CN1132857A - Detection method of core antigen of hepatitis virus - Google Patents
Detection method of core antigen of hepatitis virus Download PDFInfo
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- CN1132857A CN1132857A CN 95117915 CN95117915A CN1132857A CN 1132857 A CN1132857 A CN 1132857A CN 95117915 CN95117915 CN 95117915 CN 95117915 A CN95117915 A CN 95117915A CN 1132857 A CN1132857 A CN 1132857A
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- hepatitis
- virus
- core antigen
- hbc
- microwell plate
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Abstract
A method for detection of hepatitis virus features that enzyme-linked immunosorbent microporous plate method is used to detect core antigen of hepatitis B virus in serum. The surificial and core antigens of hepatitis B virus are simultaniously coated on microporous PVC board and the particles of hepatitis B virus are adsorbed on the board. Adding-in detergent can modify cuticulin of said virus and then crack shell of the particle, resulting in exposure of said core antigen of hepatitis B virus. The method is simple, fast and correct.
Description
The invention belongs to a kind of method that detects hepatitis virus, especially detect hepatitis B virus core antigen in the serum with enzyme linked immunological microwell plate method.
The particle of hepatitis type B virus can be divided into three kinds of forms in blood, pellet shapes, tubular and Yi Xingganyanbingdushi particle, wherein the Yi Xingganyanbingdushi particle is to have infective complete hepatitis type B virus, the Yi Xingganyanbingdushi particle is divided into core and two parts of shell, wherein the core has the antigenicity of hepatitis B virus core antigen, as everyone knows, hepatitis B virus core antigen is synthetic in liver cell nuclear, refill behind the endochylema and mix surface antigen and become complete hepatitis B virus particles, be discharged in the blood again, hepatitis B virus E antigen is hepatitis B virus core antigen P
19Polypeptide is gone into after 34 amino acid of hydroxyl terminal dissociate behind the blood from liver cell, remaining P
15.5Be hepatitis B virus E antigen, group is difficult to detect free hepatitis B virus core antigen in blood, detected hepatitis B virus core antigen in the serum with immune adherence blood cell coacervation in 1976, adopt special rabbit anti-HBs to adsorb all hepatitis B surface antibodies, again with proteinase E, α-3-mercaptoethanol and the neutral detergent protein coat that dissociates, hepatitis B virus core antigen is discharged, because the reagent that adds is more, and the susceptibility of hemagglutination method is more much lower than enzymoimmunoassay, now use and limited to, the attached the People's Hospital of Beijing medical college, adopt the principle of antigen antibody complex, in sample to be checked, add excessive anti-HBs, form the antigen-antibody immune complex, the centrifugation immune complex, take off the hepatitis type B virus shell with an amount of neutral detergent again, detect hepatitis B virus core antigen in the serum with solid-phase radioimmunoassay, though the method has solved the disturbing factor of proteinase E and α-3-mercaptoethanol, but this method operation steps is many, time is long, and there have radioactive isotope radioactive protection and radioactive waste in operation to handle again to be more a lot of than enzyme linked immunosorbent detection method trouble.
Order of the present invention be to provide a kind of easy with enzyme linked immunological microwell plate method, detect hepatitis B virus core antigen in the serum quickly and accurately.
Technical scheme of the present invention is: anti-HBs and anti-HBc are wrapped simultaneously by the Polyvinylchloride microwell plate, anti-HBs combines with Yi Xingganyanbingdushi particle surface antigen, make the Yi Xingganyanbingdushi particle be attracted on the Polyvinylchloride microwell plate, in micropore, add neutral detergent then, detergent acts on the hepatitis type B virus glutelin, the glutelin sex change, the cracking of Yi Xingganyanbingdushi particle shell, expose hepatitis B virus core antigen, free hepatitis B virus core antigen is adsorbed by the anti-HBc on the Polyvinylchloride microwell plate, in micropore, add the anti-HBc peroxidase of peroxidase labelling and the substrate reactions of adding again, finally reach the purpose that detects hepatitis B virus core antigen.Use P
HThe anti-HBs of 96005mol/l sulfate damping fluid preparation, anti-HBc double antibody coating buffer is used P
HThe anti-HBc horseradish peroxidase bond of 74mol/l phosphate buffer preparation is used P
HThe neutral detergent of the 2%-10% of 74001mol/l phosphate buffer preparation, the cleansing solution of employing polysorbas20, colour developing liquid A is a hydrogen peroxide, and colour developing liquid B is a tetramethyl benzidine, and stop buffer adopts 2mol/l sulfuric acid.
Originally with bright characteristics: easy, quick, accurate, be directly diagnosis apace clinically, observation and screening hepatitis B patient provide and have detected index more accurately.
