CN117512061A - 一种血清中高密度脂蛋白胆固醇的测定试剂 - Google Patents
一种血清中高密度脂蛋白胆固醇的测定试剂 Download PDFInfo
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Abstract
本发明提供了一种血清中高密度脂蛋白胆固醇的检测试剂盒，属于生化检测技术领域。本发明提供的血清中高密度脂蛋白胆固醇的检测试剂盒包括胆固醇氧化酶、胆固醇酯酶、脂肪醇聚氧乙烯醚、牛血清白蛋白、Tween‑80、青霉素、甘油、β‑环糊精。使用较少的样本量便可对目标物质实现灵敏、准确的检测，且减小了受样本基质影响的可能性。
Description
技术领域
本发明涉及生化检测技术领域，尤其涉及一种血清中高密度脂蛋白胆固醇的测定试剂。
背景技术
高密度脂蛋白(high density lipoprotein,HDL)是密度最大的脂蛋白，它将胆固醇从周围组织转运到肝脏进行再循环或以胆酸的形式排泄。现有技术研究表明血浆中HDL和动脉粥样硬化的发生成负相关，其检测广泛用于预防或诊断缺血性心脏病，并且，血浆中高密度脂蛋白胆固醇(high densitylipoprotein-cholesterol,HDL-C)的水平高低还是冠心病、心脑血管病、肝炎、肝硬化等疾病的非常重要的临床检验指标。
测定高密度脂蛋白胆固醇的方法有超速离心法、电泳法以及沉淀法，超速离心法的分离操作需要较长的时间，且仪器本身价格昂贵，不适合日常检查；电泳法需要使用琼脂糖凝胶等作为载体分离，会由于电泳支持介质的不同而导致分离效果不同，也会因使用条件或使用检测试剂的不同而产生差异等，而且在定量方面也存在问题；沉淀法是使用多聚阴离子和二价金属离子试剂沉淀大颗粒的脂蛋白，然后离心，再用其上清液检测其含量，结果不够准确。
目前已经有相关专利报道了可对高密度脂蛋白胆固醇快速检测的产品，尽管所报道的产品均适合用于家庭快速诊断，但由于此类产品均是基于显色原理，而显色本身需要反应层生成较多的产物以使颜色达到一定的深度变化才能被检测，这使得这些产品进行检测时需要较高的样本量，同时，显色本身涉及到酶反应，这增加了产品涉及的酶的种类及用量。再者，酶反应受样本基质影响很大，从而影响检测结果的准确性。
发明内容
本发明的目的在于提供一种血清中高密度脂蛋白胆固醇的测定试剂，使用较少的样本量便可对目标物质实现灵敏检测，且减小了受样本基质影响的可能性。
为了实现上述发明目的，本发明提供以下技术方案：
本发明提供了一种血清中高密度脂蛋白胆固醇的检测试剂盒，所述试剂盒包括胆固醇氧化酶、胆固醇酯酶、脂肪醇聚氧乙烯醚、牛血清白蛋白、Tween-80、青霉素、甘油、β-环糊精。
优选的，所述试剂盒中各成分的百分比为胆固醇氧化酶、胆固醇酯酶总共68～75％、脂肪醇聚氧乙烯醚7～10％、牛血清白蛋白15～19％、Tween-803～3.5％、青霉素1.5～1.8％、甘油0.5～0.8％，β-环糊精0.5～1％，以上成分总量为100％。
优选的，所述胆固醇氧化酶、胆固醇酯酶、脂蛋白脂酶的比例为2～5：5～2：1～3。
优选的，所述试剂盒还包括含有量子点的硝酸纤维素膜。
本发明提供的检测试剂盒准确性高且具有较宽的线性范围。通过加入能被高密度脂蛋白胆固醇的氧化产物双氧水淬灭的量子点，检测被测物中高密度脂蛋白胆固醇的量。由于极微量的双氧水便可引起量子点淬灭，因此可使用较少的样本量对目标物质实现灵敏检测，也使得试纸受样本基质影响的可能性变小。
具体实施方式
下面结合实施例对本发明提供的技术方案进行详细的说明，但是不能把它们理解为对本发明保护范围的限定。
实施例1
本实施例的高密度脂蛋白胆固醇的检测试剂盒各组分的质量百分比如下：
胆固醇氧化酶35％、胆固醇酯酶36.5％、脂肪醇聚氧乙烯醚7.5％、牛血清白蛋白15％、Tween-803％、青霉素1.8％、甘油0.7％、β-环糊精0.5％；用0.01mol/LPBS(pH 7.4)将曲拉通X-100配制成3.0g/L的浓度，将硝酸纤维素膜(Millipore 135)浸于其中30s，之后在37℃下干燥24h，再用划膜机将量子点划于处理后的硝酸纤维素膜上，干燥即可。
检测步骤：将试剂盒的试剂成分在37℃条件下保温3h，血清样本和试剂按1:100的体积比混合，37℃条件下反应1h，取10μL反应液滴至硝酸纤维素膜上，5min后用荧光免疫分析仪测定荧光强度。
