CN112063686B - 一种肝功能复合质控品及其制备方法和应用 - Google Patents
一种肝功能复合质控品及其制备方法和应用 Download PDFInfo
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Abstract
本发明涉及一种肝功能复合质控品及其制备方法和应用，其中所述复合质控品包括：α‑L‑岩藻糖苷酶、腺苷脱氨酶和5′‑核苷酸酶，其制备方法包括：以新鲜猪肝为原料，通过研磨匀浆、破碎离心、盐析提纯、超滤冻干等工艺得到一种同时含有三种酶的肝功能复合质控品。通过对制备工艺参数的优化，不仅避免了制备过程中酶失活，同时减少了成品中的杂质，使目标酶纯度提高。进一步通过冻干工艺得到稳定性更好的复合质控品冻干粉，并且对酶液的保护剂进行优化，使复合质控品冻干粉的热稳定性和长期稳定性增强，从而得到了一种制备方法简单快速，成本低廉，且酶活性、均一性和稳定性高的肝功能复合质控品。
Description
技术领域
本发明属于生化检测试剂技术领域，具体涉及一种肝功能复合质控品及其制备方法和应用。
背景技术
体外诊断试剂的质控品是实现体外诊断试剂临床质量控制的重要工具，其质量的好坏直接关系到体外诊断试剂的诊断结果。质控品分为液体和冻干两种形态，其中液态质控品操作方便，但是稳定性较差，冻干质控品的稳定性相对更好，使用前需要复溶即可用于检测。
腺苷脱氨酶(ADA)是一种与机体细胞免疫活性有重要关系的核酸代谢酶，ADA活性是反映肝损伤的敏感指标，主要用于监测和诊断肝脏疾病。5′-核苷酸酶(5′-NT)广泛存在于肝脏和各种组织中，血清中5′-NT活性升高主要见于肝胆胰系统疾病及某些恶性病变，因此具有较高的诊断价值。α-L-岩藻糖苷酶(AFU)是一种酸性水解酶，在诊断肝细胞癌中敏感性好，阳性率高，AFU是早期原发性肝癌诊断的有效指标。ADA、5′-NT和AFU均为肝功能检测项目，目前市面上都是针对单个检测项目的质控品，然而单个质控品的使用会造成操作上的不方便，并且现有的质控品所采用的酶多为纯酶，酶的制备工艺过程繁琐，操作复杂，耗时长且价格高昂，因此急需一种成本低廉、制备方法简单同时使用方便且性能稳定的肝功能复合质控品。
发明内容
本发明针对现有技术存在的缺陷，提供了一种肝功能复合质控品及其制备方法和应用，以猪肝为原料，通过研磨匀浆、破碎离心、盐析提纯、超滤冻干等一系列工艺，得到了一种成本低廉、使用方便、稳定性和均一性好的肝功能复合质控品。
为实现上述目的，本发明采用的技术方案是：
一种肝功能复合质控品，所述复合质控品包括：α-L-岩藻糖苷酶、腺苷脱氨酶、5′-核苷酸酶，所述复合质控品的制备方法包括：
S1、取新鲜猪肝，加入猪肝重量1/5～1/10的提取液捣碎得到第一混合液；
S2、向第一混合液中加入第一混合液质量8～10倍的提取液，匀浆得到第二混合液；
S3、向第二混合液中加入体积比为0.1％～0.5％的去垢剂并超声处理，得到第三混合液；
S4、将第三混合液进行离心，弃上清，用沉淀质量1/5～1/10的提取液复溶沉淀得到第四混合液；
S5、向第四混合液中加入盐析药品，其中盐析药品的加入量为40～60g/L，于2-8℃放置3-6h，获得第五混合液；
S6、将第五混合液离心，收集上清即为第六混合液，向第六混合液加入到可截留大小10～30KDa蛋白的超滤管中，于2-8℃放置10-20h后，收集滤出液即为所述肝功能复合质控品。
本发明以新鲜猪肝为原料，通过研磨匀浆、超声破碎离心、盐析提纯、超滤提纯得到含有α-L-岩藻糖苷酶、腺苷脱氨酶、5′-核苷酸酶的肝功能复合质控品，通过对制备工艺的优化，可维持混合液中所需酶，即α-L-岩藻糖苷酶、腺苷脱氨酶、5′-核苷酸酶的活性和稳定性，同时使其他酶失活并除去体系中其他杂质，即通过简单快速的制备工艺即可得到同时含有三种活性、稳定性和纯度均较高的肝功能相关酶的复合质控品。
进一步的，步骤S5中所述盐析药品的加入量为50g/L。由于不同的蛋白质进行盐析沉淀提纯时，对于所需盐的浓度不同，通过调节加入盐析药品的浓度可使体系中其他杂质沉淀，而所需的三种酶仍保留在上清液中，从而与溶液中其他杂质蛋白分离开来，以得到较高浓度的酶。
