WO2022100077A1 - 一种灵敏度高的抗Ro52抗体检测试剂盒 - Google Patents
一种灵敏度高的抗Ro52抗体检测试剂盒 Download PDFInfo
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
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Definitions
- the invention relates to the technical field of clinical in vitro detection, in particular to an anti-Ro52 antibody detection kit with high sensitivity.
- SS-A antigen is a small ribonucleoprotein composed of an RNA molecule and two different proteins (molecular weights of 52 and 60kDa, respectively).
- Anti-SS-A 60kDa antibody is specific for Sjögren's syndrome and systemic lupus erythematosus. higher, while the antibody against SS-A 52kDa alone does not have disease specificity and is prone to cross-react with other antibodies.
- anti-SSA/Ro antibodies are highly specific for Sjögren's syndrome and SLE, especially the immunoreactivity of anti-Ro antibodies with 60KD Ro protein (Ro60) and 52KD Ro protein (Ro52), respectively SLE and primary Sjögren's syndrome.
- SS-A/Ro antibodies are found in 85%-95% of Sjögren's syndrome patients. Therefore, this antibody is the main indicator to identify the disease.
- Anti-Ro52 antibody has higher specificity for primary Sjögren's syndrome than anti-Ro60 antibody. Ro52 autoantibodies can be detected alone in more than 60% of primary Sjögren's syndrome patients, while only 5% of SLE patients can detect this antibody alone.
- the methods commonly used in laboratories to detect anti-Ro52 antibodies mainly include enzyme-linked immunosorbent assay (ELISA), indirect immunofluorescence (IFF), immunoblotting test (IBT), and linear immunoassay. method (Line immunoassay, LIA) and so on.
- ELISA enzyme-linked immunosorbent assay
- IFF indirect immunofluorescence
- IBT immunoblotting test
- LIA linear immunoassay
- Indirect immunofluorescence method can be used for semi-quantitative determination, but the results need to be observed by fluorescence microscope, the objectivity of the results is insufficient, the standardization is difficult, the technical procedures are complicated, and it is not suitable for the detection of high-throughput specimens.
- Linear immunoassays are mainly used for the screening of major disease categories, which are relatively poorly targeted, and are not suitable for high-throughput sample detection.
- ELISA detection can be used for high-throughput sample determination, and its sensitivity is also high. It is widely recognized at present, but the operation is cumbersome, requiring multiple additions and washings, and is easily affected by temperature and incubation conditions. to operate.
- the detection method of commercial anti-Ro52 antibody detection kits on the market is mainly enzyme-linked immunosorbent assay. After multiple sample addition and washing steps, it takes about three hours to complete the entire experimental process.
- the operation of scientific and technical personnel in the laboratory is easily limited by personnel and venues, and at the same time, it is easily affected by various environmental conditions such as temperature and incubation time, which brings a lot of inconvenience to the experiment.
- the invention provides an anti-Ro52 antibody detection kit with high sensitivity. Compared with the widely used ELISA kits in the market, the kit can be detected by using a conventional biochemical analyzer. It is easy to operate, and can be fully automated for batch samples. It has high analytical sensitivity and good stability, which is beneficial to the reagents. application in clinical practice.
- the kit of the present invention is carried out on an automatic biochemical analyzer with dual-reagent function, and its specific technical scheme is as follows:
- the present invention provides an anti-Ro52 antibody detection kit with high sensitivity, which is composed of reagent R1 and reagent R2, wherein:
- composition and content of the reagent R1 are as follows:
- composition and content of the reagent R2 are as follows:
- the buffer is one or a combination of MES buffer and PBS buffer, and the pH value of the reagent buffer is 4-6.
- composition and content of the reagent R1 are as follows:
- composition and content of reagent R2 are as follows:
- composition and content of the reagent R1 are as follows:
- composition and content of reagent R2 are as follows:
- composition and content of the reagent R1 are as follows:
- composition and content of reagent R2 are as follows:
- the high-sensitivity anti-Ro52 antibody detection kit uses an automatic biochemical analyzer to measure the anti-Ro52 antibody by a two-point end-point method, and the main detection wavelength is 570 nm.
- the calibrator used in the present invention is the calibrator produced by Randox, UK.
- the quality control material used in the present invention is the quality control material produced by the British Randox Company.
