis of trace amounts. The amount of active material causes an obstacle. SUMMARY OF THE INVENTION The object of the present invention is to provide a magnetic separation direct chemiluminescence reagent for the above-mentioned deficiencies in the prior art, in particular, magnetic materials using isoluminol derivatives as labels and nano magnetic beads as separation materials. Chemical direct luminescent reagent. Another object of the present invention is to use the above-described magnetic separation direct chemiluminescence reagent test method. In order to achieve the above object, the present invention is achieved by the following technical solutions: the magnetic separation direct chemiluminescence reagent comprises a luminescent label, a separation reagent, a FITC-antibody conjugate and a standard antigen or antibody, wherein the luminescent label is different The luminol derivative, the isoluminol derivative structure is:

Wherein: R1 is C ₂ _3⁄4 , C ₃ H ₇ or C ₄ , and R ₂ is NH ₂ - (CH ₂ ) ₄ , 3⁄4- (CH ₂ ) ₆ , N 3⁄4- ( CH ₂ ) 8 or Leg ₂ - ( C3⁄4 ) ₁₀ Preferably, ABEI and AHEEI, wherein R1 is C ₂ H ₅ , R ₂ is NH ₂ - (CH ₂ ) ₄ or NH ₂ - (CH ₂ ) ₆ ;

所述的分离试剂是羊抗 FITC包被的纳米磁性微珠所述的纳米磁性微珠，其是里面包覆有 Fe₃0₄或 Fe₂0₃,外面含有 - 0H，- C00H，- H₂活性基团的微珠，优选 -C00H和- H₂，其活性基团的含量是 0.05-0.5 eqm/g; 所述的羊抗 FITC包被的纳米磁性微珠是在单抗或抗原上标记。本发明采用的以下步骤进行分析测试的： The separating reagent is a nano magnetic microbead described by a goat anti-FITC coated nano magnetic microbead, which is coated with Fe ₃ 0 ₄ or Fe ₂ 0 ₃ and contains - 0H, - C00H, - H outside. ₂ reactive group microbeads, preferably -C00H and -H ₂ , having a reactive group content of 0.05-0.5 eqm/g; said goat anti-FITC coated nanomagnetic microbeads are on the monoclonal antibody or antigen mark. The following steps are carried out by the present invention for analysis and testing:

A、将分别标记有异鲁米诺衍生物、 FITC的单克隆抗体和待测血清混勾温育； A, the monoclonal antibody labeled with isoluminol derivative, FITC and the serum to be tested are separately incubated;

B、待免疫反应完成后，再加入羊抗包被 FITC多克隆抗体的免疫纳米磁性微珠； B. After the immune reaction is completed, the immune nano magnetic beads coated with the FITC polyclonal antibody are added to the goat;

C、在外加磁场的作用下，将抗原 -抗体复合物特异性分离，用洗液清洗后将底部试管放入测量室内； C. Under the action of an external magnetic field, the antigen-antibody complex is specifically separated, and after washing with a washing liquid, the bottom tube is placed in the measuring chamber;

D、用自动注射泵泵入激发底物 NaOH和 ¾0_2; D, using an automatic syringe pump to pump the substrate NaOH and 3⁄40 _2;

E、用光电倍增管计数试管内混合产物发出的光子数量，用分析仪分析得出待测物的结果。 E. Count the number of photons emitted by the mixed product in the test tube with a photomultiplier tube, and analyze the result of the test object with an analyzer.

本发明中，所述异鲁米诺衍生物发光标记物的单克隆抗体是通过将异鲁米诺衍生物发光标记物与二氯硫化碳或 N-羟基琥珀酸联接后，再与单克隆抗体联接而得，所述的 C步骤中，试管底部是以纳米磁性微珠为载体。所述的磁分离直接化学发光免疫分析测试方法可分为夹心法和竞争法，其设计式分别见图 1和图 2，在图中 A *和 Ag*分别表示在 Ab fl Ag上标记异鲁米诺衍生物 ABEI或 AHEI， Add Ab™分别表示在 Ab₂ Ab上标记 FITC小分子， FITC为异硫氰酸荧光磺， In the present invention, the monoclonal antibody of the isoluminol derivative luminescent label is obtained by linking an illuminating marker of an isoluminol derivative with carbon dichloride or N-hydroxysuccinic acid, and then with a monoclonal antibody. In the C step, the bottom of the test tube is a nano magnetic microbead as a carrier. The magnetic separation direct chemiluminescence immunoassay test method can be divided into a sandwich method and a competition method, and the design formulas thereof are shown in FIG. 1 and FIG. 2 respectively. In the figure, A* and Ag* respectively indicate that the label is on the Ab fl Ag. The Mino derivatives ABEI or AHEI, Add AbTM indicate that FITC small molecules are labeled on Ab ₂ Ab, and FITC is fluorescein isothiocyanate.

