WO2022100240A1 - 一种抗链球菌溶血素o检测试剂盒 - Google Patents
一种抗链球菌溶血素o检测试剂盒 Download PDFInfo
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6854—Immunoglobulins
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
- G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
Definitions
- the invention relates to the technical field of clinical in vitro detection reagents, in particular to an anti-streptolysin O detection kit.
- Streptolysin is an exotoxin produced by group A streptococcus, referred to as anti-"O" or ASO, which can dissolve red blood cells and has a toxic effect on various cells of the body. After the human body is infected with hemolytic streptococcus, a large number of anti-streptolysin O antibodies can appear in the serum. ASO determination is very valuable for the diagnosis of group A streptococcus infection, and helps to diagnose diseases caused by hemolytic streptococcus such as rheumatoid, Diseases such as acute glomerular disease, scarlet fever, and tonsillitis, the presence and amount of which can reflect the severity of the infection. If the ASO titer does not decrease, it indicates that there may be recurrent infection or chronic infection; after multiple determinations, if the antibody titer gradually decreases, the disease is in remission.
- ASO anti-"O
- Anti-streptolysin O detection kit (latex immunoturbidimetric assay) is an analytical method that does not require sample pretreatment, requires less technology and equipment, and has higher precision and specificity, because this method does not require expensive
- the equipment can be automated and can measure a large number of specimens, so it has been widely promoted in clinical practice.
- the invention provides an anti-streptolysin O detection kit.
- the kit is consistent with the performance indicators such as the accuracy, linear range and stability of the conventional kit, the analytical sensitivity and anti-interference ability are improved by the buffer system and enzyme activity maintenance, which is beneficial to the clinical application of the reagent. Promote the application.
- the method of this kit for the detection of anti-streptolysin O is latex immunoturbidimetry, which mainly adsorbs the antibody peptide bond on the latex particle, and binds the specific antibody in the diluted human serum to the antigen through incubation. After incubation, the resulting change in absorbance is proportional to the concentration of specific antibody in the test sample.
- the present invention is achieved through the following measures:
- An anti-streptolysin O detection kit is characterized in that, it comprises reagent R1, R2 and calibrator, wherein the composition and content of reagent R1 are:
- composition and content of reagent R2 are:
- the buffer in the reagent R1 is one or more of a glycine buffer with a pH value of 2.25-5.0 and a citric acid-sodium citrate buffer.
- the biological enzyme is one or more of protease, lipase, antithrombin, catalase, bilirubin oxidase, and ascorbate oxidase.
- composition and concentration of the biological enzyme are:
- the anti-streptolysin O detection kit of the invention adopts the latex immunoturbidimetric method, and the optimized reaction system such as ⁇ -ketoglutaric acid, gentamicin, NADH, L-leucine and the like is added to the reagent, which can significantly improve the The sensitivity of the reagent; adding one or more biological enzymes can effectively avoid the interference of hemoglobin, heparin and bilirubin, and greatly enhance the anti-interference ability of the reagent; the accuracy and stability of the reagent are good, high sensitivity, strong anti-interference, It is easy to use and can fully meet clinical needs.
- the optimized reaction system such as ⁇ -ketoglutaric acid, gentamicin, NADH, L-leucine and the like is added to the reagent, which can significantly improve the The sensitivity of the reagent; adding one or more biological enzymes can effectively avoid the interference of hemoglobin, heparin and bilirubin, and greatly enhance the anti-interference
- Fig. 1 is the correlation curve diagram of Example 1 of the anti-streptolysin O detection kit of the present invention and Comparative Example 1;
- Fig. 2 is the correlation curve diagram of Example 2 of the anti-streptolysin O detection kit of the present invention and Comparative Example 1;
- Fig. 3 is the correlation curve diagram of Example 3 of the anti-streptolysin O detection kit of the present invention and Comparative Example 1;
- Fig. 4 is the correlation curve diagram of Example 4 of the anti-streptolysin O detection kit of the present invention and Comparative Example 1;
- the anti-streptolysin O detection kit of the present embodiment includes reagents R1, R2 and calibrators, and the reagents are composed as follows:
- composition and composition of reagent R1 are as follows:
- composition and composition of reagent R2 are as follows:
- the calibrator is composed of calibrators produced by Beijing Jiuqiang Company.
