CN114381495B - 一种芳香基硫酸酯酶反应试剂、试剂盒及其应用 - Google Patents
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Abstract
本发明涉及医学检验技术领域,尤其涉及一种芳香基硫酸酯酶反应试剂、试剂盒及其应用。所述芳香基硫酸酯酶反应试剂包括:4‑硝基邻苯二酚硫酸盐、缓冲液、稳定剂和表面活性剂;所述缓冲液包括:乙酸钠和/或甘氨酸;所述稳定剂包括:羟乙基淀粉和/或葡聚糖;所述表面活性剂包括:吐温20和月桂醇聚氧乙烯醚硫酸钠。本发明提供的芳香基硫酸酯酶反应试剂具有较高的稳定性,在可以稳定保存12个月以上的同时,还能在一定程度上提高反应速度,提高检测效率。
Description
技术领域
本发明涉及医学检验技术领域,尤其涉及一种芳香基硫酸酯酶反应试剂、试剂盒及其应用。
背景技术
芳香基硫酸酯酶(Arylsulphatase,ARS,EC.3.1.6.3)的分类名为:芳香基-硫酸酯磺基水解酶(Aryl-sul-phate sulphohydrolase),又名硫酸酯酶(Sulphatase)或硫酸脑苷脂硫酸酯酶(Sulfatilate sulfatase),是一类催化硫酸酯水解的蛋白家族,作用于酯键,催化芳香基硫酸酯水解,生成相应的酚类和硫酸盐。
各种恶性肿瘤患者尿中ARS活性明显升高(其中以粒性白血病最为显著),但血清酶活性正常。除血液病外,以结直肠癌、胃癌、膀胱癌病人酶活性最高,其次为乳腺癌、宫颈癌、前列腺癌、肾癌、和皮肤癌,病人尿中ARS活性为对照组的10-15倍。尿ARS-A活性升高的阳性率与肿瘤所波及的范围有关,局限性原发性肿瘤与发生转移者的阳性率分别为61%和92%。
膀胱癌病人尿中ARS活性平均可达到对照值的40倍,ARS-A升高的阳性率为100%,ARS-B为82.8%,高于尿LD(60.7%)和ALP(35.8%)。
结肠癌和直肠癌病人尿中ARS-A活性升高的阳性率(42.9%)低于ARS-B(71%),其酶活性高低与肿瘤累及范围以及肿瘤生长的速度有关。若同时测定尿ARS-A和血中的癌胚抗原,则有助于结肠癌和直肠癌的诊断,以及病情的发展和疗效观察。
鉴于ARS在尿中浓度较高,取材方便且无创伤,更易推广,尤其是在对大规模人群筛选时选用。ARS活性单独或联合检测,在癌症筛查和疗效监测过程中具有潜在的应用价值,值得临床推广应用。
测定ARS活性有层析法(操作繁琐)、透析法(费时,需26-50 h),均不适于临床常规应用;荧光测定法较为敏感,利用甲基伞形酰硫酸酯作底物,经ARS水解后生成的甲基伞形酮是荧光物质,可用荧光计测定其荧光度,此法试剂昂贵、设备特殊,难以普及;放射免疫法是利用纯制的人尿ARS-A作抗原,用131I标记酶抗体结合酶,除去游离酶后再测定ARS-A抗体复合物的放射性,此法适于科研单位进行酶的微量测定。
陈铁河等在《沉淀法分离尿中芳香基硫酸酯酶及其活性测定》中利用沉淀剂将ARS分子从尿液中沉淀出来,而抑制酶活性的物质仍然留在上清中,从而使ARS摆脱抑制物的干扰,使其催化活性得以发挥。以5-硝基-α-羟基苯硫酸酯(NCS)为底物,经ARS水解产生5-硝基儿茶酚(NC),后者在碱性条件下成红色,比色测定即可求得ARS活性,活性的计算采用NC标准液或标准曲线按照每升尿液生成5-硝基-α羟基酚的µmol/min为活性单位(U/L)计算获得,但是不能直接给出结果且37℃水浴温育60 min,反应时间长。
