CN1327224C - 热应激蛋白71抗体的检测方法 - Google Patents

热应激蛋白71抗体的检测方法 Download PDF

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CN1327224C
CN1327224C CNB2003101116948A CN200310111694A CN1327224C CN 1327224 C CN1327224 C CN 1327224C CN B2003101116948 A CNB2003101116948 A CN B2003101116948A CN 200310111694 A CN200310111694 A CN 200310111694A CN 1327224 C CN1327224 C CN 1327224C
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pbs
heat stress
stress protein
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nitrocellulose filter
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CN1635377A (zh
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邬堂春
陈�胜
肖成峰
何启强
杨杪
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Tongji Medical College of Huazhong University of Science and Technology
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Tongji Medical College of Huazhong University of Science and Technology
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Abstract

一种检测热应激蛋白71抗体的方法，将分离的热应激蛋白71电转移至硝酸纤维素膜上，丽春红S染色，将含HSP71抗原的硝酸纤维素滤膜条裁成2×2mm2的膜片，分装入96孔酶标板，PBS清洗30分钟，之后用封闭液于37℃封闭1小时，随后的步骤同ELISA方法，本发明方法将特别适用于基层研究单位及大范围人群血浆抗体滴度的测定。

Description

热应激蛋白71抗体的检测方法
技术领域
本发明涉及化学检测方法，特别是蛋白质检测方法。
背景技术
传统检测热应激蛋白71抗体是用经典的蛋白质检测方法，即Western blot法，其不足之处是此方法过于复杂，不适用于基层研究单位应用和大范围人群血浆抗体滴度的测定。本发明通过对抗原表达及纯化过程及抗体滴度测定反复研究，提供了一种将Western blot这一经典的蛋白质检测方法与ELISA相结合的Western blot-ELISA间接法用于检测血浆中热应激蛋白71抗体的滴度，使检测变得方便简单，将特别适用于基层研究单位及大范围人群血浆抗体滴度的测定。
发明内容
本发明的创新之处就是将已知抗原吸附于硝酸纤维素膜，而不是将其包被在酶标板上。将含有已知抗原的硝酸纤维素膜切分为2×2mm2的小片，分别加入96孔酶标板，PBS清洗30分钟，之后用封闭液于37℃封闭1小时，随后的步骤同ELISA方法。以辣根过氧化物酶标记抗抗体检测为例，阳性结果为硝酸纤维素膜的一面的中间有一条肉眼清晰可见的棕色条带，另一面则没有或极弱。该方法较传统的ELISA间接法更为灵敏可靠，所得结果相当直观，仅用目测即可十分准确地判断阴阳性和抗体滴度，免去了必须依赖酶标仪，结果判断必须人为界定一个参考值等主观因素，将特别适用于基层研究单位及大范围人群血浆抗体滴度的测定。本发明方法检测血浆热应激蛋白71抗体的具体步骤是：
1)将分离的热应激蛋白71电转移至硝酸纤维素膜上，丽春红S染色，将含HSP71抗原的硝酸纤维素滤膜条裁成2×2mm2的膜片，分装入96孔酶标板；
2)每孔加入200μl PBS清洗30分钟，吸去PBS；
3)加入封闭液，37℃封闭1小时或4℃封闭过夜；
4)按1∶10、1∶20、1∶40、1∶80稀释待检血浆，各加入200μl稀释液于酶标孔中，记下加样顺序。37℃温育2小时或4℃过夜，使相应抗体与硝酸纤维素滤膜上HSP71抗原结合；
5)200μl PBS-0.05％Tween 20洗涤10分钟，重复6次；
6)每孔加入200μl 1∶500稀释的辣根过氧化物酶标记羊抗人IgG，37℃温育2小时；
7)200μl PBS-0.05％Tween 20洗涤10分钟，重复8次；
8)100μl DAB显色液显色5-10分钟；
9)立即吸出显色液，用PBS-0.05％Tween 20洗涤3次以终止显色反应，判断结果。
阳性结果为硝酸纤维素膜的一面的中间有一条肉眼清晰可见的棕色条带，另一面则没有或极弱。阴性则无棕色条带。见附图1
附图说明
图1为本发明方法检测血浆HSP71抗体滴度结果
具体实施方式
下面是用本发明方法检测血浆热应激蛋白71的具体实施方法。
(一)抗原的准备
用SDS-PAGE方法从细菌表达物中分离热应激蛋白71(heat stressprotein 71，HSP71)，电转移至硝酸纤维素膜上，丽春红S染色，剪裁下含HSP71抗原的宽2mm的硝酸纤维素膜条，分为2×2mm2的小片，加入96孔酶标板，PBS清洗30分钟后，封闭液于37℃封闭1小时，备用。
(二)血浆HSP71抗体滴度的检测
1)将含HSP71抗原的硝酸纤维素滤膜条裁成2×2mm2的膜片，分装入96孔酶标板。
2)每孔加入200μl PBS清洗30分钟，吸去PBS。
3)加入封闭液，37℃封闭1小时或4℃封闭过夜。
4)按1∶10、1∶20、1∶40、1∶80稀释待检血浆，各加入200μl稀释液于酶标孔中，记下加样顺序。37℃温育2小时或4℃过夜，使相应抗体与硝酸纤维素滤膜上HSP71抗原结合。
5)200μl PBS-0.05％Tween 20洗涤10分钟，重复6次。
6)每孔加入200μl 1∶500稀释的辣根过氧化物酶标记羊抗人IgG，37℃温育2小时。
7)200μl PBS-0.05％Tween 20洗涤10分钟，重复8次。
8)100μl DAB显色液显色5-10分钟，37℃温育可加快显色速度。
DAB显色液
    DAB                6mg
    0.3％NiCl2     1ml
0.01M Tris-Cl 9ml
(pH7.6)
30％H2O2      10μl
DAB显色液临用前配制。
9)立即吸出显色液，用PBS-0.05％Tween 20洗涤3次以终止显色反应，亮光下判断结果。阳性结果为硝酸纤维素膜的一面的中间有一条肉眼清晰可见的棕色条带，另一面则没有或极弱。阴性则无棕色条带。见附图1。