表示在纳米磁珠表面联接抗 FITC抗体。 This indicates that an anti-FITC antibody is linked to the surface of the nanomagnetic beads.

根据本发明所述的磁分离直接化学发光分析方法，其技术效果有以下几方面：

According to the magnetic separation direct chemiluminescence analysis method of the present invention, the technical effects are as follows:

1、采用异鲁米诺衍生物作为发光标记物，彻底克服了传统的吖啶酯的容易水解的缺陷，作为直接化学发光模式，不但分析速度快，接近全自动直接化学发光的 180个测试 /小时，完全避免了酶促发光的酶活性易下降的缺陷； 1. Using isoluminol derivatives as luminescent markers, it completely overcomes the defects of easy hydrolysis of traditional acridinium esters. As a direct chemiluminescence mode, not only the analysis speed is fast, but also close to 180 tests of fully automatic direct chemiluminescence/ Hours, completely avoiding the defect that the enzyme activity of enzymatic luminescence is easy to decrease;

2、采用纳米免疫磁性微珠作为抗体的载体和抗原-抗体免疫复合物的分离试剂，大大缩短免疫反应的时间，其中分离免疫反应时间仅需 5分钟，整个免疫反应时间仅需 20分钟，且测试结果的高准确率、高精密度； 2. The nano-immunomagnetic microbeads are used as the carrier of the antibody and the separation reagent of the antigen-antibody immune complex, which greatly shortens the time of the immune reaction, wherein the isolation reaction time takes only 5 minutes, and the entire immune reaction time takes only 20 minutes, and High accuracy and high precision of test results;

3、 FITC和羊抗 FITC抗体包被纳米磁珠的桥联抗体免疫设计技术，大大简化了整个试剂***的免疫学设计，在纳米磁珠表面强大蛋白吸附容量特性支持下，使得纳米免疫磁性微珠成为所有测试项目的公用试剂，简化了生产程序。附图说明 3, FITC and goat anti-FITC antibody coated nano-magnetic beads bridging antibody immunological design technology, greatly simplify the immunological design of the entire reagent system, under the support of strong magnetic protein adsorption capacity characteristics of nano-magnetic beads surface, make nano-immunomagnetic micro Beads became a common reagent for all test projects, simplifying the production process. DRAWINGS

图 1是磁分离直接化学发光免疫夹心法设计图； Figure 1 is a schematic diagram of a magnetic separation direct chemiluminescence immunospinning method;

图 2是磁分离直接化学发光免疫竞争法设计图 Figure 2 is a design diagram of magnetic separation direct chemiluminescence immunocompetition

