CN103604918A - Luminescent substrate, use of luminescent substrate and detection kit containing luminescent substrate - Google Patents

Abstract

The invention discloses a luminescent substrate, a use of the luminescent substrate and a detection kit containing the luminescent substrate. The luminescent substrate has high sensitivity and high stability. The luminescent substrate comprises a solution A and a solution B. The solution A comprises 0.5-8mmol/L of luminal, 0.5-10mmol/L of p-lodophenol, and 0.1mol/L of a Tris-HCl buffer solution. The solution B comprises 1-10mmol/L of urea peroxide and 0.1mol/L of the Tris-HCl buffer solution. The kit comprises a monoclonal antibody-coated microporous plate, HRP-labeled monoclonal antibodies, a freeze-dried standard, a sample diluent, a concentrated washing liquid and the chemiluminescent substrate.

Description

Translated fromChinese

一种发光底物及应用和含该发光底物的检测试剂盒Luminescent substrate and its application and detection kit containing the luminescent substrate

技术领域technical field

本发明公开了一种发光底物，本发明还公开了该发光底物的应用以及含该发光底物的检测试剂盒。The invention discloses a luminescent substrate, and also discloses the application of the luminescent substrate and a detection kit containing the luminescent substrate.

背景技术Background technique

近十年来标记免疫分析技术的研究和应用发展迅速，已广泛应用于生物医学基础理论研究及临床疾病诊断各领域。用于检测血清学指标的方法主要包括放射性同位素免疫分析、酶联免疫吸附法和化学放光免疫分析。In the past ten years, the research and application of marker immunoassay technology has developed rapidly, and has been widely used in various fields of biomedical basic theory research and clinical disease diagnosis. The methods used to detect serological indicators mainly include radioisotope immunoassay, enzyme-linked immunosorbent assay and chemiluminescence immunoassay.

目前，临床领域应用最为广泛的定量免疫检测方法主要是化学发光免疫分析（CLIA），是一种敏感微量免疫测定法，兼有放射免疫（RIA）高灵敏度和酶联免疫（ELISA）易操作等优点，同时又克服了放射免疫和酶联免疫各自的缺点，是临床免疫检测最理想的方法之一。At present, the most widely used quantitative immunoassay method in the clinical field is mainly chemiluminescence immunoassay (CLIA), which is a sensitive micro-immunoassay method, which combines high sensitivity of radioimmunoassay (RIA) and easy operation of enzyme-linked immunoassay (ELISA), etc. Advantages, while overcoming the respective shortcomings of radioimmunoassay and ELISA, it is one of the most ideal methods for clinical immunoassay.

化学发光免疫分析法根据标记物的不同可分为三大类，即化学发光免疫分析、化学发光酶免疫分析和电化学发光免疫分析。其中以辣根过氧化物酶（HRP）标记的化学发光酶免疫分析(Chemiluminescence Enzyme Immol/Lunoassay，CLEIA)是以HRP标记生物活性物质(如酶标记的抗原或抗体)进行免疫反应，免疫反应复合物上的酶再作用于发光底物，激发化学发光底物产生光信号，利用发光信号测定仪进行发光测定。HRP有许多发光底物，但最常用的发光底物是鲁米诺及其衍生物。在CLEIA中，使用HRP酶标抗体进行免疫反应后，在碱性条件下，HRP和氧化剂(H2O2)作用于发光底物鲁米诺使其发生化学反应进而发光，利用光信号测定仪可以测量这种光信号。但是这种传统的化学发光体系(HRP-H2O2-luminol)存在发光强℃低、不易测量等缺点。后来，研究发现在发光系统中加入增强发光剂，如含有取代基的酚类化合物(如4-(3-噻吩基)酚类化合物)、萘酚、取代的溴酸化合物、乙酰苯胺类化合物等，可以大大增强发光信号，并在较长的时间内保持稳定，便于重复测量，从而提高分析灵敏度和准确性。Chemiluminescence immunoassays can be divided into three categories according to different markers, namely, chemiluminescence immunoassay, chemiluminescence enzyme immunoassay and electrochemiluminescence immunoassay. Among them, the chemiluminescent enzyme immunoassay (Chemiluminescence Enzyme Immol/Lunoassay, CLEIA) labeled with horseradish peroxidase (HRP) uses HRP-labeled biologically active substances (such as enzyme-labeled antigens or antibodies) to carry out immune reactions, and the immune response complex The enzyme on the substance acts on the luminescent substrate again, and excites the chemiluminescent substrate to generate a light signal, and the luminescent signal is measured by a luminescent signal detector. There are many luminescent substrates for HRP, but the most commonly used luminescent substrates are luminol and its derivatives. In CLEIA, after immunoreaction with HRP enzyme-labeled antibody, under alkaline conditions, HRP and oxidant (H2O2) act on the luminescent substrate luminol to cause a chemical reaction and then luminescence, which can be measured using an optical signal detector kind of light signal. However, this traditional chemiluminescence system (HRP-H2O2-luminol) has disadvantages such as low luminous intensity ℃ and difficult measurement. Later, it was discovered that enhancing luminescent agents were added to the luminescent system, such as phenolic compounds containing substituents (such as 4-(3-thienyl)phenolic compounds), naphthol, substituted bromic acid compounds, acetanilide compounds, etc. , can greatly enhance the luminescent signal and keep it stable for a long time, which is convenient for repeated measurement, thereby improving the sensitivity and accuracy of analysis.