- the detection method of this embodiment using an automatic biochemical analyzer (such as Hitachi 7180 automatic analyzer) to experimentally verify the analytical performance of the kit prepared in the above embodiment, the specific operation is to place the above reagent R1 (reagent 1) in the On the corresponding reagent position, place normal saline, calibrator and sample in the corresponding position of the sample tray, and then add R2 (reagent 2).
- R1 reagent 1
- R2 reagent 2
- the anti-streptolysin O detection kit of this embodiment includes reagents R1, R2 and calibrators, and the reagents are composed as follows:
- composition and composition of reagent R1 are as follows:
- composition and composition of reagent R2 are as follows:
- the calibrator is composed of calibrators produced by Beijing Jiuqiang Company.
- the sample concentration is 20U/mL
- the specific operation method is the same as that of embodiment 1.
- the anti-streptolysin O detection kit of the present embodiment includes reagents R1, R2 and calibrators, and the reagents are composed as follows:
- composition and composition of reagent R1 are as follows:
- composition and content of reagent R2 are:
- the calibrator is composed of calibrators produced by Beijing Jiuqiang Company.
- Step 1 Take 5 equal parts of the sample, add different interfering substances, make the concentration in the sample meet the requirements of Table 2-2, and form the sample measured in the embodiment;
- step 2 The specific implementation method of step 2 is the same as that of embodiment 1.
- the anti-streptolysin O antibody detection kit of this embodiment includes reagents R1, R2 and calibrators, and the reagents are composed as follows:
- composition and composition of reagent R1 are as follows:
- composition and content of reagent R2 are:
- the calibrator is composed of calibrators produced by Beijing Jiuqiang Company.
- the specific operation steps are to evenly divide the above reagents into 13 groups.
- the amount of reagents in each group is composed of 20mL of R1 and 5mL of R2, placed in a refrigerator at 2-8°C, and a group of reagents are taken out on the same day of each month, and then follow the implementation
- the operation steps of Example 1 detect ASO quality control substance (target value is 182 ⁇ mol/L).
- a test kit with excellent accuracy recognized in the market, its composition includes calibrator, reagent 1, and reagent 2.
- the detection method of this comparative example is the same as the detection method of Examples 1-4 (but the composition and ratio of the comparative example reagents remain different. change) to measure the linearity, sensitivity, immunity and stability data of the comparative example.
- the detection method of sensitivity is to test a sample with a known concentration of 20 U/mL and record the rate of change in absorbance ( ⁇ A/min). Experiments were carried out using the kits of Comparative Examples and Examples 1-4, respectively. The test results are shown in Table 3-1:
- the anti-streptolysin O detection kit of the present invention optimizes the reaction system by adding ⁇ -ketoglutarate, gentamicin, NADH, L-leucine and biological enzymes to the reagent, which can not only Significantly improve the sensitivity of the reagent, can also effectively avoid the interference of hemoglobin, heparin and bilirubin, greatly enhance the anti-interference ability of the reagent.
- the detection results of the kit of the present invention have good correlation, and the clinical detection sample results are consistent, which can meet the application requirements of the market for products, and has high sensitivity and good anti-interference performance. It is a more stable and good anti-streptococcus Hemolysin O detection kit.
