CN1352392A - Method and reagent for directy detecting serum hepatitis B virus core antigen - Google Patents
Method and reagent for directy detecting serum hepatitis B virus core antigen Download PDFInfo
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- CN1352392A CN1352392A CN 00131131 CN00131131A CN1352392A CN 1352392 A CN1352392 A CN 1352392A CN 00131131 CN00131131 CN 00131131 CN 00131131 A CN00131131 A CN 00131131A CN 1352392 A CN1352392 A CN 1352392A
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Abstract
A reagent for directly detecting serum hepatitis B virus core antigen comprises mainly double-antibody coating carrier with both MC-anti-HBc and MC-anti-HBs, promoter, cracking agent and HRP-HBc. The detecting process includes adding sample to be detected to double-antibody coating carrier; adding promoter, cracking agent and HRP-anti-HBc; adding substrate to develop color, adding terminating liquid to terminate reaction; and color matching to judge result. The present invention has a positive coincidence rate with HBV-DNA probe of 91.4%, meets the requirement of practical clinical detection, and is simple, fast, practical and suitable for use in basic medical organization.
Description
The present invention relates to a kind of method and reagent of direct detection serum hepatitis B virus core antigen.
Hepatitis type B virus (HBV) cAg (HBcAg) is present in the liver cell nuclear of the core of hepatitis B particle,Dane (Dane particle) and hepatitis B patient, is that hepatitis type B virus is directly duplicated index.Because little free HBcAg in the serum is so can not detect with conventional method.Someone once disclosed a kind of method that detects hepatitis B virus core antigen, hepatitis B surface antibody and hepatitis B virus core antibody are wrapped simultaneously by to microwell plate (for example polystyrene micropore plate), the hepatitis B surface antibody combines with Yi Ganbingdushi particle surface antigen, on microwell plate, add decomposition agent then, the cracking of Yi Ganbingdushi particle shell, expose hepatitis B virus core antigen, free hepatitis B virus core antigen is adsorbed by the hepatitis B virus core antibody on the microwell plate, the hepatitis B virus core antibody that in microwell plate, adds peroxidase labelling again, the substrate reactions of peroxidase and adding finally reaches the purpose that detects hepatitis B virus core antigen.Said method, aspect coated antibody, what adopt is promptly many anti-bag quilts of immunoglobulin (Ig) at HBsAg and HBcAg, all professional and technical personnel know, how anti-as coated antibody, low, the poor specificity of its antibody titer usually easily produces very heavy background in experimentation, the sensitive end of its testing result, is low, and operating process is long.In addition, said method is with many anti-bag quilts, catches the limited in one's ability of a hepatitis B material in the serum, is difficult to reach the actual clinical test requirements.
Purpose of the present invention be exactly the problem that exists at above-mentioned prior art, provide a kind of can reach actual clinical examination requirements, operating process short, be fit to former method and the reagent of direct detection hepatitis B virus core nuclear that each different medical unit uses.
Technical scheme of the present invention is: a kind of method of direct detection serum hepatitis B virus core antigen, may further comprise the steps: with monoclonal surface antibody MC-anti--HBs and monoclonal core antibody MC-be anti--HBc wraps simultaneously by on solid phase carrier, after washing, patting dry, add serum to be checked, add promoter resisting-HBs then, the washing back adds decomposition agent NP-40 solution, the washing back adds enzyme labeling thing HRP-and resists-HBc, the washing back adds the colour developing of substrate colour developing liquid, add the stop buffer cessation reaction, the colorimetric result of determination.
Above-mentioned HRP-is anti--and the working concentration of HBc is 1: 300～1500, MC-is anti--and the working concentration of HBc is 1: 300～3000, MC-is anti--and the working concentration of HBs is 1: 200～1000, promoter resists-HBs0.3～400mg/ml, and decomposition agent NP-40 solution is made up of 1～9%NP-40,0.05～1.5% mercaptoethanol, 0.05～1.0M pH7.5Tris damping fluid.
The present invention also provides a kind of reagent of direct detection serum hepatitis B virus core antigen, mainly consists of: the microwell plate with monoclonal surface antibody and monoclonal core antibody double antibody bag quilt; Promoter resists-HBs; Decomposition agent NP-40 solution; Enzyme labeling core antibody HRP-resists-HBc.
In the mentioned reagent MC-anti--dense the crossing of work of HBc is 1: 300～3000; MC-is anti--and the working concentration of HBs is 1: 200～1000; HRP-is anti--and the working concentration of HBc is 1: 300～1500; Promoter is anti--and HBs is 0.3～400mg/ml; Decomposition agent NP-40 solution is made up of the Tris damping fluid of 1～9%NP-40,0.05～15% mercaptoethanol, 0.05～1.0M pH7.5.