Operation steps of the present invention is described in detail in detail below:
1, gets 100ul anti-HBs and anti-HBc mixed liquor bag by the Polyvinylchloride microwell plate, place 37 ℃ of water bath water-baths two hours, place 4 ℃ of refrigerator twenty four hours again, take out from refrigerator, with cleansing solution flushing five times, clean free surface antibody, core antibody, pat dry.
2, get 100ul yin and yang attribute contrast liquid and serum specimen to be checked, add respectively in the Polyvinylchloride microwell plate, placed in 37 ℃ of water baths water-bath 30 minutes, take out the back and wash five times with the core antibody in the flush away serum, pat dry with cleansing solution.The yin and yang attribute contrast is that observation and calculating sample result are used, in the serum specimen as have a Yi Xingganyanbingdushi particle, pellet shapes and tubular hepatitis B surface antibody particle, then combine, be attracted on the Polyvinylchloride microwell plate of solid phase with anti-HBs on the Polyvinylchloride microwell plate.
3, get the 100ul5% neutral detergent and 100ul anti-HBc horseradish peroxidase bond adds in the microwell plate simultaneously, placed in 37 ℃ of water baths water-bath 30 minutes, take out the back and wash five times to remove free anti-HBc horseradish peroxidase bond, pat dry with cleansing solution.Neutral detergent makes the Yi Xingganyanbingdushi particle open shell, hepatitis B virus core antigen comes out, free hepatitis B surface antibody combines with the anti-HBc bond on the Polyvinylchloride microwell plate, combine with the anti-HBc horseradish peroxidase that adds again, on microwell plate, form anti-HBc-hepatitis B virus core antigen-anti-HBc horseradish peroxidase bond double antibodies sandwich state, carry out this reaction in microwell plate, avoided operation steps hepatitis B virus core antigen being lost influences its accuracy more.
4, add 50ul colour developing liquid A, add 50ul colour developing liquid B again, place 37 ℃ of water baths and avoided water-bath 15 minutes, superoxide makes TMB be blue reaction under the condition that colour developing liquid A is arranged.
5, add 50ul2mol/l sulfuric acid cessation reaction, H
2SO
4Peroxidase is lost activity reach the purpose of cessation reaction, in 450nm wavelength place colorimetric, write down the absorbance in every hole at last.
The result judges: when the ratio of the absorbance of sample and negative control absorbance greater than 21, judge that hepatitis B virus core antigen is positive, otherwise, negative.
Claims (1)
1, a kind of method that detects core antigen of hepatitis virus, it is characterized in that anti-HBs and anti-HBc are wrapped simultaneously by the Polyvinylchloride microwell plate, anti-HBs combines with Yi Xingganyanbingdushi particle surface antigen, make the Yi Xingganyanbingdushi particle be attracted on the Polyvinylchloride microwell plate, in micropore, add neutral detergent then, detergent acts on the hepatitis type B virus glutelin, the glutelin sex change, the cracking of Yi Xingganyanbingdushi particle shell, expose hepatitis B virus core antigen, free hepatitis B virus core antigen is adsorbed by the anti-HBc on the Polyvinylchloride microwell plate, the anti-HBc that in micropore, adds peroxidase labelling again, the substrate reactions of peroxidase and adding finally reaches the purpose that detects hepatitis B virus core antigen.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 95117915 CN1132857A (en) | 1995-10-31 | 1995-10-31 | Detection method of core antigen of hepatitis virus |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 95117915 CN1132857A (en) | 1995-10-31 | 1995-10-31 | Detection method of core antigen of hepatitis virus |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN1132857A true CN1132857A (en) | 1996-10-09 |
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Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 95117915 Pending CN1132857A (en) | 1995-10-31 | 1995-10-31 | Detection method of core antigen of hepatitis virus |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN1132857A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1908666B (en) * | 2006-08-14 | 2010-05-12 | 武汉大学 | Enzyme linked immunity diagnose reagent kit for HB core antigen detecting in two sandwich method and application thereof |
| CN101046477B (en) * | 2007-04-29 | 2011-03-23 | 北京标凯科技有限公司 | HBV-DNA kit for quantitative enzyme-linked measurment of heaptitis B core antigen |
| CN102914652A (en) * | 2012-10-19 | 2013-02-06 | 徐连江 | Method for directly detecting hepatitis B core antigen and matched portable device |
-
1995
- 1995-10-31 CN CN 95117915 patent/CN1132857A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1908666B (en) * | 2006-08-14 | 2010-05-12 | 武汉大学 | Enzyme linked immunity diagnose reagent kit for HB core antigen detecting in two sandwich method and application thereof |
| CN101046477B (en) * | 2007-04-29 | 2011-03-23 | 北京标凯科技有限公司 | HBV-DNA kit for quantitative enzyme-linked measurment of heaptitis B core antigen |
| 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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