实施例2
本实施例的高密度脂蛋白胆固醇的检测试剂盒各组分的百分比如下：
胆固醇氧化酶35％、胆固醇酯酶35％、脂肪醇聚氧乙烯醚9％、牛血清白蛋白15％、Tween-803％、青霉素1.5％、甘油0.8％、β-环糊精0.7％；用0.01mol/LPBS(pH 7.4)将曲拉通X-100配制成3.0g/L的浓度，将硝酸纤维素膜(Millipore 135)浸于其中30s，之后在37℃下干燥24h，再用划膜机将量子点划于处理后的硝酸纤维素膜上，干燥即可。检测步骤同实施例1。
实施例3
本实施例的高密度脂蛋白胆固醇的检测试剂盒各组分的百分比如下：
胆固醇氧化酶35％、胆固醇酯酶33％、脂肪醇聚氧乙烯醚10％、牛血清白蛋白16％、Tween-803％、青霉素1.5％、甘油0.5％、β-环糊精0.5％；用0.01mol/LPBS(pH 7.4)将曲拉通X-100配制成3.0g/L的浓度，将硝酸纤维素膜(Millipore 135)浸于其中30s，之后在37℃下干燥24h，再用划膜机将量子点划于处理后的硝酸纤维素膜上，干燥即可。检测步骤同实施例1。
实施例4
标准曲线的制作
采用高密度脂蛋白胆固醇标准品，浓度分别为0.5、1.0、1.5、2.0、2.5、3.0、3.5mmol/L，使用实施例1的试剂盒，按照实施例1的检测步骤得到高密度脂蛋白胆固醇标准曲线。标准曲线方程：y＝0.194x+0.0221，R2＝0.996。可见，本发明得到的高密度脂蛋白胆固醇试剂盒是合格的。
实施例5
高密度脂蛋白胆固醇试剂盒准确度的测试
使用本发明实施例1～3中的试剂盒测定临床样本的HDL-C含量(mmol/L)(每例测定3次)，将超速离心法测得的HDL-C含量(mmol/L)作为评价该试剂盒的准确度的标准，计算相对偏差。结果如表1所示。
表1试剂盒准确度对比
从表1可以看出，本试剂盒与超速离心法测得结果的相对偏差均小于5％，使得HDL-C含量测试结果更加准确。
实施例6
为了得到最适的表面活性剂，本发明还将脂肪醇聚氧乙烯醚与聚乙烯吡咯烷酮K、烷基酚聚氧乙烯醚进行了对比(方法同实施例1)，测定不同种类的表面活性剂对试剂盒检测准确度的影响，以超速离心法测得的HDL-C含量(mmol/L)作为评价该试剂盒的准确度的标准，每项测定3次，计算平均值M(mmol/L)，结果如表2：
用聚乙烯吡咯烷酮K、烷基酚聚氧乙烯醚替换脂肪醇聚氧乙烯醚，加入的质量百分比与脂肪醇聚氧乙烯醚相同。
表2不同的表面活性剂对试剂盒准确度的影响
| 1 | 2 | 3 | M | |
| 烷基酚聚氧乙烯醚 | 1.15 | 1.12 | 1.10 | 1.12 |
| 聚乙烯吡咯烷酮K | 1.13 | 1.12 | 1.15 | 1.13 |
| 脂肪醇聚氧乙烯醚 | 1.20 | 1.22 | 1.25 | 1.22 |
| 超速离心法 | 1.23 | 1.21 | 1.20 | 1.21 |
由此可见，本发明使用脂肪醇聚氧乙烯醚作为表面活性剂，使得试剂盒检测的准确性明显提高。
以上所述仅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，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。
Claims (4)
1.一种血清中高密度脂蛋白胆固醇的检测试剂盒，其特征在于，所述试剂盒包括胆固醇氧化酶、胆固醇酯酶、脂肪醇聚氧乙烯醚、牛血清白蛋白、Tween-80、青霉素、甘油、β-环糊精。
2.根据权利要求1所述的检测试剂盒，其特征在于，所述试剂盒中各成分的百分比为胆固醇氧化酶、胆固醇酯酶总共68～75％、脂肪醇聚氧乙烯醚7～10％、牛血清白蛋白15～19％、Tween-803～3.5％、青霉素1.5～1.8％、甘油0.5～0.8％，β-环糊精0.5～1％，以上成分总量为100％。
3.根据权利要求2所述的检测试剂盒，其特征在于，所述胆固醇氧化酶、胆固醇酯酶、脂蛋白脂酶的比例为2～5：5～2：1～3。
4.根据权利要求1～3任一项所述的检测试剂盒，其特征在于，所述试剂盒还包括含有量子点的硝酸纤维素膜。
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