CN114076741B - 一种甘胆酸检测试剂盒的制备方法 - Google Patents
一种甘胆酸检测试剂盒的制备方法 Download PDFInfo
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Abstract
本发明属于体外诊断试剂技术领域。针对胶乳增强免疫比浊法检测甘胆酸存在的试剂盒制备工艺复杂的问题,提供一种甘胆酸检测试剂盒的制备方法。包括步骤:将磷酸盐缓冲液、盐离子、离子螯合剂等依次溶于纯水中,调pH至6.0‑7.4,加入甘胆酸‑蛋白偶联物,搅拌恒温孵育得试剂R1;取聚苯乙烯胶乳颗粒于HEPES缓冲液中,摇匀之后加入现配的EDC和NHS溶液活化胶乳,搅拌加入缓冲液和鼠抗甘胆酸单抗,偶联后置于搅拌器上,加入硼酸硼砂缓冲液、封闭剂DB1130等得试剂R2;向纯水中加入硼酸和硼砂,溶解后加入盐离子、保护剂等得校准品稀释液。制备工艺简单,试剂批间差降低,稳定性好,灵敏度高,重复性好。
Description
技术领域
本发明属于体外诊断试剂技术领域,具体涉及一种甘胆酸检测试剂盒的制备方法。
背景技术
血清甘胆酸(CG)是肝脏合成、储存于胆囊,是胆酸与甘氨酸结合二次的结合型胆酸之一, 在体内的正常代谢途径为肝-胆-肠-静脉-肝的循环,绝大多数经门静脉被肝脏重新吸收。
正常情况下,外周血中CG含量非常少,正常成人无论空腹或餐后,其血清CG浓度均稳定在低水平。当肝细胞受损时,肝脏对CG的重吸收下降,致使血中CG含量增高,如急 (慢)性肝炎,肝硬化、肝癌;当胆囊堵塞致使胆汁郁滞时,肝脏内的CG不能排泄到肠道 内,致返流至血液循环的CG增高;因此,血清CG含量的高低能直接反馈肝胆系统的生理 功能是否正常。正常情况下,孕妇在怀孕中后期,由于婴儿增大,压迫肝脏,影响肝脏胆汁 的分泌而导致CG升高;另外患有胆汁淤积症的孕妇会出现皮肤瘙痒及轻度黄疸,对胎儿影 响较大,易引起早产、胎儿宫内窘迫死亡和新生儿窒息,因此孕妇血清CG的检测对孕妇和 胎儿的健康具有重要的诊断意义。
目前甘胆酸测定方法主要使用放射免疫分析法(RIA),化学发光免疫分析法(CLIA)、酶 联免疫吸附法(ELISA),均相酶免疫分析法和胶乳增强免疫比浊法。放射免疫法步骤繁琐,试 剂价格昂贵需要有专业放免设施,且存在放射污染;化学发光法灵敏度较高,但是试剂稳定 性较差,成本较高市场推广有一定压力;酶联免疫法一般用于半定量测定,且操作繁琐,检 测时间长,重复性较差,不利于临床检验以及急诊需求;均相酶免疫分析法试剂操作简单快速,但稳定性差、灵敏度不高;现有胶乳增强免疫比浊法灵敏度高、重复性好,但其试剂盒 的制备工艺复杂,耗费人力物力。
发明内容
针对胶乳增强免疫比浊法检测甘胆酸存在的试剂盒制备工艺复杂的问题,本发明提供一 种甘胆酸检测试剂盒的制备方法,无需离心和超声分散等步骤,制备工艺简单,使制备后的 胶乳可以直接参与检测,节省时间的同时还有效降低了试剂的批间差,具有良好的稳定性,灵敏度高,重复性好。
本发明是通过以下技术方案实现的:
一种甘胆酸检测试剂盒的制备方法,包括以下步骤:
(1)试剂R1的配制
将磷酸盐缓冲液、盐离子、离子螯合剂、促凝剂、防腐剂、表面活性剂依次分别溶于纯 水中,搅拌均匀,调pH至6.0-7.4,再加入甘胆酸-蛋白偶联物,搅拌均匀后置于37℃烘箱孵 育一晚即得试剂R1,试剂R1的组成如下
磷酸盐缓冲液:0.01-0.1M
盐离子:20-40g/L
离子螯合剂:1.5-4g/L
促凝剂:5-30g/L
防腐剂:0.7-1.5g/L
表面活性剂:10-20g/L
甘胆酸-蛋白偶联物:1-4mg/L;
(2)试剂R2的配制
