CN1388378A - Tubercle mycobaterium detecting reagent - Google Patents
Tubercle mycobaterium detecting reagent Download PDFInfo
- Publication number
- CN1388378A CN1388378A CN 02113890 CN02113890A CN1388378A CN 1388378 A CN1388378 A CN 1388378A CN 02113890 CN02113890 CN 02113890 CN 02113890 A CN02113890 A CN 02113890A CN 1388378 A CN1388378 A CN 1388378A
- Authority
- CN
- China
- Prior art keywords
- antibody
- antigen
- mycobacterium
- bacillus
- tubercle
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Landscapes
- Peptides Or Proteins (AREA)
Abstract
The tubercle mycobacterium detecting reagent kit can be used to detect antibody and antigen of tubercle mycobacterium in the clinical serological diagnosis of tuberculosis. The detecting antigens include two specific tubercle mycobacterium antigens ESAT-6 and CFP-10, which are obtained through gene engineering measures and are purified with fusion protein. The detecting antibody is IgG obtained through immunizing animal with the said antigen to produce immunological serum and purifying. The present invention has strong specificity and can distinguish true tubercle mycobacterium infection from sensitization caused by inoculation of bacille calmette guerin and contact with non-tubercle mycobacterium.
Description
1. technical field the invention belongs to medical immunology diagnostics technical field, specifically is detection kit and the application in clinical diagnosis lungy thereof about a kind of Much's bacillus.
2. background of invention tuberculosis is a kind of communicable disease that is caused by Much's bacillus, once occurs being very popular in the whole world.Along with the appearance of effective antituberculotic, taking a turn for the better appears in the tuberculosis popularity.But, surplus in the of nearly ten year over owing to the occur together reasons such as increase of infection and movement of population of appearance, HIV and the tubercle bacillus of tubercle bacillus multi-drug resistant bacterial strain, the tuberculosis epidemic situation is gone up once again, degradating trend has appearred in global tuberculosis situation.WHO announced " the tuberculosis whole world emergency circumstance " in 1993, and WHO in 1998 reaffirms again to contain that action lungy is very urgent.The whole world has 2,000,000,000 people to be subjected to mycobacterium tuberculosis infection approximately at present, existing 2,000 ten thousand tuberculosis patients, and wherein 95% in developing country.Annual neopathy number is 800-1000 ten thousand, and death toll is about 3,000,000, has surpassed the summation of acquired immune deficiency syndrome (AIDS), malaria, diarrhoea, tropical disease death toll.The tuberculosis popularity of China is also very serious always, it is one of the high burden of 22 tuberculosis in whole world country, according to the 4th the national tuberculosis epidemiological random sampling survey resulting estimate that carried out in 2000, China has 4,500,000 active tuberculosis patients approximately, the tuberculosis patient numerical digit occupies the second place of the world, account for 1/4th of global tuberculosis patient, the infectiousness pulmonary tuberculosis patient is about 1,500,000, has every year 12.7 ten thousand people to die from tuberculosis approximately.Tuberculosis has become one of main global disease of current threat human health, also is to cause dead common cause by single infectious agent.The key that reduces tuberculosis mortality rate is early diagnosis, thus formulation that can be accurately and timely rationally, the therapeutic scheme of science.
The main means of diagnosis of tuberculosis have smear staining microscopy and separation and Culture, tuberculin skin test and the serology detection etc. of pathogen at present.The acid-fast stain characteristic that smear staining microscopy method is based on tubercle bacillus reaches the purpose of calibrating, and sensitivity is low, the shortcoming of poor specificity but this method exists.The isolated culture inspection often needs 4-8 week or longer time, often incurs loss through delay clinical diagnosis and treatment.Tulase purified protein derivative (PPD) is the reagent that is widely used in diagnosis of tuberculosis at present, but owing to contain the common antigen molecule of many Mycobacteriums (comprising pathogenic mycobacterium, environment mycobacterium and Bacille Calmette-Guerin) among the PPD, therefore the poor specificity of PPD diagnosis of tuberculosis, actually or can not accurately distinguish the PPD test findings positive because bcg vaccination, with due to the real mycobacterium tuberculosis infection of the sensitization that causes after multiple non tuberculous Mycobacterium in the environment contacts.That the serology detection method has is quick, easy, be convenient to promote, the characteristics that need not special exact instrument are a kind of very potential diagnosis of tuberculosis means, but currently used diagnostic antigen (as PPD, 38KD, 30KD, 14KD, 19KD albumen etc.) exists the shortcoming that specificity is relatively poor, false positive rate is high, is badly in need of a kind of have strong specificity and immunogenic diagnostic antigen.