具体实施方式 detailed description

本发明是采用人工合成异鲁米诺衍生物作为发光标记物，直接标记在抗原或抗体上，通过 CSC1₂活化异鲁米诺苯环侧链上的 NH₂，形成异硫氰酸酯衍生物，通过其中的 -N二 OS键直接与抗原或抗体上胺基联接，分离纯化，去除未链接的异鲁米诺- N=C=S，异鲁米诺的化学发光反应是在金属 Mn²⁺、 Fe²⁺、 CIO—等存在下，激发试剂 NaOH造成的碱性环境，通过异鲁米诺衍生物与激发试剂 ¾0₂的氧化反应，产生波长为 400- 600nm的可见光，构成一个典型的直接化学发光反应。本发明中，纳米磁性微珠表面基团的测定，是将磁珠分散在 10— ² M Nacl溶液中，采用电位滴定法来测量磁珠表面的一 C00H或 -NH₂含量，滴定液用 5X 10一 ³M NaOH溶液，采用双平行线法求得滴定终点，计算出每克磁珠的表面羧基、胺基或羟基含量为 0. 05-0. 5eqm/g o 本发明中分析测试方法所用的分析仪，包括电源电路、自动注射泵 1和 2、测量室、发光室、光电倍增管计数器和输出***，同时还配置有计算机与中文界面的 Windows控制软件，可进行资料录入、结果汇总、质量控制、结果储存和结果查讯等功能，可完成多种分析模式的编程，定量或定性报告结果，自动生成并储存、更新功能，两点自动修正标准曲线。实施例 1 The invention adopts artificial synthesis of isoluminol derivatives as luminescent markers, which are directly labeled in the antigen Or on the antibody, the NH ₂ on the side chain of the isoluminol ring is activated by CSC1 _{2 to} form an isothiocyanate derivative, and the -N di-OS bond is directly linked to the antigen or the amine group on the antibody, and is separated and purified. , removing unlinked isoluminol-N=C=S, the chemiluminescence reaction of isoluminol is an alkaline environment caused by the excitation reagent NaOH in the presence of metals Mn ²⁺ , Fe ²⁺ , CIO-, etc. The visible light having a wavelength of 400-600 nm is generated by the oxidation reaction of the isoluminol derivative with the excitation reagent 3⁄40 ₂ to form a typical direct chemiluminescence reaction. In the present invention, the nano magnetic beads measuring surface groups, the beads are dispersed in 10- ² M Nacl solution, measuring a C00H or -NH ₂ content of the bead surface by potentiometric titration with a titrant 5X 10 a ³ M NaOH solution, dual endpoint titration obtain the parallel line method, calculated per gram of bead surface carboxyl, amino or hydroxyl group content of 0. 05-0. / go analytical test method of the present invention used in 5eqm The analyzer includes power supply circuit, automatic injection pump 1 and 2, measuring room, illuminating chamber, photomultiplier tube counter and output system. It is also equipped with Windows and control software for computer and Chinese interface. Data entry, result summary, quality Control, result storage and result inquiry, can complete a variety of analysis mode programming, quantitative or qualitative report results, automatically generate and store, update functions, two points automatically correct the standard curve. Example 1

1、异鲁米诺衍生物标记的单克隆抗体的制备：取 3. 2inmolABEI三个试管，分别溶于 0. 2ml二次水中， 3. 5mmolCSCl2溶于 0. 3mlDMF 中，二者混合均匀，室温反应 2h，分别取 lOmganti- CEA- α 、 anti-AFP- α 和 10mg anti- PSA- α 单克隆抗体，用 PH9. 5碳酸盐缓冲液调体积到 1ml,加入上述活化的 ABEI溶液，混匀后，室温反应 20h，过 G- 25凝胶柱纯化； 2、 FITC标记 CEA、 AFP、 PSA单克隆抗体 1. The preparation of the monoclonal antibody labeled with the isoluminol derivative: Take 3 in 2 AB of ABAI, dissolved in 0. 2 ml of secondary water, 3. 5 mmol of CSCl 2 dissolved in 0.3 ml of DMF, the two are evenly mixed, room temperature After 2 h of reaction, take 10 mg of anti-CEA-α, anti-AFP-α and 10 mg of anti-PSA-α monoclonal antibody, adjust the volume to 1 ml with PH9.5 buffer solution, add the above activated ABEI solution, and mix. After that, it was reacted at room temperature for 20 h, and purified by a G-25 gel column; 2, FITC labeled CEA, AFP, PSA monoclonal antibody

取 lOmg抗 CEA- β、 AFP- β和 PSA- β单克隆抗体，用 ΡΗ9. 5碳缓调体积到 lml, 加入 FITClOOug,置室温反应 20小时，过 G- 25凝胶柱纯化； Take lOmg anti-CEA-β, AFP-β and PSA-β monoclonal antibodies, use ΡΗ9.5 carbon to adjust the volume to lml, add FITClOOug, let the reaction at room temperature for 20 hours, and purify through G-25 gel column;

3、纳米磁性微珠表面包被羊抗 FITC多克隆抗体的制备： 3. Preparation of nano-magnetic microbead surface coated sheep anti-FITC polyclonal antibody:

纳米磁性微珠的制备是按中国专利制备，专利号为 CN92105584. 6, 磁珠的表面为含有- NH₂的基团，其含量是 0. 17eq/g; 取 10ml磁珠，浓度为 30mg/ml，用 P7. 4的 PBSO. 01M清洗二遍，最后悬浮在 lOmlO. 01MPH7. 4的 PBS中，加 50% 戊二醛溶液 10- 100 μ 1，使戊二醛终浓度为 0. 01-0. 5%，加入纯化的羊抗 FITC IgG 抗体 lOO w g- 1000 g， 37°C反应 2小时，在磁铁上去上清，用含 0. 01- 1%BSA的 0. 01MPH7. 4的 PBS清洗三遍，最后悬浮在该溶液中，加入 0. 01-0. 5%的 NaN₃ The preparation of the nano magnetic microbeads is prepared according to the Chinese patent, and the patent number is CN92105584. 6. The surface of the magnetic beads is a group containing -NH ₂ , and its content is 0.17 eq/g; 10 ml magnetic beads are obtained, and the concentration is 30 mg/ The glutaraldehyde concentration is 0. 01-, and the glutaraldehyde solution is added to a solution of 10% glutaraldehyde in a PBS solution. 0. 01MPH7. 4 PBS, 0. 01- 1% BSA was added to the supernatant. The 5% of NaN _{3 is} added.

4、磁分离直接化学发光试剂制备及结果分析 4, magnetic separation direct chemiluminescence reagent preparation and results analysis

取 CEA、 AFP和 PSA标准品，血清标本各 20 μ 1，加入发光标记物单克隆抗体的 ΑΒΕΙ和 FITC单克隆抗体各 40 μ 1, 混匀后在 37°C的温度下温育，水浴 15 分钟；待免疫反应发生后加入表面包被羊抗 FITC多克隆抗体的免疫纳米磁珠分离试剂 40 μ 1，混匀，水浴 5分钟，在外加磁场的作用下，上分离器分离 4分钟，倒去上清；再加应用洗液 400 μ 1，混匀，分离 4分钟，倒去上清；重复上述步骤分离一次后，直接将试管放入分析仪的测量室，自动泵泵入激发底物 NaOH和 ¾0₂发生直接发光反应，同时用光电倍增管计量光子数量，分析仪分析并打印出结果。 Take CEA, AFP and PSA standards, serum samples 20 μl each, add luminescent label monoclonal antibody ΑΒΕΙ and FITC monoclonal antibody 40 μl each, mix and incubate at 37 ° C, water bath 15 Minutes; after the immune reaction occurs, add 40 μl of immunomagnetic nanobead separation reagent coated with surface anti-FITC polyclonal antibody, mix well, and bath for 5 minutes. Under the action of external magnetic field, the upper separator is separated for 4 minutes. Remove the supernatant; add the washing solution 400 μ 1, mix, separate for 4 minutes, pour off the supernatant; repeat the above steps to separate once, put the test tube directly into the measuring chamber of the analyzer, and automatically pump the pump into the excitation substrate A direct luminescence reaction occurs between NaOH and 3⁄40 ₂ , while the number of photons is measured by a photomultiplier tube, and the analyzer analyzes and prints the result.

选择 2 0份临床标本，按上述实验过程进行分析测试，本实例中，原发性早 ***类 1例， ***肥大例，正常人 1 1例，其结果列于表 1中。Select 20 clinical specimens, analyze and test according to the above experimental procedure. In this example, primary early There were 1 case of prostate, 1 case of prostate hypertrophy, and 11 cases of normal people. The results are shown in Table 1.

表 1 4 ^#和 6 ^#病人为原发性早期肝癌病人， A F P值持续长时间增高， 1 1 ^#和 1 4 ^#为肝癌切除术并处于放疗和化疗中的病人，其血清中 A F P浓度已经下降，但仍没有降至正常值范围，提示疗效不明显，仍有转恶的可能； 2 ^#、 8 ^# 和 6 ^#病人的 A F P值也超过正常值，但临床诊断是直肠癌，其 C E A值明显增高，尤其是 16^#， C E A更高达 1270mIU/ml，这可能说明不同肿瘤标志物间存在某种联系或交叉， 1 ^#和 8 ^#病人分别为***类和单纯***肥大，其 P S A测定值轻微升高。 Table 1 4 ^# and 6 ^# patients are primary early stage liver cancer patients, AFP values continue to increase for a long time, 1 1 ^# and 1 4 ^# for liver cancer resection and in patients with radiotherapy and chemotherapy, the serum AFP concentration has decreased, but Still did not fall to the normal range, suggesting that the efficacy is not obvious, there is still the possibility of turning evil; 2 ^# , 8 ^# and 6 ^# patients' AFP value also exceeds the normal value, but the clinical diagnosis is rectal cancer, its CEA value is significantly higher, In particular, 16 ^# and CEA are as high as 1270 mIU/ml, which may indicate that there is a certain relationship or cross between different tumor markers. The 1 ^# and 8 ^# patients are prostate and simple prostatic hypertrophy, respectively, and their PSA values are slightly elevated.

