CN113655226B - 用于房水检测的校准物基质及其制备方法和应用 - Google Patents
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Abstract
本发明提供了一种用于房水检测的校准物基质及其制备方法和应用,涉及生物检测技术领域,本发明在对房水的成分及理化性质进行大量研究的基础上,提供了一种用于房水检测的校准物基质,该校准物基质通过选择特定配比的蛋白、糖类、盐类、抗坏血酸和缓冲液作为主要成分,能够有效降低样本检测过程中的基质效应,提高房水检测体外诊断试剂的检测准确性和精密度。
Description
技术领域
本发明涉及生物监测技术领域,尤其是涉及一种用于房水检测的校准物基质及其制备方法和应用。
背景技术
体外诊断在临床中的应用价值已毋庸置疑,各检测项目在血液样本的应用已经非常成熟,随着近些年体外诊断试剂细分领域的检测水平提升,在临床精准诊疗中发挥的价值也越来越大,然而在眼科原位样本的检测领域,还缺乏针对性的体外诊断试剂产品。
根据世界卫生组织(WHO)发布的《世界视力报告》显示,全球至少有10亿人的视力受损问题尚未得到解决。2012年-2016年,我国眼科门诊患者数量已从8,740万人攀升至10,940万人,复合年增长率达5.8%,眼病发病与就诊率呈上涨趋势。
以葡萄膜炎(也被称为眼内膜炎症)为代表的眼内炎症是临床常见的致盲原因。2016年全国葡萄膜炎患者数量达400多万例,以中青年患者居多。造成葡萄膜炎的病因和类型达100余种,容易反复发作,诊断和治疗非常棘手,被称为“眼科中的一块硬骨头”。葡萄膜炎如果延误治疗时机,会使病情加重,引起白内障、青光眼、黄斑水肿、视神经萎缩、眼球萎缩等并发症,后果相当严重,最终可导致永久失明,据报道,有10%到15%的失明源于此病。引起眼内炎症的病因很多,包括病毒、细菌、真菌、自身免疫紊乱以及恶性肿瘤等,不同病因引起的眼内炎症在临床表现上常常相似,治疗上却相去甚远,如果给予错误的治疗手段,不仅可能导致患者视力快速丧失,甚至可能危及生命,因此需要采用实验室手段进行精准、快速的病因学鉴定。遗憾的是,传统的染色涂片、微生物培养和PCR均存在显著缺陷,不能满足快速、准确、全面的实验室检测需要。而当快速发展的影像学给予临床足够证据的时候,也恐怕为时过晚,临床早发现病因,早治疗和干预,才是预防大面积损伤和失明的根本。
因此对快速准确的专门针对眼内液样本的体外诊断检测产品的需求日益凸显。
有鉴于此,特提出本发明。
发明内容
本发明的目的之一在于提供一种用于房水检测的校准物基质,以至少缓解现有技术中的技术问题之一。
本发明的目的之二在于提供上述用于房水检测的校准物基质的制备方法。
本发明的目的之三在于提供上述用于房水检测的校准物基质的应用。
为了实现本发明的上述目的,特采用以下技术方案:
本发明提供了一种用于房水检测的校准物基质,每L校准物基质包括:0.03~0.8 g蛋白、0.1~2.8 g糖类、0.001~3.5 g盐类、0.01~0.8 g抗坏血酸以及余量的缓冲液。
进一步的,每L校准物基质包括:0.05~0.54 g蛋白、0.14~2.556 g糖类、0.003~3.08 g盐类、0.021~0.611 g抗坏血酸以及余量的缓冲液。
进一步的,所述蛋白包括牛血清白蛋白、鸡卵清蛋白、人血清白蛋白或钥孔血蓝蛋白中的一种或多种。
进一步的,所述糖类包括葡萄糖、果糖、蔗糖或麦茅糖中的一种或多种。
进一步的,所述盐类包括有机盐和/或无机盐;
所述有机盐包括乳酸盐;所述无机盐包括磷酸盐。
进一步的,所述缓冲液的pH为7.0~7.8;
所述缓冲液包括PBS缓冲液。
进一步的,每L校准物基质还包括0.05~1.15 g防腐剂。