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Abstract
一种抗链球菌溶血素O检测试剂盒，包含试剂R1、R2和校准品。试剂R1的成分为：氨基乙酸缓冲液、α-酮戊二酸、庆大霉素、NADH、L-亮氨酸以及一种或多种生物酶；试剂R2的成分为：链球菌素O胶乳颗粒、庆大霉素、氯化铵。试剂盒的灵敏度高，稳定性良好，抗干扰性强。
Description
本发明涉及临床体外检测试剂技术领域，具体涉及一种抗链球菌溶血素O检测试剂盒。
链球菌溶血素是A群链球菌产生的一种外毒素，简称抗“O”或ASO，能溶解红细胞，并对机体多种细胞有毒性作用。人体感染溶血性链球菌后，血清中可出现大量抗链球菌溶血素O抗体，ASO测定对于诊断A族链球菌感染很有价值，有助于诊断由溶血性链球菌引起的疾病如类风湿、急性肾小球疾病、猩红热和扁桃体炎等疾病，其存在及含量可反映感染的严重程度。如果ASO滴度不下降，提示可能存在复发性感染或慢性感染；经过多次测定，若抗体效价逐渐下降，说明病情缓解。
血清中抗链球菌溶血素O(ASO)的含量检测方法目前有溶血抑制法和乳胶法两种，但是溶血抑制法操作比较繁琐，对人员要求较高，操作环境易受外界环境等因素影响，造成对ASO含量检测的不准确。而乳胶免疫法较溶血抑制法而言操作更简便、影响因素少、灵敏度较高且检测时间短，目前在临床链球菌感染疾病辅助诊断中应用较为广泛。
抗链球菌溶血素O检测试剂盒(胶乳免疫比浊法)是一种无需预处理样本，技术和设备要求不高，而精密度和特异性更高的分析方法，由于该方法不需要昂贵的设备，可以实现自动化，且可测定大量标本，因此受到临床广泛推广。
发明内容
本发明提供了一种抗链球菌溶血素O检测试剂盒。该试剂盒与与常规的试剂盒准确度、线性范围、稳定性等性能指标一致的前提下，通过缓冲液体系和酶活性保持，提高了分析灵敏度和抗干扰能力，有利于试剂在临床上的推广应用。
基本原理
本试剂盒对于抗链球菌溶血素O检测的方法是乳胶免疫比浊法，主要是将抗体肽键吸附于乳胶粒颗粒上，通过孵育使稀释的人血清中的特异性抗体与抗原结合，经孵育后，产生的吸亮度变化量与检测样本中的特异性抗体浓度成正比。
本发明是通过以下措施实现的：
一种抗链球菌溶血素O检测试剂盒，其特征在于，它包含试剂R1、R2和校准品，其中试剂R1成分和含量为：
试剂R2成分和含量为：
链球菌素O胶乳颗粒 0.01-0.20w/v％，
庆大霉素 0.01-0.05mg/L，
氯化铵 0.1-0.5mg/L；
进一步的，所述试剂R1中的缓冲液为pH值＝2.25-5.0的氨基乙酸缓冲液、柠檬酸-柠檬酸钠缓冲液中的一种或多种。
进一步的，所述校准品和所述试剂在使用时的体积比例为校准品：R1：R2＝3μL：240μL：60μL。
进一步的，所述生物酶为蛋白酶、脂肪酶、抗凝血酶、过氧化氢酶、胆红素氧化酶、抗坏血酸氧化酶其中的一种或者多种。
进一步的，所述生物酶的组成及浓度为：
与现有技术相比，本发明的有益效果体现在以下几个方面：
本发明的抗链球菌溶血素O检测试剂盒采用乳胶免疫比浊法，在试剂中添加α-酮戊二酸、庆大霉素、NADH、L-亮氨酸等优化反应体系，可以显著提高试剂的灵敏度；添加一种或多种生物酶可以有效避免血红蛋白、肝素和胆红素的干扰，大大增强试剂的抗干扰能力；试剂的准确度和稳定性良好，灵敏度高，抗干扰性强，使用方便，完全可以满足临床需要。
图1为本发明抗链球菌溶血素O检测试剂盒实施例1与对比例1相关性曲线图；
图2为本发明抗链球菌溶血素O检测试剂盒实施例2与对比例1相关性曲线图；
图3为本发明抗链球菌溶血素O检测试剂盒实施例3与对比例1相关性曲线图；