- the present invention has the following beneficial effects:
- the high-sensitivity anti-Ro52 antibody detection kit of the present invention is determined by a two-point end-point method, respectively adding sodium chloride and polyethylene glycol of appropriate concentrations to the reagent R1, and adding a combination of glycerol and BSA of a suitable concentration to the reagent R2
- the stabilizer enhances the stability of the reagent; adding a suitable concentration of sodium azide as a preservative will not affect the accuracy of the reagent.
- the kit of the present invention improves the sensitivity of the kit by adding disodium EDTA as a catalyst and citric acid as a chelating agent into the reagent R1.
- Fig. 1 is the correlation curve diagram of Example 1 and the control group in the high-sensitivity anti-Ro52 antibody detection kit of the present invention
- Fig. 2 is the correlation curve diagram of Example 2 and the control group in the high-sensitivity anti-Ro52 antibody detection kit of the present invention
- Figure 3 is a graph of the correlation between Example 3 and the control group in the highly sensitive anti-Ro52 antibody detection kit of the present invention
- Fig. 4 is the open bottle stability curve diagram of reagent R1 in the highly sensitive anti-Ro52 antibody detection kit of the present invention
- Fig. 5 is the open bottle stability curve diagram of reagent R2 in the highly sensitive anti-Ro52 antibody detection kit of the present invention
- FIG. 6 is a schematic diagram of the specific use method of the high-sensitivity anti-Ro52 antibody detection kit of the present invention on an automatic biochemical analyzer with dual-reagent function.
- An anti-Ro52 antibody detection kit that has been approved in the market is a control reagent. It is strictly operated according to the instructions to screen positive samples.
- the detection threshold is 18IU/mL, and those greater than 18IU/mL are positive.
- the high-sensitivity anti-Ro52 antibody detection kit of this embodiment includes reagent R1 and reagent R2, wherein:
- composition and content of reagent R1 are as follows:
- composition and content of reagent R2 are as follows:
- the detection method of the present embodiment adopt a fully automatic biochemical analyzer with dual reagent function (such as Hitachi 7180 automatic analyzer, OLYMPUS AU640, etc.), and use the two-point endpoint method to measure, and the specific operations are as follows:
- the high-sensitivity anti-Ro52 antibody detection kit of this embodiment includes reagent R1 and reagent R2, wherein:
- composition and content of reagent R1 are as follows:
- composition and content of reagent R2 are as follows:
- the detection method is the same as that of Example 1.
- the high-sensitivity anti-Ro52 antibody detection kit of this embodiment includes reagent R1 and reagent R2, wherein:
- composition and content of reagent R1 are as follows:
- composition and content of reagent R2 are as follows:
- the detection method is the same as that of Example 1.
- the detection value of the kit corresponding to the comparative example is 10IU/mL
- the detection value of the kit corresponding to Example 1-3 is 10-11IU/mL
- the accurate value of the sample can still be detected; and compared with the comparative example, the kits corresponding to Examples 1-3 have higher accuracy in detecting low-value samples close to the linear lower limit.
- the detection value of the kit corresponding to the comparative example is 301IU/mL
- the detection value of the kit corresponding to Examples 1-3 is between 299-301IU/mL, which is still acceptable.
- the exact value of the sample was detected, which indicated that the kits corresponding to Examples 1-3 had higher analytical sensitivity.
- Reagents were prepared according to the formulations of Example 1, Example 2 and Example 3, respectively, and were tested in comparison with the control kit, and 40 clinical serum samples were tested simultaneously. The test results are shown in Tables 2-4.
- Example 1 The detection reagents obtained in Example 1, Example 2 and Example 3 of the present invention were evenly divided into two groups, and one group of detection reagents was tested for thermal stability at 37°C, and placed in a 37°C constant temperature water bath (only at 37°C every day). Take it out during the test, after the test, it is still sealed and put back in a 37°C water bath for 12 consecutive days); another group of test reagents is subjected to a 15-day bottle opening stability test, and the test reagents are opened and placed at 2-8°C Refrigerator (do not remove for 15 days). The reagents were detected on Hitachi 7180 automatic biochemical analysis instrument at the same time, and the standard curve was established on the instrument.
- the high-sensitivity anti-Ro52 antibody detection kit of the present invention has good accuracy, correlation, stability and sensitivity.