从表 1的数据分析来看，用本明所述的磁发光免疫分析测试方法所得出的结果有很好的临床相符性。实施例 2 异鲁米诺衍生物单克隆抗体的制备和纳米磁性微珠表面包被羊抗 FITC多克隆抗体的制备方法与实施例 1相同，本实施例所用的发光标记物是 AHEI，磁珠的表面为含有 -C00H的基团，其含量是 0. 3eq/g; From the data analysis of Table 1, the results obtained by the magnetic luminescence immunoassay test method described in the present invention have a good clinical consistency. Example 2 Preparation of Isoluminol Derivative Monoclonal Antibody and Preparation of Nanomagnetic Bead Surface Coated Sheep Anti-FITC Polyclonal Antibody The same procedure as in Example 1 was used. The luminescent label used in this example was AHEI, magnetic beads. The surface is 0. 3eq/g;

分别取 TSH、 T3、 T4、 FT3和 FT4标准品，血清标本各 20 μ 1，分别加入发光标记物单克隆抗体的 ΑΗΕΙ和 FITC单克隆抗体各 40 μ 1，混匀后在 37°C的温度下温育，水浴 15分钟；待免疫反应发生后加入表面包被羊抗 FITC多克隆抗体的免疫纳米磁珠分离试剂 40 μ 1，混匀，水浴 5分钟，在外加磁场的作用下，上分离器分离 4分钟，倒去上清；再加洗液 400 μ 1，混匀，分离 4分钟，倒去上清；重复上述步骤分离一次后，直接将试管放入分析仪的测量室，自动泵泵入激发底物 NaOH和 ¾0₂发生直接发光反应，同时用光电倍增管计量光子数量，分析仪分析并打印出结果。 TSH, T3, T4, FT3 and FT4 standards were taken separately. Serum samples were 20 μl each, and 发光 and FITC monoclonal antibodies were added to the luminescent label monoclonal antibody and 40 μl each, and then mixed at 37 ° C. Under incubation, water bath for 15 minutes; after the immune reaction occurs, add the surface-coated goat anti-FITC polyclonal antibody to the immunomagnetic nanobead separation reagent 40 μ 1, mix, water bath for 5 minutes, under the action of external magnetic field, on the separation Separate for 4 minutes, pour off the supernatant; add 400 μl of lotion, mix, separate for 4 minutes, pour off the supernatant; repeat the above steps and separate the test tube directly into the measuring chamber of the analyzer, automatic pump The pumped excitation substrate NaOH and 3⁄40 ₂ undergo a direct luminescence reaction, while the photon multiplier is used to measure the number of photons, and the analyzer analyzes and prints the results.

选择 261例临床标本，其中临床确诊甲减 37例，初诊甲亢 50例，随诊甲亢 26例，治愈甲亢 6例，甲状腺瘤 13例，甲状腺肿 5例，正常人对照 120例，年龄 18— 68岁，男 45例，凡研究对象静脉采血，分离血清， -20° C贮存，一周内完成检测，测试的结果经统列于表 2中: 261 clinical specimens were selected, including 37 cases of clinically diagnosed hypothyroidism, 50 cases of newly diagnosed hyperthyroidism, 26 cases of hyperthyroidism, 6 cases of hyperthyroidism, 13 cases of thyroid tumor, 5 cases of goiter, 120 cases of normal control, age 18-68. Aged, male 45 cases, all subjects were venous blood collection, serum separation, -20 ° C storage, one The test was completed within the week and the results of the test are listed in Table 2:

表 2

Table 2

从表 2中列出的五项激素的测定结果显示甲减 3 7例病人， TDH值均明显增高， T3、 T4、 T3、 T4、 FT3、 FT4、均明显降低，符合临床诊断。对初诊 5 0 例甲亢病人， TSH明显降低， T3、 T4、 FT3、 FT4明显增高，对治疗中的 2 6例甲亢病人的五项激测定统计分析表明， T4值全部趋于恢复或接近正常值， T3 则下降速度明显慢于 T4值，用本明所述的磁发光免疫分析测试方法所得出的结果有很好的临床相符性。 The results of the five hormones listed in Table 2 showed that the TDH values were significantly increased in 37 patients with hypothyroidism, and T3, T4, T3, T4, FT3, and FT4 were significantly lower, which was consistent with clinical diagnosis. For the newly diagnosed patients with hyperthyroidism, TSH was significantly decreased, T3, T4, FT3, and FT4 were significantly increased. Statistical analysis of five mutations in 26 patients with hyperthyroidism in treatment showed that T4 values all recovered or were close to normal values. , T3, the rate of decline is significantly slower than the T4 value, the results obtained by the magnetic luminescence immunoassay test method described in the present invention have a good clinical consistency.