Mycobacterium tuberculosis ESAT-6-6 is promptly produced by the bacterium secretion in early days at mycobacterium tuberculosis infection, and is discerned by host immune system as a kind of low-molecular-weight secretion property antigen protein.Mycobacterium tuberculosis ESAT-6 antigen protein molecule epi-position is numerous (to have B cell epitope, CD
4 +, CD
8 +T cell epitope and DTH epi-position), and only in pathogenic mycobacterium, express, and be not present in Bacille Calmette-Guerin and other non-pathogenic mycobacterium, thereby be a kind of extremely promising tuberculosis specific diagnostic antigen.CFP-10 also is a kind of secretion antigen, and gene and the ESAT-6 of coding CFP-10 are adjacent, are in the same operon, all are positioned at the genomic RD1 section of Much's bacillus, lack equally in Bacille Calmette-Guerin.Coupling ESAT-6 and CFP-10 diagnosis tubercle bacillus affection can improve the susceptibility and the specificity that detect.
3. summary of the invention the present invention is by genetic engineering means, with mycobacterium tuberculosis ESAT-6-6 and CFP-10 albumen at vivoexpression, purifying and prepare the antibody and the antigen detecting agent box of Much's bacillus; And the fusion that ESAT-6 and CFP-10 amalgamation and expression are produced purified after, the antibody and the antigen detecting agent box of preparation Much's bacillus.This kit can detect the antibody and the antigen of Much's bacillus specifically, can distinguish be because bcg vaccination and with sensitization that causes after multiple non tuberculous Mycobacterium in the environment contacts or real m tuberculosis infection, can be used for clinical serodiagnosis lungy.
4. embodiment 1) classify template as with Much's bacillus H37Rv genome sequence, design the primer of ESAT-6 and CFP-10 gene respectively, clone ESAT-6 and CFP-10 gene.2) by genetic engineering means, the ESAT-6 and the CFP-10 gene end of cloning added joining peptide, obtain antigen-4 fusion protein gene.3) change each clone gene over to expression vector, express corresponding protein, carry out identification and analysis by gel electrophoresis and Western Blotting.4) the separation and purification of protein technology of employing standard, each expressing protein of purifying.As: centrifugal, ion-exchange chromatography, hydrophobic chromatography, affinity chromatography etc.5) each expressing protein adopts the immune programme for children immune animal of standard, as: rabbit, cavy, goat etc.Prepare two kinds of corresponding antibody, and its IgG of purifying.6), determine the concentration of envelope antigen (detection albumen), and be equipped with other related reagents that preparation mycobacterium tuberculosis antibody ELISA detects (indirect method) kit through the square formation titration.7), determine the concentration of coated antibody (detection protein antibodies), second antibody (the enzyme mark detects protein antibodies), and be equipped with other related reagents that preparation antigen of mycobacterium tuberculosis ELISA detects (double antibody sandwich method) kit through the square formation titration.
