CN110824176A - 中性粒细胞明胶酶相关脂质运载蛋白检测试剂盒 - Google Patents
中性粒细胞明胶酶相关脂质运载蛋白检测试剂盒 Download PDFInfo
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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Abstract
本发明公开了一种中性粒细胞明胶酶相关脂质运载蛋白检测试剂盒,包括试剂R1、试剂R2以及校准品,试剂R1为中性粒细胞明胶酶相关脂质运载蛋白与抗人中性粒细胞明胶酶相关脂质运载蛋白抗体反应缓冲液,试剂R2为结合有中性粒细胞明胶酶相关脂质运载蛋白抗体的纳米微球溶液,校准品由缓冲液、稳定剂、防腐剂以及中性粒细胞明胶酶相关脂质运载蛋白参考标准品组成;纳米微球溶液中含有纳米颗粒,金纳米颗粒包括大直径纳米颗粒和小直径纳米颗粒,大直径纳米颗粒的直径为220±3nm,小直径纳米颗粒的直径为83±1.5nm,且纳米颗粒和抗体重量之比为60‑75:10‑19;试剂R1包括含有重量体积比0.5%的牛血清白蛋白、重量体积比0.3%的三氮化钠和重量体积比0.45%聚氧乙烯去水山梨醇单月桂酸酯的缓冲液;试剂R1还包括变性剂,变性剂为盐酸胍、异硫氰酸胍、硫氰酸胍、尿素、十二烷基硫酸钠中的一种或多种。
Description
技术领域
本发明涉及一种中性粒细胞明胶酶相关脂质运载蛋白检测试剂盒。
背景技术
中性粒细胞明胶酶相关脂质运载蛋白(NGAL)是lipocalin的一种,最初是在激活中性粒细胞中被发现的一种小分子量分泌性蛋白,现代研究表明,NGAL是诊断急性肾损伤的最有效生物学标志之一,也是早期糖尿病肾病的有效标志物之一。1993年,Kjeldsen等在研究中性粒细胞中92kD的MMP-9时发现了一种25kD的新蛋白。进一步研究发现,这种新蛋白是一种由178个氨基酸残基组成的单体,既能够自身聚合形成46kD的同源二聚体,也能够与MMP-9聚合形成135kD的异源二聚体,是一种新的lipocalin,因此被命名为neutropilgelatinase-associated lipocalin,简称为NGAL。NGAL具有强大的功能,除了作为载脂家族成员具有结合并运输疏水性小分子的功能外,还与炎症、胚胎发育、免疫应答、趋化作用、信号转导以及多种肿瘤的发生与发展过程相关。NGAL可消炎、抗炎,可促进肾脏祖细胞向早期肾小管上皮细胞分化,可修复N-钙黏蛋白,上调保护酶血红素加氧酶和抑制细胞死亡。一般分为抽静脉血和尿液两种方式。静脉血标本收集:清晨抽静脉血5ml,EDTA.2Na(乙二胺四乙酸二钠)抗凝2h,4℃,1000r/min离心10min,取上清液再次4℃,1800r/min离心10min,去除血小板,取上清液存于-80℃冰箱保存。尿液标本收集:晨起清洁中段尿5ml,置无致热源及内毒素的试管中,4000r/min离心5min,取上清液置于Eppendrof管中,冻存于-20℃冰箱中,避免反复冻融。目前已有胶乳增强免疫透射比浊法测定人血浆或尿液中NGAL浓度的试剂盒,但这些试剂盒无法同时满足宽线性范围和高灵敏度的要求,因此需要稀释样本才能测定高浓度NGAL,这在使用上带来了不便。
发明内容
本发明的目的是为了克服现有技术的不足,提供了一种中性粒细胞明胶酶相关脂质运载蛋白检测试剂盒。
本发明是通过以下技术方案实现:
一种中性粒细胞明胶酶相关脂质运载蛋白检测试剂盒,包括试剂R1、试剂R2以及校准品,试剂R1为中性粒细胞明胶酶相关脂质运载蛋白与抗人中性粒细胞明胶酶相关脂质运载蛋白抗体反应缓冲液,试剂R2为结合有中性粒细胞明胶酶相关脂质运载蛋白抗体的纳米微球溶液,校准品由缓冲液、稳定剂、防腐剂以及中性粒细胞明胶酶相关脂质运载蛋白参考标准品组成;纳米微球溶液中含有纳米颗粒,金纳米颗粒包括大直径纳米颗粒和小直径纳米颗粒,大直径纳米颗粒的直径为220±3nm,小直径纳米颗粒的直径为83±1.5nm,且纳米颗粒和抗体重量之比为60-75:10-19;试剂R1包括含有重量体积比0.5%的牛血清白蛋白、重量体积比0.3%的三氮化钠和重量体积比 0.45%聚氧乙烯去水山梨醇单月桂酸酯的缓冲液;试剂R1还包括变性剂,变性剂为盐酸胍、异硫氰酸胍、硫氰酸胍、尿素、十二烷基硫酸钠中的一种或多种。