本发明还提供了上述用于房水检测的校准物基质的制备方法,取配方量的各组分混合均匀,得到所述校准物基质。
进一步的,取配方量的蛋白、糖类、盐类以及抗坏血酸,和/或防腐剂加入到少量缓冲液中预溶解、混匀,然后与余量的缓冲液混合均匀,得到所述校准物基质。
此外,本发明还提供了上述用于房水检测的校准物基质在作为房水检测校准品和质控品中的应用。
与现有技术相比,本发明具有如下有益效果:
本发明在对房水的成分及理化性质进行大量研究的基础上,提供了一种用于房水检测的校准物基质,该校准物基质通过选择特定配比的蛋白、糖类、盐类、抗坏血酸和缓冲液作为主要成分,能够有效降低样本检测过程中的基质效应,提高房水检测体外诊断试剂的检测准确性和精密度。
具体实施方式
下面将结合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前提下所获得的所有其他实施例,都属于本发明保护的范围。
如无特殊说明,对本发明中的术语定义如下:
基质效应:被测量以外的某种样品特性对被测量的值的影响。
基质:除了分析物之外的所有组成成分。
目前,为满足临床需要,亟需能够快速准确的针对眼内液样本的体外诊断检测产品。然而,好的产品性能离不开质量控制,相对稳定的眼内液基质溶液,是做好产品质量控制必备的条件。
基于此,本发明提供了一种用于房水检测的校准物基质,每L校准物基质包括:0.03~0.8 g蛋白、0.1~2.8 g糖类、0.001~3.5 g盐类、0.01~0.8 g抗坏血酸以及余量的缓冲液。
本发明在对房水的成分及理化性质进行大量研究的基础上,提供了一种用于房水检测的校准物基质,该校准物基质通过选择特定配比的蛋白、糖类、盐类、抗坏血酸和缓冲液作为主要成分,能够有效降低样本检测过程中的基质效应,提高房水检测体外诊断试剂的检测准确性和精密度。
可以理解的是,每单位校准物基质中包含了缓冲液,以及溶解于缓冲液中的配方量的蛋白、糖类、盐类和抗坏血酸。
本发明考虑到不同的待测样本本身存在的个体差异,对校准物基质中的蛋白、糖类、盐类及抗坏血酸的含量进行了合理选择,其中,每L校准物基质中蛋白的含量例如可以为,但不限于0.03 g、0.05 g、0.1 g、0.2 g、0.3 g、0.4 g、0.5 g、0.6 g、0.7 g或0.8 g;糖类的含量例如可以为,但不限于0.1 g、0.5 g、1 g、1.5 g、2 g、2.5 g或2.8 g;盐类的含量例如可以为,但不限于0.001 g、0.01 g、0.1 g、1 g、2 g、3 g或3.5 g;抗环血酸的含量例如可以为,但不限于0.01 g、0.1 g、0.2 g、0.3 g、0.4 g、0.5 g、0.6 g、0.7 g或0.8 g。
通过对上述配方中各组分含量的合理调整和优化,在一些优选的实施方式中,每L校准物基质包括:0.05~0.54 g蛋白、0.14~2.556 g糖类、0.003~3.08 g盐类、0.021~0.611g抗坏血酸以及余量的缓冲液。
可选地,所述蛋白包括牛血清白蛋白、鸡卵清蛋白、人血清白蛋白或钥孔血蓝蛋白中的一种或多种。可以理解的是,可以选择单独的牛血清白蛋白、单独的鸡卵清蛋白、单独的人血清白蛋白或单独的钥孔血蓝蛋白作为本发明校准物基质中的蛋白;也可以选择其中的两种共同作为本发明校准物基质中的蛋白,例如同时选择牛血清白蛋白和鸡卵清蛋白,或者同时选择鸡卵清蛋白和人血清白蛋白;也可以选择其中的三种或更多种共同作为本发明校准物基质中的蛋白,例如同时选择牛血清白蛋白、鸡卵清蛋白和人血清白蛋白,或者同时选择牛血清白蛋白、鸡卵清蛋白、人血清白蛋白和钥孔血蓝蛋白。