图4为本发明抗链球菌溶血素O检测试剂盒实施例4与对比例1相关性曲线图；
图5为本发明抗链球菌溶血素O检测试剂盒实施例1-4稳定性验证实验检测结果与对比例1检测结果曲线图。
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
本实施例的抗链球菌溶血素O检测试剂盒包括试剂R1、R2和校准品，所述试剂组成如下：
其中试剂R1的成分和组成如下：
试剂R2的成分和组成如下：
链球菌素O胶乳颗粒 0.01w/v％，
庆大霉素 0.01mg/L，
氯化铵· 0.1mg/L；
校准品组成为北京九强公司生产的校准品。
本实施例的检测方法：采用自动生化分析仪(如日立7180全自动分析仪)对上述实施例中制得的试剂盒分析性能进行实验验证，具体操作是将上述试剂R1(试剂1)放置到对应的试剂位上，在样品盘的对应位置放置好生理盐水、校准品和样本，然后加入R2(试剂2)，具体操作如表1：
表1
实施例2
本实施例的抗链球菌溶血素O检测试剂盒包括试剂R1、R2和校准品， 所述试剂组成如下：
其中试剂R1的成分和组成如下：
试剂R2的成分和组成如下：
链球菌素O胶乳颗粒 0.20w/v％，
庆大霉素 0.05mg/L，
氯化铵· 0.5mg/L；
校准品组成为北京九强公司生产的校准品。
本实施例的检测方法中样本浓度为20U/mL，具体操作方式同实施例1。
实施例3
本实施例的抗链球菌溶血素O检测试剂盒包括试剂R1、R2和校准品，所述试剂组成如下：
其中试剂R1的成分和组成如下：
试剂R2成分和含量为：
链球菌素O胶乳颗粒 0.01w/v％，
庆大霉素 0.01mg/L，
氯化铵 0.1mg/L；
校准品组成为北京九强公司生产的校准品。
本实施例的检测方法具体操作步骤如下：
步骤1：取样本5等份，加入不同的干扰物质，使其在样本中的浓度达到表2-2的要求，形成实施例所测样本；
步骤2的具体实施方法同实施例1。
计算：相对偏差(％)＝(干扰样本的测定均值－对照样本的测定均值)/对照样本的测定均值×100％。
实施例4
本实施例的抗链球菌溶血素O抗体检测试剂盒包括试剂R1、R2和校准品，所述试剂组成如下：
试剂R1的成分和组成如下：
试剂R2成分和含量为：
链球菌素O胶乳颗粒 0.20w/v％，
庆大霉素 0.05mg/L，
氯化铵 0.5mg/L；
校准品组成为北京九强公司生产的校准品。
具体操作步骤是将上述试剂均匀分装13组，每组的试剂量组成为20mL的R1，5mL的R2，放置到2-8℃冰箱中，每月的同一天取出一组试剂，然后按照实施例1的操作步骤检测ASO质控品(靶值为182μmol/L)。
对比例1
市场上获得认可的一种准确度优异的试剂盒，其组成包括校准品、试剂1、试剂2，本对比例检测方法同实施例1-4的检测方法(但对比例试剂组成及比例保持不变)，以测得对比例的线性、灵敏度、抗干扰性及稳定性数据。
线性数据及灵敏度实验
用本发明的抗链球菌溶血素O检测试剂盒实施例1-4测试其线性相关数据，其结果如图1，图中可看出实施例1-4与对比例的线性相关系数R
2接近1，线性相关较好。
灵敏度的检测方法是测试已知浓度在20U/mL的样本，记录吸光度变化率(ΔA/min)。分别利用对比例、实施例1-4的试剂盒进行实验。检测结果如表3-1所示：
表2-1灵敏度实验结果
通过上述检测数据可以看出，实施例1-4检测结果计算出的ΔA值明显高于对比例中的ΔA，即一定时间内吸光度的变化量更大，说明本发明试剂盒添加的α-酮戊二酸、庆大霉素、NADH、L-亮氨酸可以显著提高试剂的灵敏度。
抗干扰性试验
