CN111157748A - 胃泌素17定量检测试剂盒 - Google Patents
胃泌素17定量检测试剂盒 Download PDFInfo
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Abstract
本发明公开了胃泌素17定量检测试剂盒,包括酶标液、胃泌素17标准品、包被抗胃泌素17单克隆抗体的免疫磁珠、化学发光底物液、洗涤液;本发明将化学发光法进行了改进,将胃泌素‑17单克隆抗体包被在磁珠微球表面,捕捉样本中的胃泌素‑17,在加入酶标胃泌素‑17单克隆抗体试剂后,形成固相‑抗体‑抗原‑酶标抗体夹心免疫复合物;本发明具有灵敏度高,线性相关性良好,检测结果准确,可靠性高,发光时间长、效果好的特点。
Description
技术领域
本发明涉及体外诊断领域,具体涉及胃泌素17定量检测试剂盒。
背景技术
胃泌素是一种重要的胃肠激素,它主要由G细胞分泌,G细胞是典型的开放型细胞,以胃窦部最多,其次是胃底、十二指肠和空肠等处,人胰岛的D细胞亦能分泌胃泌素,胃泌素对调节消化道功能和维持其结构完整有重要作用。人体中,95%以上有生物活性的胃泌素是α-酰胺化胃泌素,主要含两种异构体G-17和G-34,其中80%~90%是G-17。G-17仅由胃窦部G细胞分泌,因此G-17是反映胃黏膜损伤情况的重要指标。
血清胃泌素的检测,可以用于辅助检查很多的疾病,血清胃泌素升高可见于胃泌素瘤、胃癌、胃窦细胞增生、十二指肠溃疡、萎缩性胃炎等疾病;血清胃泌素降低可能是由于甲状腺功能减退、浅表性胃炎、十二指肠溃疡等疾病造成的,对于胃部疾病的检测意义很大。
胃泌素17检测是一种通过抽取人静脉血2ml(空腹),测定其中胃泌素17含量从而辅助诊断胃粘膜疾病的方法,是一项无创、无痛、安全、经济的胃病检测方法,胃泌素17检测能够筛查胃部疾病及癌前病变,阻断胃癌的进展;判断胃酸水平,提示胃食管反流病风险。因而,胃泌素17(G-17)的检测具有十分重要的意义。
发明内容
本发明的目的在于提供胃泌素17定量检测试剂盒,化学发光效果好、灵敏度高、相关性良好。
为实现上述目的,本发明的技术方案提供胃泌素17定量检测试剂盒,包括酶标液、胃泌素17标准品、包被抗胃泌素17单克隆抗体的免疫磁珠、化学发光底物液、洗涤液。
所述酶标液为HRP标记抗胃泌素17单克隆抗体。
所述胃泌素17标准品为含有不同量胃泌素G17抗原的BSA蛋白溶液。
所述洗涤液是含有吐温-20和ProCline-300的缓冲液。
所述包被胃泌素17单克隆抗体的免疫磁珠为标记有抗胃泌素-17单克隆抗体的磁珠。
所述抗胃泌素17单克隆抗体包被免疫磁珠的方法如下:
(1)洗涤磁珠:取100μl磁珠,将其稀释成10mg/ml的磁珠浓度,从中吸取100μl,磁分离,去上清,用洗涤液1ml洗涤三次,磁分离,去上清;
(2)活化:取洗涤完成的磁珠加入150μl MES,再加入50μl EDC及50μl的NHS溶液,充分混匀,室温下25℃活化30min,活化完成磁分离去上清,以洗涤液重悬洗涤三次,分离去上清,得到活化的磁珠;
(3)稀释抗胃泌素17单克隆抗体:取抗胃泌素17单克隆抗体20μl,添加样本稀释液380μl,混匀;
(4)孵育:向洗涤完成后的磁珠中加入丙烯醛,经电子加速器照射使磁珠表面形成一层活性膜后,加入稀释后的抗胃泌素17单克隆抗体,37℃摇床孵育12h;
(5)封闭:将孵育后的磁珠磁分离,去上清,加入400μl封闭液,37℃摇床封闭1h,封闭结束后去除上清,洗涤液清洗3次,最终加稀释液定容到500μl。
所述封闭液是将0.01g叠氮化钠、10g牛血清白蛋白和100mL浓度为0.03mol/L磷酸盐溶液(pH=7.4)混合得到的溶液。
所述稀释液是含有1%BSA的PBS溶液。
所述化学发光底物液的配制方法如下:
(a)化学发光底物液A液配制:称取0.0094g过氧化脲、0.01g四聚磷酸钠、0.03g氟化钠、0.01g酒石酸钠、0.172g柠檬酸、0.347g柠檬酸钠,调整pH至5.0,用纯化水定容至100ml;