可选地,所述糖类包括葡萄糖、果糖、蔗糖或麦茅糖中的一种或多种。可以理解的是,可以选择单独的葡萄糖、单独的果糖、单独的蔗糖或单独的麦茅糖作为本发明校准物基质中的糖类;也可以选择其中的两种共同作为本发明校准物基质中的糖类,例如同时选择葡萄糖和果糖,或者同时选择蔗糖和麦芽糖;也可以选择其中的三种或更多种共同作为本发明校准物基质中的糖类,例如同时选择葡萄糖、果糖和蔗糖,或者同时选择葡萄糖、果糖、蔗糖和麦茅糖。
可选地,所述盐类包括有机盐和/或无机盐。可以理解的是,“和/或”意味着可以单独选择有机盐或者无机盐,也可以同时选择有机盐和无机盐。其中有机盐可以为乳酸盐,例如乳酸钙、乳酸锌、乳酸镁或乳酸钠;无机盐可以为磷酸盐,例如磷酸钙或磷酸钠。
可选地,本发明中典型的缓冲液可以为PBS缓冲液,综合泪液渗透压、pH值以及房水中钠、钾含量的考虑,选用PBS缓冲液代替纯水能够进一步降低检测过程中的基质效应。
为了进一步提升该校准物基质的稳定性,延长保存时间,节约生产成本,本发明提供的校准物基质中还可以包括防腐剂,其含量为每L校准物基质包括0.05~1.15 g防腐剂,例如可以为,但不限于0.05 g、0.1 g、0.5 g、1 g或1.15 g。具体的防腐剂可以选自叠氮钠、硫柳汞、PC-300、PC-950、BND-10或GML-2。
本发明还提供了上述用于房水检测的校准物基质的制备方法,包括取配方量的各组分混合均匀,得到所述校准物基质。
该制备方法工艺简单,易于操作,对人员及设备均无额外要求,适于生产应用。
作为优选,上述用于房水检测的校准物基质的制备方法,包括取配方量的蛋白、糖类、盐类以及抗坏血酸,和/或防腐剂加入到少量缓冲液中预溶解、混匀,然后与余量的缓冲液混合均匀,得到所述校准物基质。
通过预溶解、混匀的方式,能够在保证溶解性的基础上进一步提高校准物基质的制备效率。
具体地,以配置1L基质溶液为例,包括如下步骤:
1)向量杯中加入100 ml PBS缓冲液;
2)准确称取配方要求的人血清白蛋白、葡萄糖、抗坏血酸和乳酸钙质量体积;
3)分别将称取好的成分加入上述1)中,搅拌至完全溶解,混匀;
4)用PBS缓冲液定容至1L,得到最终的校准物基质。
此外,基于本发明提供的校准物基质的有益效果,本发明还提供了上述用于房水检测的校准物基质在作为房水检测校准品和质控品中的应用。
下面通过实施例对本发明作进一步说明。如无特别说明,实施例中的材料为根据现有方法制备而得,或直接从市场上购得。
实施例1
本实施例提供了一种用于房水检测的校准物基质,每L校准物基质包括:0.2 g人血清白蛋白、0.9 g葡萄糖、0.9 g乳酸钙、0.176 g抗坏血酸、0.2 g叠氮钠以及余量的PBS缓冲液。
实施例2
本实施例提供了一种用于房水检测的校准物基质,每L校准物基质包括:0.03 g人血清白蛋白、2.8 g葡萄糖、0.001 g乳酸钙、0.8g抗坏血酸、1.15 g叠氮钠以及余量的PBS缓冲液。
实施例3
本实施例提供了一种用于房水检测的校准物基质,每L校准物基质包括:0.8 g人血清白蛋白、0.1 g葡萄糖、3.5 g乳酸钙、0.01 g抗坏血酸、0.05 g叠氮钠以及余量的PBS缓冲液。
实施例4
本实施例提供了一种用于房水检测的校准物基质,每L校准物基质包括:0.3 g牛血清白蛋白、0.2 g果糖、0.05 g磷酸钙、0.1 g抗坏血酸、0.05 g PC-300以及余量的PBS缓冲液。
实施例5
本实施例提供了一种用于房水检测的校准物基质,与实施例1的区别在于不含有防腐剂。
对比例1
本对比例提供了一种用于房水检测的校准物基质,每L校准物基质包括:0.02 g人血清白蛋白、3 g葡萄糖、4 g乳酸钙、0.008 g抗坏血酸、1.22 g叠氮钠以及余量的PBS缓冲液。
对比例2
本对比例与实施例1的区别在于,不含有人血清白蛋白。