原发性肝癌，尤其是肝细胞癌（Hepatocellular Carcinoma），作为世界上最常见的恶性肿瘤之一，也是世界上各种实体肿瘤中预后效果较差的恶性肿瘤之一，并具有与乙肝、肝硬化等密切联系的疾病特点，尤其近年其患病率在我国呈逐年上升趋势。因此，肝癌的早期预警技术研究对于预防肝癌、预测肝癌的发病趋势具有非常重要的研究价值和社会意义。目前临床上广泛应用的肝癌诊断标志物AFP，但是对于早期肝癌的诊断，其敏感性、特异性均不理想。因此在临床上希望获得比甲胎蛋白特异性和灵敏度更高的新血清学指标，或者能够与AFP进行互补的指标。其中高尔基体蛋白73（GolgiProtein73，GP73）GP73是近年研究发现的最有可能成为更好的肝癌诊断尤其是早期肝癌诊断的血清标志物。对于早期肝癌（T1：单个结节，直径<2cm及T2：单个结节，直径位于2cm～5cm之间或者小于三个结节，每个直径<3cm）的诊断，GP73的敏感性显著优于AFP，而且AFP水平低于20μg/L的HCC患者中，多达57％的人群GP73水平显著升高，这表明HCC患者GP73水平显著升高，因此对于早期肝癌的诊断，GP73优于AFP。Primary liver cancer, especially hepatocellular carcinoma (Hepatocellular Carcinoma), as one of the most common malignant tumors in the world, is also one of the malignant tumors with poor prognosis among various solid tumors in the world. The characteristics of closely related diseases such as cirrhosis, especially in recent years, its prevalence has been increasing year by year in our country. Therefore, research on early warning technology of liver cancer has very important research value and social significance for preventing liver cancer and predicting the incidence trend of liver cancer. At present, AFP is widely used clinically as a diagnostic marker for liver cancer, but its sensitivity and specificity are not ideal for the diagnosis of early liver cancer. Therefore, it is hoped to obtain a new serological index with higher specificity and sensitivity than alpha-fetoprotein in clinical practice, or an index that can complement AFP. Among them, Golgi Protein 73 (Golgi Protein 73, GP73) GP73 is the most likely to become a better serum marker for the diagnosis of liver cancer, especially the diagnosis of early liver cancer, discovered in recent years. For the diagnosis of early liver cancer (T1: single nodule, diameter <2cm and T2: single nodule, diameter between 2cm and 5cm or less than three nodules, each diameter <3cm), the sensitivity of GP73 is significantly better than AFP, and as many as 57% of HCC patients with AFP levels below 20 μg/L had significantly elevated GP73 levels, which indicated that GP73 levels were significantly elevated in HCC patients. Therefore, GP73 is superior to AFP for the diagnosis of early liver cancer.