取粒径为80-200nm的聚苯乙烯胶乳颗粒于0.01M-0.1M HEPES缓冲液中,置于37℃摇 床,200r/min,摇匀2min,之后加入现配的碳化二亚胺(EDC)和N-羟基琥珀酰亚胺(NHS)溶液,置于37℃摇床,200r/min,活化胶乳15min,在搅拌的情况下,加入HEPES缓冲液和 鼠抗甘胆酸单抗,置于37℃摇床,200r/min,偶联3h后从摇床中取出,置于搅拌器上,分别加入硼酸硼砂缓冲液、封闭剂DB1130(生产商JSR)、酪蛋白、表面活性剂,上述物质均在 加完一种物质于37℃摇床孵育30min后再加入另一种物质,最后加入防腐剂和稳定剂,在37℃ 摇床稳定一夜后即得试剂R2,试剂R2的组成如下:
鼠抗甘胆酸单抗包被胶乳颗粒:60-100mg/L
4-羟乙基哌嗪乙磺酸缓冲液:0.01M-0.1M
硼酸-硼砂缓冲液:0.01M-0.05M
封闭剂DB1130:5-20g/L
酪蛋白:5-20g/L
表面活性剂:5-20g/L
防腐剂:0.7-1.5g/L
稳定剂:50-100g/L;
(3)校准品稀释液的配制
在搅拌提交下,向纯水中加入硼酸和硼砂,充分溶解后按顺序依次加入盐离子、保护剂、 防腐剂和表面活性剂,一种物质完全溶解后再加入另一种物质,待完全溶解,校准品稀释液 配制完成,校准品稀释液的组成如下:
硼酸硼砂缓冲液:0.01M-0.05M,
盐离子:20-40g/L
保护剂:10-20g/L
防腐剂:0.7-1.5g/L
表面活性剂:10-20g/L。
进一步的,所述步骤(1)甘胆酸-蛋白偶联物中的蛋白为卵清蛋白、牛转铁蛋白、人血 清白蛋白、牛血清白蛋白及血蓝蛋白中的一种或者几种。
进一步的,所述步骤(1)和步骤(3)盐离子为氯化钠、氯化钾、氯化钙中的一种或几种。
进一步的,所述步骤(1)离子螯合剂为EGTA、EDTP、DTPA、EDTA中的一种或几种。
进一步的,所述步骤(1)促凝剂为PEG6000、PEG8000、PEG12000中的一种或几种。
进一步的,所述步骤(1)~(3)中防腐剂均为叠氮钠和/或Proclin300。
进一步的,所述步骤(1)和步骤(3)中表面活性剂均选自Tween-20、甘油、曲拉通的一种或几种;所述步骤(2)中表面活性剂选择Tween-20。
进一步的,所述步骤(2)稳定剂选自海藻糖、蔗糖和牛血清蛋白中的一种或几种。
有益效果:
(1)现有胶乳增强免疫比浊法制备工艺复杂,需要用离心或超滤的步骤去除多余的影响 试剂性能的成份,并通过超声分散实现试剂的均匀稳定,耗费人力物力,不利于工业化的制 备。本发明提供方法制备工艺简单,无需离心和超声分散等步骤,使制备后的胶乳可以直接参与检测,节省时间的同时还有效降低了试剂的批间差。
(2)本试剂盒通过将封闭剂DB1130和酪蛋白联合使用,使得试剂盒具有良好的稳定性, 37℃加速破坏过程中,空白吸光度和各校准品点的反应吸光度及样本测值基本不变。
(3)本试剂盒采用竞争法,灵敏度高,重复性好。
具体实施方式
下面结合具体实施例对本发明做进一步详细说明。
实施例1:
生产一种稳定性好的甘胆酸检测试剂盒的方法如下:
(1)试剂R1的配制:
称取100g PB缓冲液,置于搅拌器上,按顺序依次加入0.4g EDTA-2Na、0.09gNaN3、0.9g PEG8000、2.6g NaCl、1.3g KCl、2.0g蔗糖以及1.43g吐温20。一种物质完全溶解后再加入 另一种物质,待完全溶解后调节pH至7.0,加入0.25mg甘胆酸-蛋白偶联物,置37℃烘箱孵育 一晚后即为试剂R1。
(2)试剂R2的配制:
取粒径为100nm的聚苯乙烯胶乳微球150ul于200ul 0.02M pH7.8的HEPES缓冲液中,置于 37℃摇床,200r/min,摇匀2min。之后加入现配的EDC和NHS溶液中,置于37℃摇床,200r/min, 摇匀15min以活化胶乳;EDC和NHS的浓度为0.15M,添加比例为1:1。之后在搅拌的情况下,加 入2ml HEPES缓冲液(0.02M pH7.8)和抗体溶液,抗体添加量为60mg/L。置于37℃摇床, 200r/min,偶联3h。从摇床中取出,置于搅拌器上,分别加入硼酸-硼砂缓冲液(0.05M pH8.0)、 封闭剂DB1130、酪蛋白、吐温20,上述物质均在加完一种物质于37℃摇床孵育30min后再加入 另一种物质,最后加入0.15%的proclin300以及10%蔗糖,在37℃摇床孵育一夜后即得试剂R2。
(3)校准品稀释液的配制
取50g PB缓冲液置于搅拌器上,充分溶解后按顺序依次加入0.1g NaN3、1.5gNaCl、0.53g KCl、1.8g蔗糖、0.1g BSA以及1.43g吐温80。一种物质完全溶解后再加入另一种物质,待 完全溶解,校准品稀释液配制完成。
取20g上述校准品稀释液,加入0.0032g甘胆酸标准物质,配制成160mg/L的母液。根据定 标所需的校准品浓度,将校准品稀释成6个不同的浓度:0mg/L、1.25mg/L、2.5mg/L、5mg/L、 20mg/L、80mg/L待用。
实施例2:
生产一种稳定性好的甘胆酸检测试剂盒的方法如下:
(1)试剂R1的配制:
取100g Tris缓冲液置于搅拌器上,按顺序依次加入0.4g EDTA.2Na、0.09g NaN3、0.70g PEG6000、0.15g PEG12000、2.6g NaCl、1.3g KCl、2.0g BSA以及1.43g曲拉通。一种物质 完全溶解后再加入另一种物质,待完全溶解后调节pH至7.0,加入0.25mg甘胆酸-蛋白偶联物 即得试剂R1。
(2)试剂R2的配制:
取粒径为145nm的聚苯乙烯胶乳微球150ul于200ul 0.05M pH7.0 HEPES缓冲液中,置于 37℃摇床,200r/min,摇匀2min。之后加入现配的EDC和NHS溶液中,置于37℃摇床,200r/min, 摇匀15min;EDC和NHS的浓度为0.15M,添加比例为2:1。之后在搅拌的情况下,加入2ml HEPES 缓冲液(0.05M pH7.0)和抗体溶液,抗体添加量为60mg/L。之后置于37℃摇床,200r/min,孵 育3h。从摇床中取出,置于搅拌器上,分别加入硼酸硼砂缓冲液(0.05MpH8.0)、封闭剂DB1130、 酪蛋白、吐温20,上述物质在加完一种物质于37℃摇床孵育30min后再加入另一种物质,最后 加入0.15%的proclin300以及10%海藻糖,在37℃摇床稳定一夜后即得试剂R2。
(3)校准品稀释液的配制:
取50g纯水,置于搅拌器上加入0.2g硼酸和0.06g硼砂,充分溶解后按顺序依次加入 0.05g NaN3、1.5g NaCl、0.53g KCl、1.8g甘露醇、0.1g山梨醇以及1.43g曲拉通。一种物质完全溶解后再加入另一种物质,待完全溶解,校准品稀释液配制完成。
取20g上述校准品稀释液,加入0.0032g甘胆酸校准品,配制成160mg/L的母液。根据定标 所需的校准品浓度,将校准品稀释成6个不同的浓度:0mg/L、1.25mg/L、2.5mg/L、5mg/L、 20mg/L、80mg/L待用。
比较实验:比较例1,封闭剂的采用酪蛋白,其他组成与实施例1相同;比较例2,封闭剂 采用DB1130,其他组成与实施例1相同。
将配好的试剂R1/R2放入37℃水浴锅中进行试剂的加速破坏过程,分别在第3天、第5天和 第7天取出部分试剂进行检测,并记录18点和34点试剂的空白吸光度和各定标浓度点的吸光度 值。通过添加不同的封闭剂进行封闭,在加速破坏过程中发现,同时添加酪蛋白和DB1130对 试剂进行封闭,试剂空白吸光度和各定标点吸光度变化及样本测值明显要优于单独添加酪蛋 白或者DB1130组。