进一步的，步骤S5中所述盐析药品为氯化钠、氯化钾、硝酸铵、硫酸铝、氯化铵、氯化镁、硫酸镁、硫酸铵中一种或多种。
进一步的，步骤S1、S2和S4中所述提取液的pH为7.0～7.4，由20～100mmol/L Tris缓冲液，1.6～10mmol/L无机盐，1.3～8.1mmol/L金属离子螯合剂和质量体积比为0.1～5％的防腐剂组成。
进一步的，提取液中无机盐为氯化钠、氯化钾、氯化镁中的一种或多种。
进一步的，提取液中金属离子螯合剂为EDTA、HEDTA、EDTA-2Na中的一种或多种。
进一步的，步骤S3中所述去垢剂为聚乙二醇对异辛基苯基醚，牛磺胆酸钠，十二烷基磺酸钠中的一种或多种。通过加入去垢剂并配合超声破碎处理，使猪肝中的蛋白沉淀下来，与体系中其他杂质分离开来。
进一步的，步骤S3中超声处理的过程具体为：功率150W，超声60s，停歇60s，连续40次，然后停顿5分钟，再重复超声步骤，反复进行5次，直到肝脏完全破碎为止。
进一步的，所述制备方法中还包括稀释冻干步骤，具体为：将步骤S6得到的肝功能复合质控品用保护剂稀释后冻干得到冻干粉。
进一步的，所述保护剂的pH为7.0～7.5，由20～100mmol/L Tris缓冲液，0.05～1mmol/L无机盐，4～12g/L甘露醇，3～10g/L葡聚糖，8～15g/L牛血清白蛋白，0.03～0.8mmol/L EDTA和质量体积比为0.1～1％的防腐剂组成。通过对保护剂中组分进行优化，得到一种均一性和稳定性好的复合质控品冻干粉。
进一步的，所述冻干步骤具体为：将稀释后的溶液于-10～-30℃条件下预冻8h，然后于-50～-70冷冻8h，接着于气压低于20Pa条件下减压干燥13h，即得所述肝功能复合质控品。
本发明还提供了一种检测试剂盒，所述检测试剂盒包含上述复合质控品。
进一步的，所述检测试剂盒与α-L-岩藻糖苷酶检测试剂盒或腺苷脱氨酶检测试剂盒或5′-核苷酸酶检测试剂盒配套使用。
与现有技术相比，本发明的有益效果是：本发明针对现有技术中无α-L-岩藻糖苷酶、腺苷脱氨酶、5′-核苷酸酶三种肝功能检测项目的复合质控品的技术空白以及酶质控品制备工艺复杂、成本高昂的问题，提供了一种同时含有α-L-岩藻糖苷酶、腺苷脱氨酶、5′-核苷酸酶的肝功能复合质控品及其制备方法，通过对制备工艺参数的优化，不仅避免了制备过程中酶失活，同时减少了成品中的杂质，使目标酶纯度提高。进一步通过冻干工艺得到稳定性更好的复合质控品冻干粉，并且对酶液的保护剂进行优化，使复合质控品冻干粉的热稳定性和长期稳定性增强，从而得到了一种制备方法简单快速，成本低廉，且酶活性、均一性和稳定性高的肝功能复合质控品。将其与酶检测试剂盒联用，可用于验证试剂盒或检查分析仪器或检测方法的准确性和稳定性。
具体实施方式
下面将结合本发明中的实施例，对本发明的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动条件下所获得的所有其它实施例，都属于本发明保护的范围。
实施例1
复合质控品的制备过程中，加入盐析药品提纯步骤对于成品中酶活性和浓度的影响较大。其中不同的蛋白质在盐析提纯时所需的盐浓度不同，因此本实施例主要针对三种目标酶的性质，对制备方法中步骤S5进行优化，以使其他蛋白和杂质沉淀析出，从而得到较高浓度的活性酶液。
本实施例中各溶液的配制方法如下：
(1)提取液：配制50mmol/L Tris(三羟甲基氨基甲烷)缓冲液，然后向缓冲液中加入氯化钾至浓度为5mmol/L，加入EDTA至浓度为6mmol/L，再加入叠氮化钠至浓度为2g/L，调节pH至7.5，然后定容即得提取液。
本实施例的制备方法包括：
S1、取新鲜猪肝，用自来水反复冲洗去血液，并去除结缔组织，取肝脏组织切割成小于4cm的小块，入组织捣碎机，加入猪肝重量1/8的提取液，间歇捣碎约60分钟，至肝脏组织呈糊状，获得第一混合液；
S2、向第一混合液中加入第一混合液9倍质量的提取液，放入内切匀浆机中匀浆，匀浆约5分钟，匀浆过程间歇进行，直至无肉眼明显可见组织块存在为止，得到第二混合液；