发明内容Contents of the invention

针对上述问题，本发明的目的在于提供一种高灵敏度、高稳定性的化学发光底物，并且提供了该发光底物的用途以及含有该发光底物的试剂盒，该试剂盒使用方便，灵敏度高。In view of the above problems, the object of the present invention is to provide a highly sensitive, highly stable chemiluminescent substrate, and provides the use of the luminescent substrate and a kit containing the luminescent substrate, the kit is easy to use, high sensitivity high.

为解决上述技术问题，本发明提供的前一技术方案是这样的：该发光底物由A、B液组成，所述的A液包含下述组分：0.5～8mmol/L鲁米诺、0.5～10mmol/L对碘苯酚、0.1mol/LTris-HCl缓冲液；所述的B液包含下述组分：1～10mmol/L过氧化脲、0.1mol/L Tris-HCl缓冲液。In order to solve the above-mentioned technical problems, the previous technical scheme provided by the present invention is as follows: the luminescent substrate is composed of A and B liquids, and the A liquid contains the following components: 0.5-8mmol/L luminol, 0.5 ~10mmol/L p-iodophenol, 0.1mol/L Tris-HCl buffer solution; the B solution contains the following components: 1~10mmol/L carbamide peroxide, 0.1mol/L Tris-HCl buffer solution.

更优的，上述的发光底物，所述的A液包含下述组分：2.5mmol/L鲁米诺、3.0mmol/L对碘苯酚、0.1mol/L Tris-HCl；所述的B液包含下述组分：1.3mmol/L过氧化、0.1mol/L Tris-HCl。More preferably, the above-mentioned luminescent substrate, the described A solution comprises the following components: 2.5mmol/L luminol, 3.0mmol/L p-iodophenol, 0.1mol/L Tris-HCl; the described B solution Contains the following components: 1.3mmol/L peroxide, 0.1mol/L Tris-HCl.

进一步的，上述的发光底物，所述的Tris-HCll缓冲液的pH值是8.5。Further, for the above-mentioned luminescent substrate, the pH value of the Tris-HCll buffer is 8.5.

进一步的，上述的发光底物，所述的Tris-HCl缓冲液包括下述质量百分比的组分：0.05～0.3%BSA、0.01～0.05%Tween-20、0.01～0.1%proclin300，用HCl调节pH至8.5。Further, for the above-mentioned luminescent substrate, the Tris-HCl buffer solution includes the following components in mass percentage: 0.05-0.3% BSA, 0.01-0.05% Tween-20, 0.01-0.1% proclin300, the pH is adjusted with HCl to 8.5.

本发明的另外一个技术方案是该发光底物作为制备ELISA定量检测试剂盒的应用。Another technical scheme of the present invention is the application of the luminescent substrate as the preparation of an ELISA quantitative detection kit.

本发明的最后一个技术方案是含有该发光底物的试剂盒，所述的试剂盒包括包被有单克隆抗体的微孔板、HRP标记的单克隆抗体、冻干标准品、样品稀释液、浓缩洗涤液和化学发光底物。The last technical scheme of the present invention is a kit containing the luminescent substrate, said kit including microwell plates coated with monoclonal antibodies, HRP-labeled monoclonal antibodies, freeze-dried standards, sample diluents, Concentrate the wash solution and chemiluminescence substrate.

进一步的，上述的试剂盒，所述的单克隆抗体为GP73特异性单克隆抗体5F10；所述的HRP标记的单克隆抗体为GP73单克隆抗体5B12*HRP。Further, in the above kit, the monoclonal antibody is GP73-specific monoclonal antibody 5F10; the HRP-labeled monoclonal antibody is GP73 monoclonal antibody 5B12*HRP.