本发明实施例子所述试剂的测试项目以甲状腺激素和肿瘤标志物的测定为例，但本发明并不限于测试这两种项目。工业实用性本发明的磁分离直接化学发光分析方法，采用异鲁米诺衍生物作为发光标记物，彻底克服了传统的吖啶酯的容易水解的缺陷，作为直接化学发光模式，不但分析速度快，接近全自动直接化学发光的 180个测试 /小时，完全避免了酶促发光的酶活性易下降的缺陷；采用纳米免疫磁性微珠作为抗体的载体和抗原- 抗体免疫复合物的分离试剂，大大缩短免疫反应的时间，其中分离免疫反应时间仅需 5分钟，整个免疫反应时间仅需 20分钟，且测试结果的高准确率、高精密度； FITC和羊抗 FITC抗体包被纳米磁珠的桥联抗体免疫设计技术，大大简化了整个试剂***的免疫学设计，在纳米磁珠表面强大蛋白吸附容量特性支持下，使得纳米免疫磁性微珠成为所有测试项目的公用试剂，简化了生产程序。 The test items of the reagents according to the examples of the present invention are exemplified by the determination of thyroid hormones and tumor markers, but the present invention is not limited to testing both items. Industrial applicability The magnetic separation direct chemiluminescence analysis method of the invention adopts an isoluminol derivative as a luminescent label, completely overcomes the defect of easy hydrolysis of the traditional acridinium ester, and as a direct chemiluminescence mode, not only the analysis speed is fast, but also close to the whole 180 tests/hour of automatic direct chemiluminescence completely avoids the defect that the enzyme activity of enzymatic luminescence is easy to decrease; using nano-immunomagnetic microbeads as a carrier for antibodies and an isolation reagent for antigen-antibody immune complexes, greatly shortening the immune response Time, in which the time of isolation of the immune reaction is only 5 minutes, the entire immune reaction time is only 20 minutes, and the test results are highly accurate and high-precision; FITC and goat anti-FITC antibody coated with nanomagnetic beads are bridged antibody immunization The design technology greatly simplifies the immunological design of the entire reagent system. Under the support of the strong protein adsorption capacity of the surface of the nanomagnetic beads, the nano-immunomagnetic microbeads become a common reagent for all test items, simplifying the production process.

Claims

权利要求 Rights request

1、磁分离直接化学发光试剂，包括发光标记物、分离试剂、 FITC-抗体联系物、标准抗原或抗体和激发底物，其特 ffi在于：所述的发光标记物是异鲁米诺衍生物，所述的异鲁米诺衍生物的结构式是- 1. A magnetic separation direct chemiluminescence reagent comprising a luminescent label, a separation reagent, a FITC-antibody conjugate, a standard antigen or antibody, and an excitation substrate, wherein the fluorochrome is: the luminol derivative is an isoluminol derivative , the structural formula of the isoluminol derivative is -

其中： R1是 C₂¾、 C₃H₇或 C₄¾, R2是丽 ₂- (CH₂) ₄、匪 ₂- (CH₂) ₆、 N¾- (CH₂) ₈或丽 ₂- (CH₂) ₁₀。 Wherein: R1 is C ₂ _3⁄4 , C ₃ H ₇ or C _{4 3⁄4} , R2 is 丽₂ - (CH ₂ ) ₄ , 匪₂ - (CH ₂ ) ₆ , N3⁄4- (CH ₂ ) ₈ or 丽₂ - (CH ₂ ) ₁₀ .