进一步地,试剂R2中还包括促凝剂、稳定剂、蔗糖和甘油,其中,稳定剂为蛋白质、PEG、糖和可溶性的碱金属和碱土金属的无机盐中的一种或多种;蛋白质为牛血清白蛋白或明胶;糖为单糖、二糖或者多糖;无机盐为卤素碱金属和碱土金属的无机盐;试剂R2含有磷酸盐缓冲液、硼酸盐缓冲液、甘氨酸缓冲液、柠檬酸-磷酸盐缓冲液、Tris 缓冲液和HEPES缓冲液中的一种或多种的组合。
进一步地,试剂R1的pH值为8.3,试剂R2的pH值为9.0。
进一步地,校准品为缓冲基质,其包括4-羟乙基哌嗪乙磺酸、氯化钠、氮化钠、乙二胺四乙酸、牛血清白蛋白、蔗糖,其余为去离子水,溶于基质的6500ng/ml 浓度的中性粒细胞明胶酶相关脂质运载蛋白中。
进一步地,抗人中性粒细胞明胶酶相关脂质运载蛋白抗体为多克隆抗体或单克隆抗体;多克隆抗体为兔抗人中性粒细胞明胶酶相关脂质运载蛋白抗体、羊抗人中性粒细胞明胶酶相关脂质运载蛋白抗体或鸡抗人中性粒细胞明胶酶相关脂质运载蛋白抗体的一种;单克隆抗体为鼠抗人中性粒细胞明胶酶相关脂质运载蛋白抗体。
与现有的技术相比,本发明具有以下有益效果:能够极大地提高了低浓度中性粒细胞明胶酶相关脂质运载蛋白时抗原抗体反应速率,从而提高了试剂的灵敏度,纳米微球表面包被抗体后,其亲和性相对较低,在高抗原浓度下仍可形成抗原抗体复合物,从而提高了试剂的线性范围;将大直径纳米微球和小直径纳米微球混合在同一缓冲液体系后,由于大直径与中性粒细胞明胶酶相关脂质运载蛋白与抗原结合力强,这样在低中性粒细胞明胶酶相关脂质运载蛋白浓度下,试剂也具有较高的灵敏度,当中性粒细胞明胶酶相关脂质运载蛋白浓度较高时,抗原可与小颗粒结合形成免疫复合物,这样试剂具有宽的线性范围;具有灵敏度高,特异性强,稳定性好的特点,反应时间短,显色反应时间小,平衡时间小,可用于检测血清或血浆中中性粒细胞明胶酶相关脂质运载蛋白含量,适用于临床全自动生化分析仪;反应后不产生沉淀,便于进行清洗,延长了其使用寿命,且能够降低中性粒细胞明胶酶相关脂质运载蛋白检测试剂盒的制造成本。
具体实施方式
为了使本发明的目的、技术方案及优点更加清楚明白,以下对本发明进行进一步详细说明。应当理解,此处所描述的具体实施例仅仅用以解释本发明,并不用于限定本发明。
一种中性粒细胞明胶酶相关脂质运载蛋白检测试剂盒,包括试剂R1、试剂R2以及校准品,试剂R1为中性粒细胞明胶酶相关脂质运载蛋白与抗人中性粒细胞明胶酶相关脂质运载蛋白抗体反应缓冲液,试剂R2为结合有中性粒细胞明胶酶相关脂质运载蛋白抗体的纳米微球溶液,校准品由缓冲液、稳定剂、防腐剂以及中性粒细胞明胶酶相关脂质运载蛋白参考标准品组成;纳米微球溶液中含有纳米颗粒,金纳米颗粒包括大直径纳米颗粒和小直径纳米颗粒,大直径纳米颗粒的直径为220±3nm,小直径纳米颗粒的直径为83±1.5nm,且纳米颗粒和抗体重量之比为60-75:10-19;试剂R1包括含有重量体积比0.5%的牛血清白蛋白、重量体积比0.3%的三氮化钠和重量体积比 0.45%聚氧乙烯去水山梨醇单月桂酸酯的缓冲液;试剂R1还包括变性剂,变性剂为盐酸胍、异硫氰酸胍、硫氰酸胍、尿素、十二烷基硫酸钠中的一种或多种。试剂R2中还包括促凝剂、稳定剂、蔗糖和甘油,其中,稳定剂为蛋白质、PEG、糖和可溶性的碱金属和碱土金属的无机盐中的一种或多种;蛋白质为牛血清白蛋白或明胶;糖为单糖、二糖或者多糖;无机盐为卤素碱金属和碱土金属的无机盐;试剂R2含有磷酸盐缓冲液、硼酸盐缓冲液、甘氨酸缓冲液、柠檬酸-磷酸盐缓冲液、Tris 缓冲液和HEPES缓冲液中的一种或多种的组合。试剂R1的pH值为8.3,试剂R2的pH值为9.0。