11、进一步的，上述的试剂盒，所述的样品稀释液为pH7.4的含1%BSA、5%胎牛血清、0.1%Proclin300的0.01mol/L Tris-HCl缓冲液；所述的浓缩洗涤液为含0.05%Tween-20的PBST浓缩洗涤液。11. Further, in the above kit, the sample diluent is 0.01mol/L Tris-HCl buffer containing 1% BSA, 5% fetal bovine serum, and 0.1% Proclin300 at pH 7.4; the concentrated The washing solution was concentrated washing solution in PBST containing 0.05% Tween-20.

与现有现有技术相比，本发明提供的技术方案具有如下优点：Compared with the existing prior art, the technical solution provided by the present invention has the following advantages:

1、本发明提供的发光底物，光信号强，灵敏度高，稳定性好，并且成本低；1. The luminescent substrate provided by the present invention has strong optical signal, high sensitivity, good stability and low cost;

2、本发明提供的试剂盒，利用该底物进行化学发光ELISA定量检测GP73蛋白含量，大大提高了检测GP73的灵敏度和特异性，可用于监测肝癌的早期诊断、肝硬化以及慢性肝炎的发展程度，为肝癌的预防、诊断以及治疗提供了更有力的支持。2. The kit provided by the present invention uses the substrate to perform quantitative detection of GP73 protein content by chemiluminescent ELISA, which greatly improves the sensitivity and specificity of detecting GP73, and can be used to monitor the early diagnosis of liver cancer, liver cirrhosis and the development degree of chronic hepatitis , providing stronger support for the prevention, diagnosis and treatment of liver cancer.

附图说明Description of drawings

图1是GP73化学发光定量检测试剂盒灵敏性研究曲线图；Figure 1 is a graph showing the sensitivity research curve of the GP73 chemiluminescence quantitative detection kit;

图2是实施例1中的化学发光底物与市场上同类化学发光底物对比分析示意图；Fig. 2 is a schematic diagram of comparative analysis of the chemiluminescent substrate in Example 1 and similar chemiluminescent substrates on the market;

图3本发明提供的试剂盒的临床样品研究示意图。Fig. 3 is a schematic diagram of clinical sample research of the kit provided by the present invention.

具体实施方式Detailed ways

下面结合具体实施方式，对本发明的权利要求做进一步的详细说明，但不构成对本发明的任何限制，任何人在本发明权利要求范围内所做的有限次的修改，仍在本发明的权利要求保护范围内。The claims of the present invention will be described in further detail below in conjunction with specific embodiments, but this does not constitute any limitation to the present invention. Anyone who makes limited modifications within the scope of the claims of the present invention is still in the claims of the present invention within the scope of protection.

实施例1化学发光底物液的配制The preparation of embodiment 1 chemiluminescence substrate liquid

底物配方如下：The substrate formulation is as follows:

配方1：Recipe 1:

发光剂溶液A液：鲁米诺2.5mmol/L、对碘苯酚3.0mmol/L、pH8.5的Tris-HCl缓冲液；Luminescent agent solution A liquid: 2.5mmol/L luminol, 3.0mmol/L p-iodophenol, Tris-HCl buffer solution with pH 8.5;

氧化剂溶液B液：过氧化脲1.3mmol/L、pH8.5的Tris-HCl缓冲液；Oxidant solution B solution: carbamide peroxide 1.3mmol/L, Tris-HCl buffer solution with pH 8.5;

配方2：Recipe 2:

发光剂溶液A液：鲁米诺0.5mmol/L、对碘苯酚5.0mmol/L、pH8.5的Tris-HCl缓冲液；Luminescent agent solution A: luminol 0.5mmol/L, p-iodophenol 5.0mmol/L, Tris-HCl buffer solution with pH 8.5;

氧化剂溶液B液：过氧化脲1mmol/L、pH8.5的Tris-HCl缓冲液；Oxidant solution B liquid: carbamide peroxide 1mmol/L, Tris-HCl buffer solution with pH 8.5;

配方3：Recipe 3:

发光剂溶液A液：鲁米诺8mmol/L、对碘苯酚10mmol/L、pH8.5的Tris-HCl缓冲液；Luminescent agent solution A liquid: luminol 8mmol/L, p-iodophenol 10mmol/L, Tris-HCl buffer solution with pH 8.5;