Abstract
一种灵敏度高的抗Ro52抗体检测试剂盒，包括试剂R1和试剂R2，其中，试剂R1的成分如下：缓冲液、聚乙二醇、氯化钠、防腐剂、催化剂、螯合剂；试剂R2的成分如下：缓冲液、包被Ro52抗原的胶乳、复合稳定剂、防腐剂，其能够有效增强分析灵敏度、稳定性。
Description
本发明涉及临床体外检测技术领域，具体涉及一种灵敏度高的抗Ro52抗体检测试剂盒。
SS-A抗原是由一个RNA分子和两种不同的蛋白质(分子量分别为52和60kDa)组成的小核糖核蛋白，抗-SS-A 60kDa蛋白的抗体对于干燥综合症和系统性红斑狼疮的特异性更高，而单独抗SS-A 52kDa的抗体不具有疾病的特异性，并且容易与其它抗体发生交叉反应。
在鉴别诊断中，抗SSA/Ro抗体对干燥综合症和SLE具有极高的特异性，尤其是抗Ro抗体与60KD Ro蛋白(Ro60)和52KD Ro蛋白(Ro52)的免疫反应，分别用于辨别SLE和原发性干燥综合症。SS-A/Ro抗体见于85％-95％的干燥综合症病人。因此，此抗体是鉴别此病的主要指标。抗Ro52抗体与抗Ro60抗体相比，对原发性干燥综合症具有更高的特异性。在超过60％的原发性干燥综合症病人中可单独检测到Ro52自身抗体，而只有5％的SLE病人可单独检测到此抗体。
目前实验室常用的检测抗Ro52抗体的方法主要有酶联免疫吸附剂试验(enzymelinked immunosorbent assay，ELISA)、间接免疫荧光法(indirect immunoflurescence，IFF)、免疫印迹法(immunoblottingtest，IBT)、线性免疫分析法(Line immuno assay，LIA)等。
免疫印迹法虽综合了SDS-PAGE的高分辨力和ELISA法的高特异性和敏感性，但操作相对复杂，且试剂具有较强的毒性和污染性。
间接免疫荧光法可作半定量测定，但需通过荧光显微镜观察结果，结果判定的客观性不足，标准化困难，技术程序比较复杂，也不适合高通量标本的检测。
线性免疫分析法主要用于疾病大类的筛查，针对性相对较差，同时也 不适合高通量样本的检测。
ELISA法检测可用于高通量标本测定，灵敏度也较高，目前被广泛认可，但操作比较繁琐，需多次加样和洗涤，且易受温度和孵育条件的影响，需在专业实验室里进行操作。
目前市场上商品化的抗Ro52抗体检测试剂盒的检测方法主要为酶联免疫吸附法，经过多次加样和洗涤步骤，完成整个实验过程需三小时左右，需酶标仪，亦需要专业免疫学技术人员在实验室进行操作，易受人员和场地的限制，同时易受各种温度和孵育时间等环境条件因素的影响，给实验带来诸多不便。
发明内容
本发明提供了一种灵敏度高的抗Ro52抗体检测试剂盒。该试剂盒与市场上广泛使用的酶联免疫吸附法试剂盒相比，使用常规生化仪即可实现检测，操作简便，可进行批量样本全自动化检测，分析灵敏度高，稳定性好，有利于试剂在临床上的推广应用。
本发明的试剂盒在具有双试剂功能的自动生化分析仪上进行，其具体技术方案如下：
本发明提供一种灵敏度高的抗Ro52抗体检测试剂盒，由试剂R1和试剂R2组成，其中：
所述试剂R1的成分和含量如下：
所述试剂R2的成分和含量如下：
进一步的，所述缓冲液为MES缓冲液、PBS缓冲液中的一种或两种组合，所述试剂缓冲液的PH值为4-6。
进一步的，所述试剂R1和试剂R2的体积比为试剂R1：试剂R2＝3:1。
进一步的，所述试剂R1的成分和含量如下：
试剂R2的成分和含量如下：
进一步的，所述试剂R1的成分和含量如下：
试剂R2的成分和含量如下：
进一步的，所述试剂R1的成分和含量如下：
试剂R2的成分和含量如下：
所述的灵敏度高的抗Ro52抗体检测试剂盒使用全自动生化分析仪，利用两点终点法对抗Ro52抗体进行测定，检测主波长为570nm。
所述的灵敏度高的抗Ro52抗体检测试剂，试剂R1和试剂R2在使用时的比例为试剂R1：试剂R2＝225：75。