S3、向第二混合液中加入体积比为0.3％的十二烷基磺酸钠用于抽提膜蛋白，用超声破碎仪进行超声处理，具体为：功率150W，超声60s，停歇60s，连续40次，然后停顿5分钟，再重复前述超声步骤，反复进行5次，直到肝脏完全破碎为止，获得第三混合液；
S4、将第三混合液9000rpm离心80分钟，完全去除上清，用沉淀质量1/8的提取液复溶沉淀得到第四混合液；
S5、在搅拌过程中，向第四混合液中加入盐析药品，待盐析药品完全溶解后，于2-8℃放置3-6h，获得第五混合液；
S6、将第五混合液9000rpm离心60分钟，收集上清即为第六混合液，向第六混合液加入到可截留大小10～30KDa蛋白的超滤管中，于2-8℃放置10-20h，收集滤出液即为所述肝功能复合质控品。
其中将第四混合液共分为7组，每组中加入的盐析药品具体如下：
将7组制备得到的复合质控品分别用来源于武汉生之源生物科技股份有限公司的α-L-岩藻糖苷酶测定试剂盒(CNPF底物法)、腺苷脱氨酶测定试剂盒(过氧化物酶法)和5′-核苷酸酶测定试剂盒(过氧化物酶法)进行测定，测定最终得到的质控品中α-L-岩藻糖苷酶(AFU)、腺苷脱氨酶(ADA)和5′-核苷酸酶(5′-NT)三种酶的浓度(单位均为U/L)，结果如表1所示。
表1盐析药品对酶浓度的影响
| 组1 | 组2 | 组3 | 组4 | 组5 | 组6 | 组7 | |
| AFU浓度 | 30 | 135 | 515 | 603 | 545 | 607 | 613 |
| ADA浓度 | 42 | 257 | 985 | 1205 | 1078 | 1189 | 1207 |
| 5′-NT浓度 | 51 | 298 | 1043 | 1397 | 1242 | 1403 | 1388 |
根据表1的测定结果，盐析药品的浓度对于最终获得的复合质控品中各酶的浓度有显著影响，其中盐析药品浓度过低会导致部分杂质未析出除去，成品中杂质过多则目标酶的浓度显著降低，而盐析药品浓度过高会导致目标酶部分沉淀，进而降低了成品中目标酶的浓度。且盐析药品的种类对于酶浓度无显著影响。其中当盐析药品的加入量为40～60g/L时，得到的酶浓度较高，满足使用需求，进一步的，当盐析药品的加入量为50g/L时，制备得到的复合质控品中三种酶的浓度最高。
实施例2
为使复合质控品的稳定性提高，本实施例在实施例1中所述制备方法的基础上，将实施例1组4得到的肝功能复合质控品用保护剂稀释后，通过冻干工艺得到复合质控品冻干粉，以显著提高试剂的稳定性。
其中保护剂的配方为：配制50mmol/L Tris缓冲液，依次加入氯化钠至浓度为0.5mmol/L，甘露醇至浓度为8g/L，葡聚糖至浓度为6g/L，牛血清白蛋白至浓度为10g/L，EDTA至浓度为0.5mmol/L和叠氮化钠至浓度为0.5g/L，调节pH至7.4，定容即得保护剂。
将实施例1组4得到的肝功能复合质控品用上述保护剂稀释20倍后，等量分装至西林瓶中，然后将每个西林瓶于-20℃条件下预冻8小时，然后放入-60℃的冻干机冷井内8小时，然后从冷井中取出预冻好的质控品，立即放入干燥层减压干燥13小时(其中气压＜20Pa)，得到的冻干粉即为复合质控品冻干粉。
实施例3
本实施例与实施例2的不同之处在于：其中保护剂的配方为：20mmol/L Tris缓冲液，0.05mmol/L氯化钠，4g/L甘露醇，3g/L葡聚糖，8g/L牛血清白蛋白，0.03mmol/L EDTA和0.1g/L叠氮化钠。
实施例4
本实施例与实施例2的不同之处在于：其中保护剂的配方为：100mmol/L Tris缓冲液，1mmol/L氯化钠，12g/L甘露醇，10g/L葡聚糖，15g/L牛血清白蛋白，0.8mmol/L EDTA和1g/L叠氮化钠。
评价方案
对实施例2-4获得的复合质控品冻干粉分别进行均一性、热稳定性和长期稳定性的评价，其中复合质控品冻干粉需复溶于蒸馏水中再进行测定。
进行评价时所采用的试剂盒为来源于武汉生之源生物科技股份有限公司的α-L-岩藻糖苷酶测定试剂盒(CNPF底物法)、腺苷脱氨酶测定试剂盒(过氧化物酶法)和5′-核苷酸酶测定试剂盒(过氧化物酶法)。