氧化剂溶液B液：过氧化脲10mmol/L、pH8.5的Tris-HCl缓冲液；Oxidant solution B liquid: carbamide peroxide 10mmol/L, Tris-HCl buffer solution with pH 8.5;

配方4：Recipe 4:

发光剂溶液A液：鲁米诺5mmol/L、对碘苯酚0.3mmol/L、pH8.5的Tris-HCl缓冲液；Luminescent agent solution A liquid: luminol 5mmol/L, p-iodophenol 0.3mmol/L, Tris-HCl buffer solution with pH 8.5;

氧化剂溶液B液：过氧化脲5mmol/L、pH8.5的Tris-HCl缓冲液；Oxidant solution B solution: carbamide peroxide 5mmol/L, Tris-HCl buffer solution with pH 8.5;

配方1中Tris-HCl缓冲液、A液和B液的配置方法为：The configuration method of Tris-HCl buffer solution, solution A and solution B in formula 1 is as follows:

缓冲液的配制：取Tris12.1g，牛血清白蛋白1.0g，Tween-200.5ml，Proclin-3001.0ml，用800ml双蒸水溶解，用浓盐酸调节溶液的pH至8.5，用双蒸水定容至1000ml，即为0.1mol/LTris-HCl缓冲液，含有0.1%BSA、0.05%Tween-20、0.05%Proclin300，pH8.5。Preparation of buffer: take Tris12.1g, bovine serum albumin1.0g, Tween-200.5ml, Proclin-3001.0ml, dissolve in 800ml double-distilled water, adjust the pH of the solution to 8.5 with concentrated hydrochloric acid, and distill to volume with double-distilled water To 1000ml, that is 0.1mol/LTris-HCl buffer solution, containing 0.1%BSA, 0.05%Tween-20, 0.05%Proclin300, pH8.5.

发光剂溶液A液的配制：分别称取鲁米诺0.4429g，对碘苯酚0.6600g，其中鲁米诺先用0.1M的氢氧化钠溶解，对碘苯酚用二甲基甲酰胺（DMF）溶解，然后将上述两种溶液用Tris-HCl缓冲液定容至1000ml，0.22um滤膜过滤，4℃避光保存。Preparation of luminescent agent solution A: Weigh 0.4429g of luminol and 0.6600g of p-iodophenol, among which luminol is first dissolved with 0.1M sodium hydroxide, and p-iodophenol is dissolved with dimethylformamide (DMF) , and then the above two solutions were adjusted to 1000ml with Tris-HCl buffer solution, filtered through a 0.22um filter membrane, and stored at 4°C in the dark.

氧化剂溶液B液的配制：称取过氧化脲0.1200g，用Tris-HCl缓冲液溶解，并定容至1000ml，0.22um滤膜过滤，4℃避光保存。Preparation of Oxidant Solution B: Weigh 0.1200g of carbamide peroxide, dissolve it in Tris-HCl buffer solution, and dilute to 1000ml, filter through a 0.22um membrane filter, and store in the dark at 4°C.

配方2至4中的Tris-HCl缓冲液、A液和B液的制备方法与配方1中的Tris-HCl缓冲液、A液和B液制备方法类似，只需根据具体物质量浓度调节各个组分的用量。The preparation method of Tris-HCl buffer solution, A solution and B solution informula 2 to 4 is similar to the preparation method of Tris-HCl buffer solution, A solution and B solution in formula 1, only need to adjust each group according to the specific substance concentration points of use.

实施例2Example 2

本发明提供的试剂盒包括包被有单克隆抗体的微孔板、HRP标记的单克隆抗体、冻干标准品、样品稀释液、浓缩洗涤液和实施例1中任意一种配方的化学发光底物。The kit provided by the present invention includes a microwell plate coated with monoclonal antibody, HRP-labeled monoclonal antibody, freeze-dried standard substance, sample diluent, concentrated washing solution and the chemiluminescence base of any one of the formulations in Example 1. things.