表1比较例1加速破坏试验
表2比较例2加速破坏试验
表3实施例1加速破坏试验
以上所述的实施例仅是对本发明的优选实施方式进行描述,并非对本发明的范围进行限 定,在不脱离本发明设计精神的前提下,本领域普通技术人员对本发明的技术方案作出的各 种变形和改进,均应落入本发明权利要求书确定的保护范围内。
Claims (8)
1.一种甘胆酸检测试剂盒的制备方法,其特征在于,包括以下步骤:
(1)试剂R1的配制
将磷酸盐缓冲液、盐离子、离子螯合剂、促凝剂、防腐剂、表面活性剂依次分别溶于纯水中,搅拌均匀,调pH至6.0-7.4,再加入甘胆酸-蛋白偶联物,搅拌均匀后置于37℃烘箱孵育一晚即得试剂R1,试剂R1的组成如下:
磷酸盐缓冲液:0.01-0.1M
盐离子:20-40g/L
离子螯合剂:1.5-4g/L
促凝剂:5-30g/L
防腐剂:0.7-1.5g/L
表面活性剂:10-20g/L
甘胆酸-蛋白偶联物:1-4mg/L;
(2)试剂R2的配制
取粒径为80-200nm的聚苯乙烯胶乳微球于0.01M-0.1M 4-羟乙基哌嗪乙磺酸(HEPES)缓冲液中,置于37℃摇床,200r/min,摇匀2min,之后加入现配的碳化二亚胺和N-羟基琥珀酰亚胺溶液,置于37℃摇床,200r/min,活化胶乳15min,在搅拌的情况下,加入HEPES缓冲液和鼠抗甘胆酸单抗,置于37℃摇床,200r/min,偶联3h后从摇床中取出,置于搅拌器上,分别加入硼酸-硼砂缓冲液、封闭剂DB1130、酪蛋白、表面活性剂,上述物质均在加完一种物质于37℃摇床孵育30min后再加入另一种物质,最后加入防腐剂和稳定剂,在37℃摇床稳定一夜后即得试剂R2,试剂R2的组成如下:
鼠抗甘胆酸单抗包被胶乳颗粒:60-100mg/L
4-羟乙基哌嗪乙磺酸缓冲液:0.01M-0.1M
硼酸-硼砂缓冲液:0.01M-0.05M
封闭剂DB1130:5-20g/L
酪蛋白:5-20g/L
表面活性剂:5-20g/L
防腐剂:0.7-1.5g/L
稳定剂:50-100g/L;
(3)校准品稀释液的配制
在搅拌提交下,向纯水中加入硼酸和硼砂,充分溶解后按顺序依次加入盐离子、保护剂、防腐剂和表面活性剂,一种物质完全溶解后再加入另一种物质,待完全溶解,校准品稀释液配制完成,校准品稀释液的组成如下:
硼酸硼砂缓冲液:0.01M-0.05M,
盐离子:20-40g/L
保护剂:10-20g/L
防腐剂:0.7-1.5g/L
表面活性剂:10-20g/L。
2.根据权利要求1所述的制备方法,其特征在于,所述步骤(1)甘胆酸-蛋白偶联物中的蛋白为卵清蛋白、牛转铁蛋白、人血清白蛋白、牛血清白蛋白及血蓝蛋白中的一种或者几种。
3.根据权利要求1所述的制备方法,其特征在于,所述步骤(1)和步骤(3)盐离子均为氯化钠、氯化钾、氯化钙中的一种或几种。
4.根据权利要求1所述的制备方法,其特征在于,所述步骤(1)离子螯合剂为EGTA、EDTP、DTPA、EDTA中的一种或几种。
5.根据权利要求1所述的制备方法,其特征在于,所述步骤(1)促凝剂为PEG6000、PEG8000、PEG12000中的一种或几种。
6.根据权利要求1所述的制备方法,其特征在于,所述步骤(1)~(3)中防腐剂均选自叠氮钠和Proclin300中的一种或两种。
7.根据权利要求1所述的制备方法,其特征在于,所述步骤(1)和步骤(3)中表面活性剂均选自Tween-20、甘油、曲拉通的一种或几种;所述步骤(2)中表面活性剂选择Tween-20。
8.根据权利要求1所述的制备方法,其特征在于,所述步骤(2)稳定剂选自海藻糖、蔗糖和牛血清蛋白中的一种或几种。
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