发明内容
为了解决现有技术存在的问题,本发明提供一种芳香基硫酸酯酶反应试剂、试剂盒及其应用。本发明通过特定成分的反应试剂相组合,得到一种稳定性较高的芳香基硫酸酯酶反应试剂,其在可以长时间保存的同时,可以在一定程度上提高反应效率。
第一方面,本发明提供一种芳香基硫酸酯酶反应试剂,包括:
4-硝基邻苯二酚硫酸盐、缓冲液、稳定剂和表面活性剂;
所述缓冲液包括:乙酸钠和/或甘氨酸;
所述稳定剂包括:羟乙基淀粉和/或葡聚糖;
所述表面活性剂包括:吐温20和月桂醇聚氧乙烯醚硫酸钠。
本发明通过缓冲液、稳定剂和表面活性剂组分的配合,增加了稳定性,提高了反应速度,缩短了反应时间。其中缓冲液可产生共轭离子对稳定试剂pH,维持样本反应时芳香基硫酸酯酶酶活最适pH的恒定;稳定剂可以增加溶液粘度,降低二氧化碳在溶液中的溶解速度,维持底物溶液的pH,助力样本反应时芳香基硫酸酯酶酶活最适pH的恒定。表面活性剂不仅可以协同稳定剂增加溶液粘度,还可以通过静电力和疏水效应等与稳定剂相互作用,从而使得混合体系的稳定性更优,保护硫酸酯键的结构稳定,能特异与芳香基硫酸酯酶反应,进而提高芳香基硫酸酯酶水解4-硝基邻苯二酚硫酸盐产生4-硝基儿茶酚的效率,在一定程度上缩短检测时间。
进一步地,包括:4-硝基邻苯二酚硫酸盐3~15份、缓冲液7.5~25.0份、稳定剂1~5份和表面活性剂0.1~0.5份。
进一步地,所述4-硝基邻苯二酚硫酸盐的浓度为0.01~0.05 mol/L;所述缓冲液的浓度为0.1~0.3 mol/L;所述稳定剂的浓度为1~5 g/L;所述表面活性剂的浓度为0.1~0.5g/L。
进一步地,所述芳香基硫酸酯酶反应试剂的pH为4.5~7.0。
进一步地,还包括防腐剂;所述防腐剂为叠氮钠、Procine 300或链霉素中的任意一种或多种,质量百分含量为0.1%~0.3%。
第二方面,本发明提供一种试剂盒,所述试剂盒包括所述芳香基硫酸酯酶反应试剂。
进一步地,还包括沉淀试剂和反应终止试剂;
所述沉淀试剂为0.1-0.5 mol/L 乙酸铅溶液;所述反应终止试剂为1~3 mol/L的NaOH溶液。
进一步地,还包括校准品和质控品。
进一步地,所述校准品和质控品包括缓冲液、稳定剂、激活剂、表面活性剂和芳香基硫酸酯酶;
所述缓冲液包括磷酸氢二钠、磷酸二氢钾、柠檬酸钠和醋酸钠;
所述稳定剂包括甘氨酸、甘油或AES中的一种或多种;
所述激活剂包括氯化钙和/或氯化钾;
所述表面活性剂包括N-月桂酰基肌氨酸钠、N-月桂酰基谷氨酸钠或N-月桂酰基丙氨酸钠中的一种或多种。
进一步地,包括:
缓冲液0.5-25份、稳定剂27.5~81份、激活剂1-10份和表面活性剂0.05-10份;还包括芳香基硫酸酯酶0~2 U/L。
进一步地,所述稳定剂包括:
甘油25~75份、甘氨酸2~5份和AES 0.5~1份。
进一步地,还包括防腐剂1-3份,所述防腐剂为叠氮钠、Procine 300或链霉素中的任意一种或多种。
进一步地,所述校准品和质控品的pH为5.0-7.0。
本发明进一步提供所述芳香基硫酸酯酶反应试剂,或所述试剂盒在检测芳香基硫酸酯酶中的应用。
本发明具备如下有益效果:
本发明通过组合不同的缓冲液、稳定剂和表面活性剂成分和浓度,得到一种芳香基硫酸酯酶反应试剂,成本低廉、原料易获得,制备方便。
本发明提供的试剂盒中的试剂具有良好的稳定性,货架稳定性为一年,且在37℃下反应10 min就能到达峰值。