下面以GP73化学发光定量检测试剂盒为例，具有说明该检测试剂盒的制备方法，也可以同样应用于其它蛋白的检测。The GP73 chemiluminescence quantitative detection kit is used as an example below to illustrate the preparation method of the detection kit, which can also be applied to the detection of other proteins.

1、固相包被板的制备1. Preparation of solid-phase coated plates

a)包被：GP73单克隆抗体5F10以2ug/ml浓度包被微孔板，100ul/孔，4℃包被18h；a) Coating: GP73 monoclonal antibody 5F10 was coated with a concentration of 2ug/ml on a microwell plate, 100ul/well, and coated at 4°C for 18h;

b)封闭：洗板2遍，拍干，加入封闭液200ul/孔，37℃1.5h。b) Blocking: wash the plate twice, pat dry, add blocking solution 200ul/well, 37°C for 1.5h.

c)封袋：弃封闭液，拍干，在37℃培养箱进行干燥1h，立即进行真空封袋。c) Seal the bag: Discard the blocking solution, pat dry, dry in a 37°C incubator for 1 hour, and immediately vacuum seal the bag.

2、标准品的制备：真核表达纯化的GP73蛋白，用样品稀释液（具体为何种物质）倍比稀释至浓度为31.25ng/ml、15.6ng/ml、7.8ng/ml、3.9ng/ml、1.95ng/ml、0.98ng/ml、0ng/ml，经0.22um滤膜过滤，每种浓度的标准品分装500ul/管，后经冷冻干燥得到冻干标准品。2. Preparation of standard products: Eukaryotically expressed and purified GP73 protein, diluted to a concentration of 31.25ng/ml, 15.6ng/ml, 7.8ng/ml, 3.9ng/ml with sample diluent (specifically what kind of substance) , 1.95ng/ml, 0.98ng/ml, and 0ng/ml were filtered through a 0.22um filter membrane, and each concentration of the standard product was divided into 500ul/tubes, and then freeze-dried to obtain a freeze-dried standard product.

3、酶标抗体的制备：GP73单克隆抗体5B12经改良的过碘酸钠法-硼氢化钠法标记辣根过氧化物酶HRP，即在低pH(pH4.4)下HRP经0.1mol/L的NaIO₄氧化形成醛化酶，醛化酶与抗体分子5B12的氨基相连形成的斯夫氏硷，可进一步用4mg/ml的NaBH₄还原得到稳定的酶标记抗体。得到的5B12*HRP用抗体稀释液即pH7.4的含1%BSA、5%胎牛血清、0.1%Proclin300的0.01mol/L Tris-HCl缓冲液按1/5000稀释，经0.22um滤膜过滤，分装12ml/管。3. Preparation of enzyme-labeled antibody: GP73 monoclonal antibody 5B12 was labeled with horseradish peroxidase HRP by the improved sodium periodate method-sodium borohydride method, that is, at low pH (pH4.4) HRP was passed through 0.1mol/ The NaIO₄ of L is oxidized to form aldolase, and the aldolase is connected to the amino group of antibody molecule 5B12 to form Schiff's base, which can be further reduced with 4 mg/ml NaBH₄ to obtain a stable enzyme-labeled antibody. The obtained 5B12*HRP was diluted 1/5000 with the antibody diluent, pH 7.4 containing 1% BSA, 5% fetal bovine serum, 0.1% Proclin300 in 0.01mol/L Tris-HCl buffer, and filtered through a 0.22um filter membrane , aliquot 12ml/tube.

4、配制浓缩洗涤液：配制20×的含0.05%Tween-20的PBST浓缩洗涤液。4. Prepare concentrated washing solution: prepare 20× PBST concentrated washing solution containing 0.05% Tween-20.

5、配制化学发光底物液：分别配制发光剂溶液A液以及氧化剂溶液B液，经0.22um滤膜过滤，后分装6ml/管，避光保存。5. Preparation of chemiluminescent substrate solution: prepare luminescent agent solution A and oxidant solution B respectively, filter through a 0.22um filter membrane, then pack into 6ml/tubes, and store in the dark.