(b)化学发光底物液B液配制:称取0.03g鲁米诺、0.01g十二烷基二甲基甜菜碱、0.05g对乙酰氨基酚、0.01g 2、4、6三(二甲氨基甲基)苯酚、0.0858g硼砂、0.0680g硼酸,调整pH至8.4,用纯化水定容至100ml。
本发明具有有益效果:本发明将化学发光法进行了改进,将胃泌素-17单克隆抗体包被在磁珠微球表面,捕捉样本中的胃泌素-17,在加入酶标胃泌素-17单克隆抗体试剂后,形成固相-抗体-抗原-酶标抗体夹心免疫复合物,化学发光底物液通过采用十二烷基二甲基甜菜碱,具有成本低廉、环保无害的优点;本发明具有灵敏度高,线性相关性良好,检测结果准确,可靠性高,发光时间长、效果好的特点。
附图说明
图1为胃泌素17标准品检测的标准曲线图。
具体实施方式
下面结合实施例,对本发明的具体实施方式作进一步详细描述。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
胃泌素17定量检测试剂盒,包括酶标液、胃泌素17标准品、包被抗胃泌素17单克隆抗体的免疫磁珠、化学发光底物液、洗涤液;所述酶标液为HRP标记抗胃泌素17单克隆抗体;所述胃泌素17标准品为含有不同量胃泌素G17抗原的BSA蛋白溶液;所述洗涤液是含有吐温-20和ProCline-300的缓冲液;所述包被胃泌素17单克隆抗体的免疫磁珠为标记有抗胃泌素-17单克隆抗体的磁珠。
所述抗胃泌素17单克隆抗体包被免疫磁珠的方法如下:
(1)洗涤磁珠:取100μl磁珠,将其稀释成10mg/ml的磁珠浓度,从中吸取100μl,磁分离,去上清,用洗涤液1ml洗涤三次,磁分离,去上清;
(2)活化:取洗涤完成的磁珠加入150μl MES,再加入50μl EDC及50μl的NHS溶液,充分混匀,室温下25℃活化30min,活化完成磁分离去上清,以洗涤液重悬洗涤三次,分离去上清,得到活化的磁珠;
(3)稀释抗胃泌素17单克隆抗体:取抗胃泌素17单克隆抗体20μl,添加样本稀释液380μl,混匀;
(4)孵育:向洗涤完成后的磁珠中加入丙烯醛,经电子加速器照射使磁珠表面形成一层活性膜后,加入稀释后的抗胃泌素17单克隆抗体,37℃摇床孵育12h;
(5)封闭:将孵育后的磁珠磁分离,去上清,加入400μl封闭液,37℃摇床封闭1h,封闭结束后去除上清,洗涤液清洗3次,最终加稀释液定容到500μl。
所述封闭液是将0.01g叠氮化钠、10g牛血清白蛋白和100mL浓度为0.03mol/L磷酸盐溶液(pH=7.4)混合得到的溶液;所述稀释液是含有1%BSA的PBS溶液。
所述化学发光底物液的配制方法如下:
(a)化学发光底物液A液配制:称取0.0094g过氧化脲、0.01g四聚磷酸钠、0.03g氟化钠、0.01g酒石酸钠、0.172g柠檬酸、0.347g柠檬酸钠,调整pH至5.0,用纯化水定容至100ml;
(b)化学发光底物液B液配制:称取0.03g鲁米诺、0.01g十二烷基二甲基甜菜碱、0.05g对乙酰氨基酚、0.01g 2、4、6三(二甲氨基甲基)苯酚、0.0858g硼砂、0.0680g硼酸,调整pH至8.4,用纯化水定容至100ml。
实施例2
本发明还包括了胃泌素17定量检测试剂盒的检测方法,步骤如下:
1)取胃泌素17标准品100μl加入反应管中,再依次加入包被抗胃泌素17单克隆抗体的免疫磁珠30μl、酶标液100μl,37℃孵育15min;
2)将孵育反应完成的反应管放在磁分离架上分离2min,去上清,加上洗涤液500μl,放在磁分离架上清洗1min;去上清,加上洗涤液300μl,放在磁分离架上清洗1min;
3)向反应管中加入100μl化学发光底物液A,1min后加入100μl化学发光底物液B,37℃避光孵育2min后,测量发光值。
检测结果如表1所示,灵敏度最低检出值0.1pmol/L;根据化学发光值与浓度作校准曲线,如图1所示,线性系数r=0.9977,线性范围1~100pmol/L。
表1
实施例3
本发明还进行了重复试验,以验证本试剂盒的准确性。
1)分别取浓度为5pmol/l的胃泌素17标准品100μl加入反应管中,再依次加入包被抗胃泌素17单克隆抗体的免疫磁珠30μl、酶标液100μl,37℃孵育15min;
