CN110132945A - 一种消除尿素干扰的化学发光法试剂盒配方 - Google Patents
一种消除尿素干扰的化学发光法试剂盒配方 Download PDFInfo
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Abstract
本发明公开了一种消除尿素干扰的化学发光法试剂盒配方，属于利用可见光，通过测试反应的结果产生颜色变化来测试材料的方法，属于免疫分析技术领域。本发明的技术方案是：化学发光法样品稀释液为含有尿素酶的Tris‑HCL缓冲液，尿素酶的用量根据血清样品体积，稀释体积，反应时间及酶动力学方程计算出酶用量，以使尿素酶促反应在测定反应前分解完尿素，本发明检测时不受内源性尿素影响，其使用方法和范围与原有化学发光法相同，经济方便易行，是一种准确性更高的化学发光检测方法。
Description
技术领域
本发明属于一种包含酶的测定方法；或是利用可见光，通过测试反应的结果产生颜色变化来测试材料的方法，特别是涉及一种应用尿素酶消除化学发光干扰的测定方法。
背景技术
化学发光免疫分析(chemiluminescence immunoassay,CLIA)是将具有高灵敏度的化学发光测定技术与高特异性的免疫反应相结合，用于各种抗原、半抗原、抗体、激素、酶、脂肪酸、维生素和药物等的检测分析技术，是继放免分析、酶免分析、荧光免疫分析和时间分辨荧光免疫分析之后发展起来的一项最新免疫测定技术。免疫学分析临床实验室常包括三种:1、直接化学发光，标记物为吖啶酯（雅培）或者ABEI（新产业）。2、酶促化学发光，标记物为碱性磷酸酶（厦门波生）或者辣根过氧化物酶（强生）。3、电化学发光，标记物为三联吡啶钌（罗氏）。在临床免疫测定中，尿素常常干扰抗原-抗体的结合而影响测定结果，实验研究表明，在6M尿素／5分钟、7M尿素／5分钟、8M尿素／5分钟和7M尿素／10分钟中，尿素对测定结果的干扰可达19％～31％，在健康人群和患者中血清中尿素的含量分别为3.1～8.0mmol/L和8.0～70mmol/L（如肾衰患者），应用化学发光法检测时血清中尿素常造成一定的干扰。
抗原抗体的结合实质上是抗原表位与抗体超变区中抗原结合点之间的结合，由于两者在化学结构和空间构型上呈互补关系，抗原决定簇和抗体分子可变区互补构型，使两分子间有较强的亲和力。空间构型互补程度不同，抗原和抗体分子之间结合力强弱也不同，互补程度高，则亲和力强，抗原抗体结合实质是分子表面的结合，犹如酶与底物的结合，是一种非共价键结合；维系这种结合是静电引力、范登华力、氢键和疏水键共同作用的结果，它使抗原抗体结合稳定并存在解离的可逆性。在实验中加入高浓度尿素后，大量的尿素分子与抗体肽链形成氢键能力比水分子强，尿素分子中氢原子与抗体的肽链中氧原子作用而形成氢键，还能破坏抗体蛋白质分子内部氢键，使抗体肽链中靠氢键维系的二级结构如螺旋、折叠、自由回转结构遭到破坏，进而降低蛋白质非极性基因与水的疏水作用，这样抗原抗体间的氢键、疏水作用大大降低，造成低亲和力抗体与抗原结合的互补程度差而发生解离，从而被洗涤液洗脱；高亲和力抗体因与抗原结合互补程度高这点破坏对其影响小，而没有被洗脱液洗脱。
尿素酶常作为生物化学试剂中工具酶测定血清尿素氮，血清中尿素在脲酶的作用下，水解生成氨和二氧化碳，氨在α-酮戊二酸和还原型辅酶Ⅰ作用下，经谷氨酸脱氢酶的作用下NADH被氧化生成NAD+,在340nm波长处下监测吸光度的变化，可计算出血清中尿素的含量。在本发明化学发光法测定中，当血清加入到含有尿素酶的稀释液中时，血清中的尿素在尿素酶的作用下，水解生成氨和二氧化碳，酸性缓冲液中易于正反应进行，使反应液中的尿素不干扰化学发光法中抗原-抗体的结合，从而不影响反应结果。特别是在透析前后患者，由于血清中尿素变化而使检验结果出现较大变异。
化学发光法试剂中，在肝炎系列检测中血清稀释液常为磷酸盐缓冲液或生理盐水，在游离甲状腺素和游离三碘甲状原氨酸检测中血清稀释液常用含有小牛血清的磷酸盐缓冲液，在本发明中，采用含有尿素酶的Tris-HCL缓冲液为样品稀释液测定梅毒螺旋体抗体为例，其干扰性明显降低。稀释液中尿素酶的用量与样品稀释比和稀释作用时间相关，根据酶动力学法计算出尿素酶的用量。
反应公式为：
尿素酶
(NH2)2CO＋3H2O 2NH3+CO2
发明内容
为了解决现有技术中血清内源性尿素干扰抗原-抗体反应而影响测定结果的问题，本发明提供一种经济方便易行，准确性更高、能够消除内源性尿素影响的化学发光测定方法。