本发明提供的试剂盒具有很高的安全性,便于实际使用,为芳香基硫酸酯酶的广泛应用奠定了基础。
附图说明
图1为本发明实验例1提供的实施例1-3和对照5的样本酶活与时间的关系图。
具体实施方式
以下实施例用于说明本发明,但不用来限制本发明的范围。
实施例1
本实施例提供一种检测芳香基硫酸酯酶的反应试剂,具体包括试剂1、试剂2和试剂3,具体成分如下:
试剂1:0.5 mol/L乙酸铅。
试剂2:0.2 mol/L乙酸钠、吐温20 0.5 g/L、月桂醇聚氧乙烯醚硫酸钠0.1 g/L、葡聚糖5 g/L、0.03 mol/L 4-硝基邻苯二酚硫酸盐、叠氮钠1 g/L,pH 5.0。
试剂3:1 mol/L氢氧化钠。
配置好后,置于2-8℃条件下保存。
实施例2
本实施例提供一种检测芳香基硫酸酯酶的反应试剂,具体包括试剂1、试剂2和试剂3,具体成分如下:
试剂1:0.4 mol/L乙酸铅。
试剂2:0.3 mol/L乙酸钠、吐温20 0.1 g/L、月桂醇聚氧乙烯醚硫酸钠0.5 g/L、羟乙基淀粉1 g/L、0.04 mol/L 4-硝基邻苯二酚硫酸盐、叠氮钠1 g/L,pH 5.0。
试剂3::2 mol/L氢氧化钠。
配置好后,置于2-8℃条件下保存。
实施例3
本实施例提供一种检测芳香基硫酸酯酶的反应试剂,具体包括试剂1、试剂2和试剂3,具体成分如下:
试剂1:0.4 mol/L乙酸铅。
试剂2:0.2 mol/L甘氨酸、吐温20 0.25 g/L、月桂醇聚氧乙烯醚硫酸钠0.25 g/L、葡聚糖2.5 g/L、0.04 mol/L 4-硝基邻苯二酚硫酸盐、叠氮钠1 g/L,pH 5.0。
试剂3:3 mol/L氢氧化钠。
配置好后,置于2-8℃条件下保存。
实验例1
本发明针对沉淀法检测芳香基硫酸酯酶进行了大量的研究,具体流程如下:
1、设置对照组
对照组1:
试剂1:0.5 mol/L乙酸铅。
试剂2:0.2 mol/L乙酸钠、月桂醇聚氧乙烯醚硫酸钠0.1 g/L 、葡聚糖5 g/L、0.03mol/L 4-硝基邻苯二酚硫酸盐、叠氮钠1 g/L,pH 5.0。
试剂3:1 mol/L氢氧化钠。
对照组2:
试剂1:0.5 mol/L乙酸铅。
试剂2:0.2 mol/L乙酸钠、吐温20 0.5 g/L、葡聚糖5 g/L、0.03 mol/L 4-硝基邻苯二酚硫酸盐、叠氮钠1 g/L,pH 5.0。
试剂3:1 mol/L氢氧化钠。
对照组3:
试剂1:0.5 mol/L乙酸铅。
试剂2:0.2 mol/L乙酸钠、吐温20 0.5 g/L、月桂醇聚氧乙烯醚硫酸钠0.1 g/L、0.03 mol/L 4-硝基邻苯二酚硫酸盐、叠氮钠1 g/L,pH 5.0。
试剂3:1 mol/L氢氧化钠。
对照组4:
试剂1:0.5 mol/L乙酸铅。
试剂2:0.2 mol/L乙酸钠、曲拉通X-100 0.5 g/L、月桂醇聚氧乙烯醚0.1 g/L、葡聚糖5 g/L、0.03 mol/L 4-硝基邻苯二酚硫酸盐、叠氮钠1 g/L,pH 5.0。
试剂3:1 mol/L氢氧化钠。
对照组5:
试剂1:0.5 mol/L乙酸铅。
试剂2:0.2 mol/L乙酸钠、葡聚糖5 g/L、0.03 mol/L 4-硝基邻苯二酚硫酸盐、叠氮钠1 g/L,pH 5.0。
试剂3:1 mol/L氢氧化钠。
测定方法如下:
按照先密后疏的原则在预期稳定时效(2-8℃保存12个月)的7个时间间隔内,测定低浓度样本和高浓度样本,每个样本分别测量3次,测量方法如下:
(1)将试剂盒、校准品、质控品、尿液样本平衡至室温(25-30℃),所有试剂必须摇匀但不能起泡。
(2)分别取20 μL去离子水、校准品、质控品、尿液样本于1.5 mL离心管,然后加入80 μL试剂1,涡旋混匀。4℃静置5 min,4000 rpm离心1 min,离心结束后,放在冰浴上,弃尽上清液保留沉淀。
(3)每个样本中加入40 μL试剂2,充分混匀,使沉淀完全溶解。
(4)所有样本放在37℃的金属浴上,反应10 min。
(5)反应结束后,取出所有的样本置于零度金属浴或冰浴上,在每个样本中快速加入200 μL试剂3,涡旋混匀,置于室温以备用。
(6)取出透明酶标板,按照200 μL/孔加入上述样本。
(7)在酶标仪515 nm处检测。
其中校准品和质控品均采用如下进行配置:
醋酸钠 10 g/L、甘油75 g/L、甘氨酸5 g/L、AES 1 g/L、氯化钙5 g/L、氯化钾10g/L、N-月桂酰基丙氨酸钠 0.1 g/L、叠氮钠1 g/L,pH为6.0,将各成分混匀后,按照需求加入芳香基硫酸酯酶,制备成液体形式的校准品/质控品。
计算测量平均值与实验首日的相对偏差。
图1是样本酶活与时间的关系图,本发明提供的实施例1-3试剂在反应10 min时达到峰值,用于后续检测,优于陈铁河等在《沉淀法分离尿中芳香基硫酸酯酶及其活性测定》中所说的30 min,反应速度快。而对照组5将其中的表面活性剂吐温和月桂醇聚氧乙烯醚成分去除后,反应20分钟后到达峰值,方才用于后续检测,提高了反应速度和检测效率。
实施例1-3稳定性结果见下表:
表1 实施例1-3稳定性检测结果
对照组1-4稳定性结果见表2。
表2 对照组1-4稳定性检测结果
从如上结果中可以看出,本发明提供的实施例1-3的试剂在2-8℃可以在12个月后仍然拥有较高的检测准确率,而对照组1-4在12个月后检测准确率的下降幅度就超过10%以上,体现出了本发明所提供的稳定性组合的优异性能。
虽然,上文中已经用一般性说明及具体实施方案对本发明作了详尽的描述,但在本发明基础上,可以对之作一些修改或改进,这对本领域技术人员而言是显而易见的。因此,在不偏离本发明精神的基础上所做的这些修改或改进,均属于本发明要求保护的范围。
Claims (3)
1.一种芳香基硫酸酯酶反应试剂,其特征在于,包括:
4-硝基邻苯二酚硫酸盐0.01~0.05 mol/L、缓冲液0.1~0.3 mol/L、稳定剂1~5 g/L和表面活性剂0.1~0.5 g/L;
所述缓冲液为:乙酸钠缓冲液或甘氨酸缓冲液;
所述稳定剂为:羟乙基淀粉或葡聚糖;
所述表面活性剂为:吐温20和月桂醇聚氧乙烯醚硫酸钠;
所述芳香基硫酸酯酶反应试剂的pH为5;
所述芳香基硫酸酯酶反应试剂还包括防腐剂;所述防腐剂为叠氮钠、Procine 300或链霉素中的任意一种或多种,质量百分含量为0.1%~0.3%。
2.一种试剂盒,其特征在于,所述试剂盒包括权利要求1所述的芳香基硫酸酯酶反应试剂。
3.根据权利要求2所述的试剂盒,其特征在于,还包括沉淀试剂和反应终止试剂;
所述沉淀试剂为0.1-0.5 mol/L乙酸铅溶液;所述反应终止试剂为1~3 mol/L的NaOH溶液。
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