2)将孵育反应完成的反应管放在磁分离架上分离2min,去上清,加上洗涤液500μl,放在磁分离架上清洗1min;去上清,加上洗涤液300μl,放在磁分离架上清洗1min;
3)向反应管中加入100μl化学发光底物液A,1min后加入100μl化学发光底物液B,37℃避光孵育2min后,测量发光值;
3)重复检测9次,利用线性方程y=3114.3x+3982.5,计算检测浓度值。
检测结果如表2所示:测定的平均值分别为4.998pmol/l,标准偏差为0.076,批内变异系数为1.5%,本试剂盒检测结果准确度高,可作为辅助诊断胃部疾病的重要工具。
表2
胃泌素17标准品(5pmol/l) | |
第一次 | 4.95 |
第二次 | 5.02 |
第三次 | 5.09 |
第四次 | 5.02 |
第五次 | 4.98 |
第六次 | 4.89 |
第七次 | 5.07 |
第八次 | 5.1 |
第九次 | 4.89 |
第十次 | 4.97 |
平均值 | 4.998 |
标准偏差 | 0.076 |
批内变异系数 | 1.5% |
尽管已经示出和描述了本发明的实施例,对于本领域的普通技术人员而言,可以理解在不脱离本发明的原理和精神的情况下可以对这些实施例进行多种变化、修改、替换和变型,本发明的范围由所附权利要求及其等同物限定。
Claims (9)
1.胃泌素17定量检测试剂盒,其特征在于,包括酶标液、胃泌素17标准品、包被抗胃泌素17单克隆抗体的免疫磁珠、化学发光底物液、洗涤液。
2.如权利要求1所述的胃泌素17定量检测试剂盒,其特征在于,所述酶标液为HRP标记抗胃泌素17单克隆抗体。
3.如权利要求1所述的胃泌素17定量检测试剂盒,其特征在于,所述胃泌素17标准品为含有不同量胃泌素G17抗原的BSA蛋白溶液。
4.如权利要求1所述的胃泌素17定量检测试剂盒,其特征在于,所述包被胃泌素17单克隆抗体的免疫磁珠为标记有抗胃泌素-17单克隆抗体的磁珠。
5.如权利要求1所述的胃泌素17定量检测试剂盒,其特征在于,所述抗胃泌素17单克隆抗体包被免疫磁珠的方法如下:
(1)洗涤磁珠:取100μl磁珠,将其稀释成10mg/ml的磁珠浓度,从中吸取100μl,磁分离,去上清,用洗涤液1ml洗涤三次,磁分离,去上清;
(2)活化:取洗涤完成的磁珠加入150μlMES,再加入50μlEDC及50μl的NHS溶液,充分混匀,室温下25℃活化30min,活化完成磁分离去上清,以洗涤液重悬洗涤三次,分离去上清,得到活化的磁珠;
(3)稀释抗胃泌素17单克隆抗体:取抗胃泌素17单克隆抗体20μl,添加样本稀释液380μl,混匀;
(4)孵育:向洗涤完成后的磁珠中加入丙烯醛,经电子加速器照射使磁珠表面形成一层活性膜后,加入稀释后的抗胃泌素17单克隆抗体,37℃摇床孵育12h;
(5)封闭:将孵育后的磁珠磁分离,去上清,加入400μl封闭液,37℃摇床封闭1h,封闭结束后去除上清,洗涤液清洗3次,最终加稀释液定容到500μl。
6.如权利要求5所述的胃泌素17定量检测试剂盒,其特征在于,所述封闭液是将0.01g叠氮化钠、10g牛血清白蛋白和100mL浓度为0.03mol/L磷酸盐溶液(pH=7.4)混合得到的溶液。
7.如权利要求5所述的胃泌素17定量检测试剂盒,其特征在于,所述稀释液是含有1%BSA的PBS溶液。
8.如权利要求1或5所述的胃泌素17定量检测试剂盒,其特征在于,所述洗涤液是含有吐温-20和ProCline-300的缓冲液。
9.如权利要求1所述的胃泌素17定量检测试剂盒,其特征在于,所述化学发光底物液的配制方法如下:
(a)化学发光底物液A液配制:称取0.0094g过氧化脲、0.01g四聚磷酸钠、0.03g氟化钠、0.01g酒石酸钠、0.172g柠檬酸、0.347g柠檬酸钠,调整pH至5.0,用纯化水定容至100ml;
(b)化学发光底物液B液配制:称取0.03g鲁米诺、0.01g十二烷基二甲基甜菜碱、0.05g对乙酰氨基酚、0.01g 2、4、6三(二甲氨基甲基)苯酚、0.0858g硼砂、0.0680g硼酸,调整pH至8.4,用纯化水定容至100ml。
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