6、组装为成品试剂盒，置于2-8℃保存。6. Assemble into a finished kit and store at 2-8°C.

实施例3本发明试剂盒的使用方法Embodiment 3 The using method of kit of the present invention

以实施例2制备的GP73化学发光定量检测试剂盒的具体操作如下：The specific operations of the GP73 chemiluminescent quantitative detection kit prepared in Example 2 are as follows:

1、试剂、样本制备1. Reagent and sample preparation

a）试剂盒从4℃取出，室温平衡放置20～30min；a) Take out the kit from 4°C, and place it at room temperature for 20-30 minutes;

b）冻干标准品每管加入500ul纯水进行充分溶解；b) Add 500ul of pure water to each tube of freeze-dried standard substance to fully dissolve;

c）浓缩洗涤液用纯水以1/20稀释成1×的洗涤液。c) Dilute the concentrated washing solution with pure water at 1/20 to make 1× washing solution.

d）使用前将待测样本置于室温平衡20-30min，轻轻摇动混匀，若样本出现沉淀物，应离心除去沉淀后使用，检测所用标本应符合试剂盒的检测范围，若浓度过高，可用样品稀释液稀释后再行检测。d) Before use, put the sample to be tested at room temperature to balance for 20-30 minutes, shake gently to mix well, if there is sediment in the sample, it should be centrifuged to remove the precipitate before use, the sample used for detection should meet the detection range of the kit, if the concentration is too high , can be diluted with sample diluent before detection.

2、操作步骤2. Operation steps

a）取出包被板，分别加入标准品以及待测样品，100ul/孔，37℃孵育50min；a) Take out the coated plate, add the standard and the sample to be tested respectively, 100ul/well, incubate at 37°C for 50min;

b）包被板拍干，加入酶标二抗5B12*HRP，100ul/孔，37℃孵育30min；b) Pat the coated plate dry, add enzyme-labeled secondary antibody 5B12*HRP, 100ul/well, incubate at 37°C for 30min;

c）洗板5遍，加入等体积混匀配制好的化学发光底物，100ul/孔，1min内进行Luminescence读值检测；c) Wash the plate 5 times, add the prepared chemiluminescent substrate with equal volume and mix well, 100ul/well, and detect the Luminescence reading within 1min;

d）以各标准品的发光值和相应浓度绘制标准曲线，得出发光值与浓度的关系方程式，将待测标本的发光值代入公式计算GP73蛋白含量。d) Draw a standard curve based on the luminescence value and the corresponding concentration of each standard substance, and obtain the relationship equation between the luminescence value and the concentration, and substitute the luminescence value of the sample to be tested into the formula to calculate the GP73 protein content.

实施例4GP73化学发光定量检测试剂盒的灵敏性研究Example 4 Sensitivity Research of GP73 Chemiluminescence Quantitative Detection Kit

使用实施例2制备的试剂盒，按实施例3所述步骤进行检测，其结果如表1和图1所示。可以看出本发明试剂盒绘制的标准曲线线性关系良好，检测范围在0.98～31.25ng/ml。Using the kit prepared in Example 2, the detection was carried out according to the steps described in Example 3, and the results are shown in Table 1 and Figure 1. It can be seen that the standard curve drawn by the kit of the present invention has a good linear relationship, and the detection range is 0.98-31.25 ng/ml.

表1Table 1

实施例5本发明化学发光底物与市场上同类化学发光底物对比分析Embodiment 5 Comparative analysis of the chemiluminescent substrate of the present invention and similar chemiluminescent substrates on the market