The present invention is owing to adopt monoclonal antibody bag quilt, and not only tire height, specificity of its monoclonal antibody is good, and in the experimentation, background is clean, the detection sensitivity height.And the promoter of using among the present invention resists-HBs, be to utilize that institute's matters of containing biological activities shows antibody with the monoclonal that is coated in advance on the solid phase in the promoter, form a kind of special immune complex with hepatitis B Dane particle, thereby solid phase hepatitis B particle to greatest extent, make corresponding the increasing of HBsAg burst size after the cracking, and then cAg positive rate of the present invention is significantly improved.The inventor carries out cAg to clinical 164 routine hepatitis B virus e antigen positive patient serum and detects, as a result, add promoter resisting-HBs and detect positive 120 examples of cAg, positive rate 73.2%, do not add promoter and detect positive 44 examples of cAg, positive rate 26.8%.The present invention is used to detect serum hepatitis B virus core antigen and the positive coincidence rate of HBV-DNA probe is 91.4% approaching molecular biological HBV-DNA probe level, reach the actual clinical examination requirements, and because the present invention is simple to operate, fast, practical, be fit to each different medical unit and use.
The invention will be further described below in conjunction with embodiment.
The Dane particle of hepatitis B contains HBcAg, and the outer quilt of Dane particle is HBsAg, and if any the Dane particle, it is free through the decomposition agent cracking HBcAg to be exposed in the serum.The site that has two energy and antibodies according to the formation of antigen antibody complex and antigen of hepatitis B virus at least, MC-is resisted-HBs, MC-is anti--and HBc wraps simultaneously by on solid phase carrier (microwell plate), can make anti--HBs on HBsAg in the sample and the carrier form solid phase HBsAg anti--the HBs immune complex, the HBsAg of this compound is if having the Dane particle, in microwell plate through the decomposition agent cracking, HBcAg promptly is released in the hole and with the anti--HBc of original solid phase in the hole and combines, forming solid phase HBcAg resists-the HBc immune complex, the HBcAg of this compound can combine with the anti--HBc enzyme labeling thing that adds, and forms double-antibody sandwich.Add substrate colour developing liquid and can carry out the colorimetric result of determination.But since above formations HBsAg resist-the HBs immune complex is limited, makes that the HBcAg burst size after the cracking is less, its positive rate is lower.The present invention is through thousands of experimental exploring, with anti--HBs as promoter, can make HBsAg in the serum and the immobilization MC-on the carrier anti--HBs forms more immune complex, make also corresponding the increasing of HBcAg burst size after the cracking, thereby recall rate is improved greatly, and testing result is near molecular biological HBV-DNA probe level.
Embodiment:
Bag is by double antibody: according to a conventional method with MC-anti--HBs and MC-be anti--HBc wraps simultaneously by on solid phase carrier (Polyvinylchloride microwell plate or polystyrene micropore plate), MC-is anti--and the working concentration of HBc is 1: 300～3000, and MC-is anti--and the working concentration of HBs is 1: 200～1000; With cleansing solution washing 3 times, 3 minutes/time, pat dry; Sample to be checked is added the double antibody bag by in the microwell plate, addition 100 μ l/ holes, 37 ℃ are following 1 hour; Microoscillator vibration 1 minute is put in the anti--HBs50 μ l/ hole that adds promoter 0.3～400mg/ml, puts l hour for 37 ℃; With cleansing solution washing (ditto), add the decomposition agent 100 μ l/ holes of l～9%NP-40,0.05～1.5% mercaptoethanol, 0.05～1.0M, PH7.5Tris damping fluid composition, 37 ℃ 1 hour; With cleansing solution washing (ditto), enzyme-added label HRP-resists-HBc (working concentration 1: 300～1500) 100 μ l/ holes, 37 ℃ 1 hour, wash 4 times with cleansing solution, 3 minutes/time, pat dry; Every hole adds l00 μ l substrate colour developing liquid, vibrates room temperature 5～20 minutes 1 minute; Add 2mol/L H
2SO
450 μ l/ hole cessation reactions, the colorimetric result of determination.
Above-mentioned cleansing solution can adopt 1.0ml 25% Tween-20 to face with before washing in the physiological saline of 500ml 0.85～0.9%.
Above-mentioned substrate buffer solution can be prepared routinely.
Above-mentioned promoter also can adopt anti--HBs and MC-anti--potpourri of HBs.
Operating process of the present invention is short, and easy background is clean fast, and negative, positive contrast is obvious, can with hepatitis B " and two half " synchronous detection, practical, be convenient to the clinical expansion utilization.