采用的技术方案是：样品稀释液采用含有尿素酶的Tris-HCL为缓冲液，本发明以安图生物测定梅毒螺旋体抗体为例配置样品稀释液，采用日本东洋纺尿素酶，其米氏常数为1.05×10-2M。具体参数如下：血清样品体积为10μL，稀释液体积300μL，稀释作用时间为5分钟，根据酶动力学方程计算出工具酶用量，在1升Tris-HCL缓冲液溶解41.66mL尿素酶溶液。
本发明与现有技术相比有如下优点：本发明配制的化学发光法样品稀释液不受血清中内源性尿素的干扰，特别是肾衰患者，由于高浓度的尿素造成非特异性干扰而影响化学发光法进行测定，本发明方法能够显著降低尿素的干扰，其使用方法和范围与原有的化学发光法相同，不会增加实验人员的负担，经济方便易行，是一种准确性更高的检测方法。
具体实施方式：
下面通过采用安图A2000PLUS化学发光分析仪测定梅毒螺旋体抗体实施例对本发明做进一步详细说明。
实施例1
试剂的组成：
a. 梅毒螺旋体抗体试剂为郑州安图生物工程股份有限公司配套试剂，采用双抗原夹心法原理进行检测，用梅毒螺旋体抗原包被磁微粒，辣根过氧化物酶标记梅毒螺旋体抗原制备酶结合物，通过免疫反应形成抗原-抗体-抗原-酶复合物，该复合物催化发光底物发出光子，发光强度与梅毒螺旋体抗体的含量成正比。
b.样品稀释液为本发明配置稀释液，每升Tris-HCl 缓冲液内含Tris150 mmol(pH6.0)溶解41.66mL尿素酶溶液，200µl Proclin-300防腐剂，尿素酶的终浓度为≥1000U/L。
实施例2
试剂的组成：
a. 梅毒螺旋体抗体试剂为郑州安图生物工程股份有限公司配套试剂。
b.样品稀释液为郑州安图生物工程股份有限公司配套试剂。
实施例3
1.检测对象：肾衰患者66例，其中非梅毒感染患者50例，男性28例，平均年龄38.5岁；女性22例，平均年龄34.5岁。梅毒感染患者16例，其中男性9例，平均年龄44.5岁；女性7例，平均年龄36.5岁。
2.方法：采用实施例1和实施例2分别测定肾衰患者梅毒螺旋体抗体，比较透析前后患者前后发光值的改变。
3.仪器： AutoLumo A2000 PLUS化学发光检测仪
4.结果
4.1 采用实施例1和实施例2分别测定非梅毒感染肾衰患者透析前后发光值比较如表1：
表1 本两种方法测定非梅毒感染肾衰患者透析前后发光值（RLU）比较
组别 例数 实施例1 实施例2
透析前组 50 3804±563 3347±685
透析后组 50 3935±545 3560±765
4.2 采用实施例1和实施例2分别测定测定梅毒感染肾衰患者透析前后发光值比较如表2：
表2 本两种方法测定梅毒感染肾衰患者透析前后发光值（RLU）比较
组别 例数 实施例1 实施例2
透析前组 16 46798084±9884630 42675108±1043795
透析后组 16 47759760±9887608 44876908±1563908
通过表1和2统计学分析得知：采用实施例2在测定非梅毒感染组肾衰患者前后，发光值结果有显著性差异，t=11.98, P=0.0338；在测定梅毒感染组肾衰患者前后，发光值结果有高度显著性差异，t=79.89, P=0.0091。采用实施例1在测定非梅毒感染组肾衰患者前后，发光值结果无显著性差异，t=1.04, P=0.5338；在测定梅毒感染组肾衰患者前后发光值结果无显著性差异，t=2.05, P=0.3776。
经过以上统计学分析可看出，本发明采用含有尿素酶的化学发光法稀释液可以有效地降低血清中内源性尿素的干扰，特别是高尿素血症患者使测定结果具有一致性和可靠性，具有推广应用前景和应用价值。
Claims (3)
1.一种消除尿素干扰的化学发光法试剂盒配方，其特征在于样品稀释液中含有尿素酶，利用尿素酶可以分解血清中的尿素，来消除尿素对抗原-抗体亲和力的干扰，样品稀释液为含有尿素酶的Tris-HCL缓冲液，根据血清样品体积，稀释体积，反应时间及酶动力学方程计算出酶用量，以使尿素酶促反应在测定反应前降解尿素，使尿素不干扰化学发光法测定。
2.根据权利要求1所述的一种消除尿素干扰的化学发光法试剂盒配方，其特征在于每升样品稀释液中溶解41.66mL尿素酶溶液，200µl Proclin-300防腐剂，尿素酶的终浓度为尿素酶的终浓度为≥1000U/L。
3.根据权利要求1所述的一种消除尿素干扰的化学发光法试剂盒配方，其特征在于样品稀释液中Tris-HCl缓冲液的浓度为150mmol/L，pH值为6.0±0.2。
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