对比例3
本对比例与实施例1的区别在于,不含有乳酸钙。
对比例4
本对比例与实施例1的区别在于,不含有抗坏血酸。
实验例
本实验例以白介素-6(IL-6)检测试剂盒(生产厂家:罗氏(Roche),批号:54022601,规格:100T)及其检测系统作为基质效应的验证系统:
1)验证本发明实施例1-5及对比例1-4提供的校准物基质与实际临床IL-6阴性房水样本的基质检测差异是否显著;
2)用本发明实施例1-5及对比例1-4提供的校准物基质和人体房水样本分别配置两个不同浓度的标准品同步检测,验证检测差异是否显著。
实验准备:
1)备好检测用的试剂和仪器,仪器提前预热准备,试剂提前平衡至室温;
2)准备本发明实施例1-5及对比例1-4提供的校准物基质各8份,房水样本8份,用于空白样本检测;
3)用本发明实施例1-5及对比例1-4提供的校准物基质和房水样本分别制备两个高、低不同浓度的标准品样本各8份(标品浓度分别为10ppt和100ppt),用于加标样本检测。
实验方法:
同样的人员和环境条件下,根据白介素-6(IL-6)检测试剂盒检测方法要求,对上述准备好的样本进行检测,记录每例样本检测值。
评价方法:
分别对两组空白样本、两组浓度为10ppt的样本和两组浓度为100ppt的样本进行独立样本t检验,双尾p>0.05,两组数据差异无统计学意义,可接受;双尾p<0.05,两组数据差异有统计学意义,不可接受。
实验数据和验证结果:
实验数据见表1,t检验数据见表2~表4。
表1 检测数据
表2 空白样本t检验数据
表3 浓度10ppt样本t检验数据
表4 浓度100ppt样本t检验数据
结论:
经过验证,本发明实施例1-5提供的校准物基质与人体真实房水相比较,基质效应对检测结果影响可接受,可用作房水检测用的校准品和质控品。
最后应说明的是:以上各实施例仅用以说明本发明的技术方案,而非对其限制;尽管参照前述各实施例对本发明进行了详细的说明,本领域的普通技术人员应当理解:其依然可以对前述各实施例所记载的技术方案进行修改,或者对其中部分或者全部技术特征进行等同替换;而这些修改或者替换,并不使相应技术方案的本质脱离本发明各实施例技术方案的范围。
Claims (8)
1.一种用于房水检测的校准物基质,其特征在于,每升校准物基质由以下成分组成:0.03~0.8 g人血清白蛋白、0.1~2.8 g糖类、0.001~3.5 g盐类、0.01~0.8 g抗坏血酸以及余量的缓冲液。
2.根据权利要求1所述的校准物基质,其特征在于,每升校准物基质由以下成分组成:0.05~0.54 g人血清白蛋白、0.14~2.556 g糖类、0.003~3.08 g盐类、0.021~0.611 g抗坏血酸以及余量的缓冲液。
3.根据权利要求1所述的校准物基质,其特征在于,所述糖类包括葡萄糖、果糖、蔗糖或麦茅糖中的一种或多种。
4.根据权利要求1所述的校准物基质,其特征在于,所述盐类包括有机盐和/或无机盐;
所述有机盐包括乳酸盐;所述无机盐包括磷酸盐。
5.根据权利要求1所述的校准物基质,其特征在于,所述缓冲液的pH为7.0~7.8;
所述缓冲液包括PBS缓冲液。
6.权利要求1-5任一项所述的用于房水检测的校准物基质的制备方法,其特征在于,取配方量的各组分混合均匀,得到所述校准物基质。
7.根据权利要求6所述的制备方法,其特征在于,取配方量的人血清白蛋白、糖类、盐类以及抗坏血酸加入到少量缓冲液中预溶解、混匀,然后与余量的缓冲液混合均匀,得到所述校准物基质。
8.权利要求1-5任一项所述的用于房水检测的校准物基质在作为房水检测校准品和质控品中的应用。
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