校准品为缓冲基质,其包括4-羟乙基哌嗪乙磺酸、氯化钠、氮化钠、乙二胺四乙酸、牛血清白蛋白、蔗糖,其余为去离子水,溶于基质的6500ng/ml 浓度的中性粒细胞明胶酶相关脂质运载蛋白中。抗人中性粒细胞明胶酶相关脂质运载蛋白抗体为多克隆抗体或单克隆抗体;多克隆抗体为兔抗人中性粒细胞明胶酶相关脂质运载蛋白抗体、羊抗人中性粒细胞明胶酶相关脂质运载蛋白抗体或鸡抗人中性粒细胞明胶酶相关脂质运载蛋白抗体的一种;单克隆抗体为鼠抗人中性粒细胞明胶酶相关脂质运载蛋白抗体。能够极大地提高了低浓度中性粒细胞明胶酶相关脂质运载蛋白时抗原抗体反应速率,从而提高了试剂的灵敏度,纳米微球表面包被抗体后,其亲和性相对较低,在高抗原浓度下仍可形成抗原抗体复合物,从而提高了试剂的线性范围;将大直径纳米微球和小直径纳米微球混合在同一缓冲液体系后,由于大直径与中性粒细胞明胶酶相关脂质运载蛋白与抗原结合力强,这样在低中性粒细胞明胶酶相关脂质运载蛋白浓度下,试剂也具有较高的灵敏度,当中性粒细胞明胶酶相关脂质运载蛋白浓度较高时,抗原可与小颗粒结合形成免疫复合物,这样试剂具有宽的线性范围;具有灵敏度高,特异性强,稳定性好的特点,反应时间短,显色反应时间小,平衡时间小,可用于检测血清或血浆中中性粒细胞明胶酶相关脂质运载蛋白含量,适用于临床全自动生化分析仪;反应后不产生沉淀,便于进行清洗,延长了其使用寿命,且能够降低中性粒细胞明胶酶相关脂质运载蛋白检测试剂盒的制造成本。
以上仅为本发明的较佳实施例而已,并不用以限制本发明,凡在本发明的精神和原则之内所作的任何修改、等同替换和改进等,均应包含在本发明的保护范围之内。
Claims (5)
1.一种中性粒细胞明胶酶相关脂质运载蛋白检测试剂盒,其特征在于:包括试剂R1、试剂R2以及校准品,试剂R1为中性粒细胞明胶酶相关脂质运载蛋白与抗人中性粒细胞明胶酶相关脂质运载蛋白抗体反应缓冲液,试剂R2为结合有中性粒细胞明胶酶相关脂质运载蛋白抗体的纳米微球溶液,校准品由缓冲液、稳定剂、防腐剂以及中性粒细胞明胶酶相关脂质运载蛋白参考标准品组成;纳米微球溶液中含有纳米颗粒,金纳米颗粒包括大直径纳米颗粒和小直径纳米颗粒,大直径纳米颗粒的直径为220±3nm,小直径纳米颗粒的直径为83±1.5nm,且纳米颗粒和抗体重量之比为60-75:10-19;试剂R1包括含有重量体积比0.5%的牛血清白蛋白、重量体积比0.3%的三氮化钠和重量体积比 0.45%聚氧乙烯去水山梨醇单月桂酸酯的缓冲液;试剂R1还包括变性剂,变性剂为盐酸胍、异硫氰酸胍、硫氰酸胍、尿素、十二烷基硫酸钠中的一种或多种。
2.如权利要求1所述的中性粒细胞明胶酶相关脂质运载蛋白检测试剂盒,其特征在于:试剂R2中还包括促凝剂、稳定剂、蔗糖和甘油,其中,稳定剂为蛋白质、PEG、糖和可溶性的碱金属和碱土金属的无机盐中的一种或多种;蛋白质为牛血清白蛋白或明胶;糖为单糖、二糖或者多糖;无机盐为卤素碱金属和碱土金属的无机盐;试剂R2含有磷酸盐缓冲液、硼酸盐缓冲液、甘氨酸缓冲液、柠檬酸-磷酸盐缓冲液、Tris 缓冲液和HEPES缓冲液中的一种或多种的组合。
3.如权利要求1中性粒细胞明胶酶相关脂质运载蛋白检测试剂盒,其特征在于:抗人中性粒细胞明胶酶相关脂质运载蛋白抗体为多克隆抗体或单克隆抗体;多克隆抗体为兔抗人中性粒细胞明胶酶相关脂质运载蛋白抗体、羊抗人中性粒细胞明胶酶相关脂质运载蛋白抗体或鸡抗人中性粒细胞明胶酶相关脂质运载蛋白抗体的一种;单克隆抗体为鼠抗人中性粒细胞明胶酶相关脂质运载蛋白抗体。
4.如权利要求1中性粒细胞明胶酶相关脂质运载蛋白检测试剂盒,其特征在于:校准品为缓冲基质,其包括4-羟乙基哌嗪乙磺酸、氯化钠、氮化钠、乙二胺四乙酸、牛血清白蛋白、蔗糖,其余为去离子水,溶于基质的6500ng/ml 浓度的中性粒细胞明胶酶相关脂质运载蛋白中。
5.如权利要求1中性粒细胞明胶酶相关脂质运载蛋白检测试剂盒,其特征在于:试剂R1的pH值为8.3,试剂R2的pH值为9.0。
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