本发明所使用的校准品为英国朗道公司生产的校准品。
本发明所使用的质控品为英国朗道公司生产的质控品。
加入生理盐水、样本或校准品15μL，再加入225μL的R1试剂预孵育5min后读取吸光度A1，之后再加入75μL的R2试剂反应5min后，读取吸光度A2，并计算ΔA。
与现有技术相比，本发明具有以下有益效果：
本发明的灵敏度高的抗Ro52抗体检测试剂盒通过两点终点法进行测定，分别在试剂R1中添加适宜浓度的氯化钠和聚乙二醇，在试剂R2中添加适宜浓度的甘油和BSA组合的稳定剂，增强了试剂的稳定性；添加适宜浓度的叠氮钠作为防腐剂，不会对试剂的准确度产生影响。本发明的试剂盒通过试剂R1中加入乙二胺四乙酸二钠作为催化剂，柠檬酸作为螯合剂，提高了试剂盒的灵敏度。
图1为本发明的灵敏度高的抗Ro52抗体检测试剂盒中实施例1与对照组的相关性曲线图；
图2为本发明的灵敏度高的抗Ro52抗体检测试剂盒中实施例2与对照组的相关性曲线图；
图3本发明的灵敏度高的抗Ro52抗体检测试剂盒中实施例3与对照组的相关性曲线图；
图4为本发明的灵敏度高的抗Ro52抗体检测试剂盒中试剂R1的开瓶稳定性曲线图；
图5为本发明的灵敏度高的抗Ro52抗体检测试剂盒中试剂R2的开瓶稳定性曲线图；
图6为本发明的灵敏度高的抗Ro52抗体检测试剂盒在具有双试剂功能的自动生化分析仪上具体使用方法示意图。
为使本发明要解决的技术问题、技术方案和优点更加清楚，下面将结合具体实施例和附图进行详细描述。
实施例1：
市场上获得认可的一种抗Ro52抗体检测试剂盒为对照试剂，按照说明书进行严格操作，筛选阳性样本，检测阈值为18IU/mL，大于18IU/mL的为阳性。
本实施例的灵敏度高的抗Ro52抗体检测试剂盒包括试剂R1和试剂R2，其中：
试剂R1的成分和含量如下：
试剂R2的成分和含量如下：
本实施例的检测方法：采用具有双试剂功能的全自动生化分析仪(如日立7180全自动分析仪、OLYMPUS AU640等)，利用两点终点法进行测定，具体操作如下：
加入生理盐水、样本或校准品15μL，再加入225μL的试剂R1预孵育5min后读取吸光度A1，之后再加入75μl的试剂R2反应5min后，读取吸光度A2，并计算ΔA。
实施例2：
本实施例的灵敏度高的抗Ro52抗体检测试剂盒包括试剂R1和试剂R2，其中：
试剂R1的成分和含量如下：
试剂R2的成分和含量如下：
检测方法同实施例1的检测方法。
实施例3：
本实施例的灵敏度高的抗Ro52抗体检测试剂盒包括试剂R1和试剂R2，其中：
试剂R1的成分和含量如下：
试剂R2的成分和含量如下：
检测方法同实施例1的检测方法。
灵敏度试验：
选取12个已知不同浓度的抗Ro52抗体样本(如表1)，然后将12个不同的样本分别使用实施例1、实施例2和实施例3中的配方配制的试剂以及对照试剂进行检测，检测结果如表1所示：
表1 灵敏度试验结果
| 理论浓度(IU/mL) | 300 | 270 | 240 | 210 | 180 | 150 | 120 | 90 | 60 | 30 | 20 | 10 |
| 对比例检测结果 | 301 | 271 | 240 | 209 | 180 | 151 | 120 | 91 | 60 | 29 | 21 | 10 |
| 实施例1检测结果 | 300 | 271 | 240 | 210 | 180 | 150 | 120 | 90 | 60 | 30 | 20 | 11 |
| 实施例2检测结果 | 299 | 270 | 241 | 209 | 180 | 151 | 121 | 90 | 60 | 30 | 20 | 10 |
| 实施例3检测结果 | 300 | 270 | 240 | 209 | 180 | 151 | 120 | 91 | 61 | 30 | 21 | 10 |