Reagent of the present invention mainly consists of: have monoclonal surface antibody and monoclonal core antibody double antibody bag quilt microwell plate (according to a conventional method with MC-anti--HBc and MC-be anti--the HBs bag is by on solid phase carrier, MC-is anti--and the working concentration of HBc is 1: 300～3000, MC-is anti--and the working concentration of HBs is 1: 200～1000), 0.3 the promoter of～400mg/ml resists-HBs, by 1～9%NP-40,0.05～1.5% mercaptoethanol, 0.05 the decomposition agent that the Tris damping fluid of～1.0MPH7.5 is formed, working concentration is l: 300～1500 enzyme labeling core antibody HRP-resists-HBc.
Claims (4)
1, a kind of method of direct detection serum hepatitis B virus core antigen may further comprise the steps:
With monoclonal surface antibody MC-anti--HBs and monoclonal core antibody MC-be anti--HBc wraps simultaneously by on solid phase carrier, after washing, patting dry, add serum to be checked, add promoter resisting-HBs then, add decomposition agent NP-40 solution after the washing, the washing back adds enzyme labeling thing HRP-and resists-HBc, and the washing back adds substrate colour developing liquid and develops the color, add the stop buffer cessation reaction, the colorimetric result of determination.
2, method according to claim 1, it is characterized in that: HRP-is anti--and the working concentration of HBc is 1: 300～1500, MC-is anti--and the working concentration of HBc is 1: 300～3000, MC-is anti--and the working concentration of HBs is 1: 200～1000, promoter is anti--and HBs is 0.3～400mg/ml, decomposition agent NP-40 solution is by 1～9%NP-40,0.05～1.5% mercaptoethanol, and 0.05～1.0M PH7.5Tris damping fluid is formed.
3, a kind of reagent of direct detection serum hepatitis B virus core antigen mainly consists of: the microwell plate with monoclonal surface antibody and monoclonal core antibody double antibody bag quilt;
Promoter resists-HBs;
Decomposition agent NP-40 solution;
Enzyme labeling core antibody HRP-resists-HBc.
4, reagent according to claim 3, it is characterized in that MC-anti--working concentration of HBc is 1: 300～3000; MC-is anti--and the working concentration of HBs is 1: 200～1000; HRP-is anti--and the working concentration of HBc is 1: 300～1500; Promoter is anti--and HBs is 0.3～400mg/ml; Decomposition agent NP-40 solution is made up of the Tris damping fluid of 1～9%NP-40,0.05～1.5% mercaptoethanol, 0.05～1.0M PH7.5.
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| Application Number | Priority Date | Filing Date | Title |
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| CN 00131131 CN1131432C (en) | 2000-11-09 | 2000-11-09 | Method and reagent for directy detecting serum hepatitis B virus core antigen |
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| CN 00131131 CN1131432C (en) | 2000-11-09 | 2000-11-09 | Method and reagent for directy detecting serum hepatitis B virus core antigen |
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| CN1352392A true CN1352392A (en) | 2002-06-05 |
| CN1131432C CN1131432C (en) | 2003-12-17 |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100582781C (en) * | 2006-06-27 | 2010-01-20 | 厦门大学 | Method for joint investigating hepatitis B virus pro S1 antigen and nuclear antigen and diagnostic kit |
| CN102043050A (en) * | 2009-10-23 | 2011-05-04 | 上海荣盛生物药业有限公司 | In vitro detection method of hepatitis B virus C antibody |
| CN102207504A (en) * | 2011-03-23 | 2011-10-05 | 北京华创远航科技有限公司 | Enzyme-linked immunosorbent assay kit, and preparation method thereof |
| CN102914652A (en) * | 2012-10-19 | 2013-02-06 | 徐连江 | Method for directly detecting hepatitis B core antigen and matched portable device |
-
2000
- 2000-11-09 CN CN 00131131 patent/CN1131432C/en not_active Expired - Fee Related
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100582781C (en) * | 2006-06-27 | 2010-01-20 | 厦门大学 | Method for joint investigating hepatitis B virus pro S1 antigen and nuclear antigen and diagnostic kit |
| CN102043050A (en) * | 2009-10-23 | 2011-05-04 | 上海荣盛生物药业有限公司 | In vitro detection method of hepatitis B virus C antibody |
| CN102207504A (en) * | 2011-03-23 | 2011-10-05 | 北京华创远航科技有限公司 | Enzyme-linked immunosorbent assay kit, and preparation method thereof |
| CN102914652A (en) * | 2012-10-19 | 2013-02-06 | 徐连江 | Method for directly detecting hepatitis B core antigen and matched portable device |
| CN102914652B (en) * | 2012-10-19 | 2015-08-19 | 徐连江 | A kind of supporting portable unit of direct-detection hepatitis B virus core antigen |
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| CN1131432C (en) | 2003-12-17 |
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