取5等份样品，加入不同的干扰物质，使其在血清中的浓度达到表2的要求。然后分别用对比例试剂盒及实施例1-4所得试剂同样测定样品中 抗链球菌溶血素O的含量，对照组测定结果与加入不同干扰物质后各组的测定结果见表2-2。
表2-2实施例试剂抗干扰性能比较
由表2-2可以看出，在样本中中加入干扰物后，胆红素≤60μmol/L、血红蛋白≤12mg/dL、肝素≤20IU/mL、EDTA≤300μmol/L时对实施例1-4试剂的测试结果相对偏差较小，没有明显干扰。而对比例3试剂在上述浓度干扰物质存在时，相对偏差都在10％以上，受到明显干扰，这表明本试剂盒通过添加本发明中所述的生物酶，优化反应缓冲体系，抗干扰性能显著提高，远远优于对比试剂。
稳定性实验
取本发明的实施例1-4检测试剂盒各13组与对比例1试剂盒，经冰箱放置一段时间后检测结果如图2所示(靶值为182μmol/L)，对比例检测结果围绕靶值浮动程度比实施例1-4更大，说明实施例1-4试剂在2-8℃储存条件下比市场上这种抗链球菌溶血素O试剂盒更加稳定。
综上所述，本发明的抗链球菌溶血素O检测试剂盒通过在试剂中添加α-酮戊二酸、庆大霉素、NADH、L-亮氨酸及生物酶优化反应体系，既可以显著提高试剂的灵敏度，也可以有效避免血红蛋白、肝素和胆红素的干扰，大大增强试剂的抗干扰能力。同类检测试剂对比，本发明试剂盒检测结果相关性好，临床检测样本结果一致，能够达到市场对产品的应用要求，并且灵敏度高、抗干扰性能好，是一种更加稳定、良好的抗链球菌溶血素O检测试剂盒。
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 (5)
- 一种抗链球菌溶血素O检测试剂盒，其特征在于，它包含试剂R1、R2和校准品，其中试剂R1成分和含量为：
试剂R2成分和含量为：链球菌素O胶乳颗粒 0.01-0.20w/v％，庆大霉素 0.01-0.05mg/L，氯化铵 0.1-0.5mg/L； - 根据权利要求1所述的抗链球菌溶血素O检测试剂盒，其特征在于，所述试剂R1中的缓冲液为pH值＝2.25-5.0的氨基乙酸缓冲液、柠檬酸-柠檬酸钠缓冲液中的一种或多种。
- 根据权利要求1所述的抗链球菌溶血素O检测试剂盒，其特征在于，所述校准品和所述试剂在使用时的体积比例为校准品：R1：R2＝3μL：240μL：60μL。
- 根据权利要求1所述的抗链球菌溶血素O检测试剂盒，其特征在于，所述生物酶为蛋白酶、脂肪酶、抗凝血酶、过氧化氢酶、胆红素氧化酶、抗坏血酸氧化酶中的一种或者多种。
- 根据权利要求4所述的抗链球菌溶血素O检测试剂盒，其特征在于，所述生物酶的组成及浓度为：
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|---|---|---|---|---|
| CN116298243A (zh) * | 2023-02-01 | 2023-06-23 | 河北艾驰生物科技有限公司 | 一种抗链球菌脱氧核糖核酸酶b测定试剂盒及其制备方法 |
| CN116298243B (zh) * | 2023-02-01 | 2023-09-08 | 河北艾驰生物科技有限公司 | 一种抗链球菌脱氧核糖核酸酶b测定试剂盒及其制备方法 |
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| WO2022100077A1 (zh) | 2022-05-19 |
| CN112485443A (zh) | 2021-03-12 |
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