将实施例1中所述底物溶液与Thermo的supersignal ELSA Femto Maximum sensitivitysubatract进行对比。按实施例2准备GP73化学发光定量检测试剂盒，分别用两个底物检测试剂盒中标准品以及质控阳性样品以及阴性样品的RLU值，分别比较分析本发明化学发光底物和进口底物检测的标准品RLU以及信噪比，其结果如表2和图3所示，可以看到：与进口底物相比，本发明底物的RLU值虽然较低，但是其信噪比P/N值高于进口底物，而且本发明底物的成本远低于进口底物。The substrate solution described in Example 1 was compared with Thermo's supersignal ELSA Femto Maximum sensitivity subatract. Prepare the GP73 chemiluminescent quantitative detection kit according to Example 2, use the RLU values of the standard substance in the two substrate detection kits and the quality control positive sample and the negative sample respectively, and compare and analyze the chemiluminescent substrate of the present invention and the imported substrate respectively The standard product RLU of detection and signal-to-noise ratio, its result is as shown in table 2 and Fig. 3, can see: compared with imported substrate, although the RLU value of substrate of the present invention is lower, its signal-to-noise ratio P/ The N value is higher than that of the imported substrate, and the cost of the substrate of the present invention is much lower than that of the imported substrate.

表2Table 2

实施例6本发明试剂盒的临床样品研究The clinical sample research ofembodiment 6 kit of the present invention

按实施例2所组装试剂盒对一批临床血清样品包括重型肝炎患者40份，肝硬化患者39份，肝癌患者66份以及正常人血清样品50份，检测血清中所含GP73蛋白含量，用GraphpadPrism5进行统计学分析，其结果如图3所示，可以看出肝病患者血清GP73含量远高于正常人血清GP73，其中重型肝炎血清中GP73含量最高，肝癌患者血清中GP73含量高于肝硬化患者血清中GP73含量但无显著性差异。A batch of clinical serum samples including 40 severe hepatitis patients, 39 liver cirrhosis patients, 66 liver cancer patients and 50 normal human serum samples were tested by the kit assembled in Example 2 to detect the GP73 protein content in the serum, using GraphpadPrism5 Statistical analysis was carried out, and the results are shown in Figure 3. It can be seen that the serum GP73 content of patients with liver disease is much higher than that of normal people, and the serum GP73 content of severe hepatitis is the highest. GP73 content but no significant difference.

Claims (10)

1. a luminous substrate, is characterized in that, A, B liquid, consists of, and described A liquid comprises following component: 0.5～8mmol/L luminol, 0.5～10mmol/L are to iodophenol, 0.1mol/L Tris-HCl damping fluid; Described B liquid comprises following component: 1～10mmol/L urea peroxide, 0.1mol/L Tris-HCl damping fluid.

2. luminous substrate according to claim 1, is characterized in that, described A liquid comprises following component: 2.5mmol/L luminol, 3.0mmol/L are to iodophenol, 0.1mol/L Tris-HCl; Described B liquid comprises following component: 1.3mmol/L urea peroxide, 0.1mol/L Tris-HCl.

3. luminous substrate according to claim 1 and 2, is characterized in that, the pH value of described Tris-HCl is 8.5.

4. luminous substrate according to claim 1 and 2, it is characterized in that, described Tris-HCl damping fluid comprises the component of following mass percent: 0.05～0.3%BSA, 0.01～0.05%Tween-20,0.01～0.1%proclin300, with HCl, regulates pH to 8.5.

5. luminous substrate claimed in claim 1 is as the application of preparation ELISA immue quantitative detection reagent box.

6. the kit containing the luminous substrate described in claim 1, it is characterized in that, described kit comprises and is coated with the microwell plate of monoclonal antibody, the monoclonal antibody of HRP mark, freeze-drying standard items, sample diluting liquid, concentrated cleaning solution and chemical luminous substrate.

7. kit according to claim 6, is characterized in that, described monoclonal antibody is GP73 monoclonal antibody specific 5F10.

8. kit according to claim 6, is characterized in that, the monoclonal antibody of described HRP mark is GP73 monoclonal antibody 5B12*HRP.

9. kit according to claim 6, is characterized in that, the 0.01mol/L Tris-HCl damping fluid containing 1%BSA, 5% hyclone, 0.1%Proclin300 that described sample diluting liquid is pH7.4.

10. kit according to claim 6, is characterized in that, described concentrated cleaning solution is the PBST concentrated cleaning solution containing 0.05%Tween-20.

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