(1)均一性评价：取各实施例中的复合质控品，用试剂盒进行检测，每个实施例检测15次，计算均值、标准差和变异系数(CV)，其中CV小于5％为均一性良好，结果如表2所示。
(2)热稳定性评价：将各实施例得到的复合质控品于37℃储存7天，并在储存第0天和第7天分别用试剂盒检测其中各酶的含量，每个实施例测定3次，计算平均值，以及第7天相比于第0天的测量值偏差(％)，偏差在10％以内，表示热稳定性好，结果如表3所示。
(3)长期稳定性：将各实施例得到的复合质控品于2-8℃储存30个月，在第6、12、18、24、30个月测定复合质控品中各酶的含量，每个实施例测定3次，计算平均值，以及各个月与第0个月的偏差(％)偏差在10％以内，表示长期稳定性好，结果如表4所示
表2均一性数据
表3热稳定性数据(37℃储存)
表4长期稳定性数据
根据表1-3的检测结果，将复合质控品用本发明所述的保护剂稀释并冻干后得到的复合质控品冻干粉，其均一性、热稳定性和长期稳定性均较优，可充分满足使用需求。更进一步的，保护剂中组分含量变化对试剂的稳定性有一定的影响，综合来看，其中使用实施例2所述的保护剂后，三种酶在37℃热处理或长期放置30个月后，其偏差值相对较低，即实施例2中所述保护剂对于AFU、ADA和5’-NT三种酶维持稳定性的效果最好。
以上所述，仅为本发明较佳的具体实施方式，但本发明的保护范围并不局限于此，任何熟悉本技术领域的技术人员在本发明揭露的技术范围内，可轻易想到的变化或替换，都应涵盖在本发明的保护范围之内。
Claims (5)
1.一种肝功能复合质控品，其特征在于，所述复合质控品包括：α-L-岩藻糖苷酶、腺苷脱氨酶、5′-核苷酸酶，所述复合质控品的制备方法包括：
S1、取新鲜猪肝，加入猪肝重量1/5～1/10的提取液捣碎得到第一混合液；
S2、向第一混合液中加入第一混合液质量8～10倍的提取液，匀浆得到第二混合液；
S3、向第二混合液中加入体积比为0.1％～0.5％的去垢剂并超声处理，得到第三混合液；
S4、将第三混合液进行离心，弃上清，用沉淀质量1/5～1/10的提取液复溶沉淀得到第四混合液；
S5、向第四混合液中加入盐析药品，其中盐析药品的加入量为40～60g/L，于2-8℃放置3-6h，获得第五混合液；
S6、将第五混合液离心，收集上清即为第六混合液，向第六混合液加入到可截留大小10～30KDa蛋白的超滤管中，于2-8℃放置10-20h后，收集滤出液即为所述肝功能复合质控品；
步骤S1、S2和S4中所述提取液的pH为7.0～7.4，由20～100mmol/L Tris缓冲液，1.6～10mmol/L无机盐，1.3～8.1mmol/L金属离子螯合剂和质量体积比为0.1～5％的防腐剂组成；
步骤S5中所述盐析药品为氯化钠、氯化钾、硝酸铵、硫酸铝、氯化铵、氯化镁、硫酸镁、硫酸铵中一种或多种；
步骤S3中所述去垢剂为聚乙二醇对异辛基苯基醚，牛磺胆酸钠，十二烷基磺酸钠中的一种或多种；
将步骤S6得到的肝功能复合质控品用保护剂稀释后冻干得到冻干粉，所述保护剂的pH为7.0～7.5，由20～100mmol/L Tris缓冲液，0.05～1mmol/L无机盐，4～12g/L甘露醇，3～10g/L葡聚糖，8～15g/L牛血清白蛋白，0.03～0.8mmol/L EDTA和0.1～1g/L防腐剂组成。
2.根据权利要求1所述的复合质控品，其特征在于，步骤S5中所述盐析药品的加入量为50g/L。
3.根据权利要求1所述的复合质控品，其特征在于，步骤S3中超声处理的过程具体为：功率150W，超声60s，停歇60s，连续40次，然后停顿5分钟，再重复超声步骤，反复进行5次，直到肝脏完全破碎为止。
4.一种检测试剂盒，其特征在于，所述检测试剂盒包含如权利要求1-3中任一项所述的复合质控品。
5.根据权利要求4所述的检测试剂盒，其特征在于，所述检测试剂盒与α-L-岩藻糖苷酶检测试剂盒或腺苷脱氨酶检测试剂盒或5′-核苷酸酶检测试剂盒配套使用。
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