从表1中可以看出，在样本浓度低至10IU/mL时，对比例对应的试剂盒检测值为10IU/mL，而实施例1-3对应的试剂盒检测值为10-11IU/mL，仍可以检测出样本的准确值；并且与对比例相比，实施例1-3对应的试剂盒检测接近线性下限的低值样本时的准确度更高。另外，在样本浓度在线性上限300IU/mL时，对比例对应的试剂盒检测值为301IU/mL，，而实施例1-3对应的试剂盒检测值在299-301IU/mL之间，仍可以检测出样本的准确值，这表明实施例1-3对应的试剂盒拥有更高的分析灵敏度。
相关性试验：
按照实施例1、实施例2和实施例3的配方分别配制试剂，与对照试剂盒进行对照检测，同时检测40个临床血清样本，检测结果如表2-表4所示。
表2 实施例1试剂相关性试验结果
表3 实施例2试剂相关性试验结果
表4 实施例3试剂相关性试验结果
由表2-表4可以看出，实施例1、实施例2和实施例3的试剂盒与对照试剂盒的相关系数为0.99以上，说明实施例1、实施例2和实施例3与对照试剂盒有极大的相关性。
稳定性试验：
将本发明实施例1、实施例2和实施例3中所得检测试剂均匀分装两组，一组检测试剂做37℃热稳定性检测试验，封闭放置在37℃恒温水浴 锅中(每天仅在检测的时候取出，检测完毕后，依然封口放回37℃水浴锅中，连续12天)；另一组检测试剂做15天开瓶稳定性检测试验，将检测试剂开瓶放置到2-8℃冰箱中(15天不取出)。将试剂同时在日立7180全自动生化分析仪器上进行检测，并在仪器上建立标准曲线。取冻干粉质控品，溶解均匀后，平均分成15份，-20℃储存，每天质控一个，并且跟踪检测结果。实施例1、实施例2和实施例3的试剂盒热稳定性跟踪监测趋势如图4所示，实施例1、实施例2和实施例3的试剂盒开瓶稳定性跟踪监测趋势如图5所示。
从图4可以看出，实施例1在37℃热稳定性实验中，7天时的稳定性仅下降6.8％，实施例2的试剂盒下降5.4％，实施例3下降4.1％，而对照例试剂盒下降了31.21％，说明本发明的试剂盒在37℃热稳定性试验中性能指标良好。
从图5可以看出，实施例1在开瓶稳定性实验中，20天时的稳定性仅下降5.37％，实施例2的试剂盒下降5.56％，实施例3下降4.98％，而对照例试剂盒下降了21.12％，说明本发明的试剂盒在开瓶稳定性试验中性能指标良好。
综上所述，本发明的灵敏度高的抗Ro52抗体检测试剂盒具有良好的准确度、相关性、稳定性和灵敏性。
以上所述是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明所述原理的前提下，还可以作出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。
Claims (6)
- 一种灵敏度高的抗Ro52抗体检测试剂盒，其特征在于，包括试剂R1和试剂R2，其中：所述试剂R1的成分和含量如下：
试剂R2的成分和含量如下：
- 根据权利要求1所述的灵敏度高的抗Ro52抗体检测试剂盒，其特征在于，所述缓冲液为MES缓冲液、PBS缓冲液中的一种或两种组合，所述试剂缓冲液的PH值为4-6。
- 根据权利要求1所述的灵敏度高的抗Ro52抗体检测试剂盒，其特征在于，所述试剂R1和试剂R2的体积比为试剂R1：试剂R2＝3:1。
- 根据权利要求1-3中任一所述的灵敏度高的抗Ro52抗体检测试剂盒，其特征在于，所述试剂R1的成分和含量如下：
试剂R2的成分和含量如下：
- 根据权利要求1-3中任一所述的灵敏度高的抗Ro52抗体检测试剂盒，其特征在于，所述试剂R1的成分和含量如下：
试剂R2的成分和含量如下：
- 根据权利要求1-3中任一所述的灵敏度高的抗Ro52抗体检测试剂盒，其特征在于，所述试剂R1的成分和含量如下：
试剂R2的成分和含量如下：
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