2、根据权利要求 1所述的磁分离直接化学发光试剂，其特征在于：所述的分离试剂是羊抗 FITC包被的纳米磁性微珠，所述的纳米磁性微珠是里面包覆有 Fe₃0₄或 Fe₂0₃，外面含有- 0H、 -C00H或-歷 ₂活性基团的微珠。 2. The magnetic separation direct chemiluminescence reagent according to claim 1, wherein: the separation reagent is a goat anti-FITC coated nano magnetic microbead, and the nano magnetic microbead is coated with Fe inside. ₃ 0 ₄ or Fe ₂ 0 ₃ , microspheres containing -0H, -C00H or _-2 active groups on the outside.

3、根据权利要求 1所述的磁分离直接化学发光试剂，其特征在于：所述的羊抗 FITC包被的纳米磁性微珠是在单抗或抗原上标记。 The magnetic separation direct chemiluminescence reagent according to claim 1, wherein the sheep anti-FITC coated nanomagnetic microbeads are labeled on the monoclonal antibody or antigen.

4、根据权利求 2或 3所述的磁分离直接化学发光试剂， ·其特征在于：所述的激发底物是 NaOH和 0₂。 . 4. The magnetic separation direct chemiluminescence reagent according to claim 2 or 3, wherein: the excitation substrate is NaOH and 0 ₂ . .

5、根据权利要求 4所述的磁分离直接化学发光试剂，其特征在于：所述的异鲁米诺发光标记物是 ABEI和 AHEI，其结构式中 R1是 C₂¾, R2是丽 ₂- (CH₂) ₄ 或腿 ₂— (CH₂) ₆。 The magnetic separation direct chemiluminescence reagent according to claim 4, wherein: the isoluminol luminescent label is ABEI and AHEEI, wherein R1 is C ₂ 3⁄4, and R2 is 丽₂ - ( CH ₂ ) ₄ or leg ₂ — (CH ₂ ) ₆ .

6、根据权利要求 5所述的磁分离直接化学发光试剂，其特征在于：所述的纳米免疫磁性微珠表面含有基团是 -C00H或- N¾。 The magnetic separation direct chemiluminescence reagent according to claim 5, wherein the surface of the nano-immunomagnetic microbead contains -C00H or -N3⁄4.

7、根据权利要求 6所述的磁分离直接化学发光试剂，其特征在于：所述的纳米免疫磁性微珠表面含有基团的含量是 0. 05-0. 5eqm/g。 0. 5eqm/g。 The content of the surface of the nano-immunomagnetic microbeads is 0. 05-0. 5eqm / g.

8、采用上述磁分离直接化学发光试剂的测试方法，其包括以下步骤：8. A test method using the above magnetic separation direct chemiluminescence reagent, comprising the steps of:

A、将分别标记有异鲁米诺衍生物、 FITC的单克隆抗体和待测血清混勾温育;A, the monoclonal antibody labeled with isoluminol derivative, FITC and the serum to be tested are separately incubated;

B、待免疫反应完成后，再加入包被羊抗 FITC多克隆抗体的免疫纳米磁性微珠； B. After the immune reaction is completed, the immunomagnetic nanobeads coated with the goat anti-FITC polyclonal antibody are added;

C、在外加磁场的作用下，将抗原 -抗体免疫复合物特异性分离，用洗液清洗后，将试管放入分析仪的测量室内； C. Under the action of an external magnetic field, the antigen-antibody immune complex is specifically separated, and after washing with a washing liquid, the test tube is placed in the measuring chamber of the analyzer;

E、用光电倍增管计数试管内混合产物发出的光子数量，分析仪分析得出待测物的浓度。 E. Count the number of photons emitted by the mixed product in the test tube with a photomultiplier tube, and analyze the analyzer to obtain the concentration of the analyte.

9、根据权利要求 8所述的磁分离直接化学发光分析测试方法，其特征在于: 异鲁米诺发光标记物的单克隆抗体是通过将异鲁米诺发光标记物与二氯硫化碳或 N-羟基琥珀酸联接后，再与单克隆抗体联接而得。 9. The magnetic separation direct chemiluminescence analysis test method according to claim 8, wherein the monoclonal antibody of the isoluminol luminescent label is obtained by using an isoluminol luminescent label with carbon dichloride or N. After the hydroxysuccinic acid is linked, it is then linked to a monoclonal antibody.

10、根据权利要求 8所述的磁分离直接化学发光分析测试方法，其特征在于：所述 C步骤中，试管底部是以纳米磁性微珠为载体。 10. The magnetic separation direct chemiluminescence analysis test method according to claim 8, wherein: in the step C, the bottom of the test tube is a nano magnetic microbead as a carrier.