Claims (1)

1.一种检测热应激蛋白71抗体的方法，包括以下步骤：
1)将分离的热应激蛋白71电转移至硝酸纤维素膜上，丽春红S染色，将含HSP71抗原的硝酸纤维素滤膜条裁成2×2mm2的膜片，分装入96孔酶标板；
2)每孔加入200μl PBS清洗30分钟，吸去PBS；
3)加入封闭液，37℃封闭1小时或4℃封闭过夜；
4)按1∶10、1∶20、1∶40、1∶80稀释待检血浆，各加入200μl稀释液于酶标孔中，记下加样顺序，37℃温育2小时或4℃过夜，使相应抗体与硝酸纤维素滤膜上HSP71抗原结合；
5)200μl PBS-0.05％Tween 20洗涤10分钟，重复6次；
6)每孔加入200μl 1∶500稀释的辣根过氧化物酶标记羊抗人IgG，37℃温育2小时；
7)200μl PBS-0.05％Tween 20洗涤10分钟，重复8次；
8)100μl DAB显色液显色5-10分钟；
9)立即吸出显色液，用PBS-0.05％Tween 20洗涤3次以终止显色反应，判断结果。
CNB2003101116948A 2003-12-31 2003-12-31 热应激蛋白71抗体的检测方法 Expired - Fee Related CN1327224C (zh)

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CN101173913B (zh) * 2007-11-29 2010-11-10 河北大学 一种食品中丽春红2r的检测方法
CN103983793A (zh) * 2014-05-29 2014-08-13 上海理工大学 一种含丽春红的蛋白质芯片点样缓冲液及其制备方法

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH08240593A (ja) * 1995-03-06 1996-09-17 Kureha Chem Ind Co Ltd Ｈｓｐ４７の検出方法及び悪性腫瘍転移性検査試薬
CN1181785A (zh) * 1995-02-20 1998-05-13 味之素株式会社 抗应激性微生物及发酵产物的制备方法
CN1192807A (zh) * 1995-08-07 1998-09-09 金伸圭 自体免疫疾病的诊断方法

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1181785A (zh) * 1995-02-20 1998-05-13 味之素株式会社 抗应激性微生物及发酵产物的制备方法
JPH08240593A (ja) * 1995-03-06 1996-09-17 Kureha Chem Ind Co Ltd Ｈｓｐ４７の検出方法及び悪性腫瘍転移性検査試薬
CN1192807A (zh) * 1995-08-07 1998-09-09 金伸圭 自体免疫疾病的诊断方法

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