EXAMPLE Example 1:ELISA indirect method detects mycobacterium tuberculosis antibody and gets blood serum sample 0.1ml to be checked, adding has been coated with in the ELISA Plate of fusion antigen, hatches 30 minutes for 37 ℃, and every block of plate is established three hole negative controls, two hole positive controls, a hole blank.Hatch end, discard the liquid in the plate hole, wash plate five times with cleansing solution, button is done.Add anti-human IgG enzyme labelled antibody, fully mixing was hatched 30 minutes for 37 ℃.The same wash plate after, add colour developing liquid 0.1ml, pat mixing, 37 ℃ of lucifuges colour developings 30 minutes.Every hole adds one of stop buffer, pats mixing, surveys with 450nm wavelength microplate reader and reads OD value, result of determination.Embodiment 2:ELISA double antibody sandwich method detects antigen of mycobacterium tuberculosis and gets blood serum sample 0.1ml to be checked, adding has been coated with in the ELISA Plate of fusion antibody (first antibody), hatches 30 minutes for 37 ℃, and every block of plate is established three hole negative controls, two hole positive controls, a hole blank.Hatch end, discard the liquid in the plate hole, wash plate five times with cleansing solution, button is done.Add second antibody (the anti-fusion antibody of rabbit), fully mixing was hatched 30 minutes for 37 ℃.The same washing adds anti-rabbit igg enzyme labelled antibody behind the plate, and fully mixing was hatched 30 minutes for 37 ℃.Wash plate, add colour developing liquid 0.1ml, pat mixing, 37 ℃ of lucifuges developed the color 30 minutes.Every hole adds one of stop buffer, pats mixing, surveys with 450nm wavelength microplate reader and reads OD value, result of determination.
Claims (9)
1. the diagnostic kit of a Much's bacillus is characterized in that adopting specific detection antigen of Much's bacillus and antibody, can be used for clinical serodiagnosis lungy.
2. described specific detection antigen of claim 1 and antibody are two species-specific antigen ESAT-6 of Much's bacillus and the potpourri of CFP-10, and with the immune serum that this hybrid antigen immune animal was produced purified and antibody.
3. described specific detection antigen of claim 2 and antibody also can be a kind of fusions, and this albumen has the antigenic determinant of ESAT-6 and CFP-10 simultaneously, and with the antibody of this protein Preparation.They can combine with antigen with the antibody of mycobacterium respectively specifically.
4. the described antigen protein of claim 2 and claim 3 is to adopt escherichia expression system, expression of recombinant virus system, insect cell expression system, yeast expression system and mammalian cell expression system etc. to express to produce purified obtaining.
5. the described antigen protein of claim 2 and claim 3 also can be used for interior Skin-test of body and the generation of external evoked IFN-r, thereby detects the infection of Much's bacillus.
6. the described Much's bacillus of claim 1 comprises mycobacterium bovis.
7. be envelope antigen with claim 2 and the described albumen of claim 3, the mycobacterium tuberculosis antibody ELISA of preparation detects (indirect method) kit.
8. the described antibody of claim 7 comprises IgG, IgM, IgA.
9. be coated antibody with claim 2 and the described antibody of claim 3, the antigen of mycobacterium tuberculosis ELISA of preparation detects (double antibody sandwich method) kit.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN 02113890 CN1388378A (en) | 2002-06-17 | 2002-06-17 | Tubercle mycobaterium detecting reagent |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN 02113890 CN1388378A (en) | 2002-06-17 | 2002-06-17 | Tubercle mycobaterium detecting reagent |
Publications (1)
Publication Number | Publication Date |
---|---|
CN1388378A true CN1388378A (en) | 2003-01-01 |
Family
ID=4742852
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN 02113890 Pending CN1388378A (en) | 2002-06-17 | 2002-06-17 | Tubercle mycobaterium detecting reagent |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN1388378A (en) |
Cited By (19)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP1674868A1 (en) * | 2004-12-21 | 2006-06-28 | Chang Gung University (a university of Taiwan) | Method and device for detection of mycobacterium tuberculosis antigens in biological fluids |
CN1305900C (en) * | 2004-03-16 | 2007-03-21 | 中国药品生物制品检定所 | Use of Esat-6 protein in diagnosis of tuberculosis |
CN100368437C (en) * | 2005-01-28 | 2008-02-13 | 中国农业科学院哈尔滨兽医研究所 | Recombinant antigen protein for diagnosing ox tuberculosis and its prepn process |
CN1866023B (en) * | 2006-05-25 | 2010-11-03 | 上海市肺科医院 | Method for synchronous detection of multiple tubercle bacillus specific secretion antigens |
CN101985474A (en) * | 2010-10-12 | 2011-03-16 | 浙江大学 | Monoclonal antibody TBCA6 resistant to mycobacterium tuberculosis CFP-10 and application |
CN101987872A (en) * | 2010-10-12 | 2011-03-23 | 浙江大学 | Monoclonal antibody TBEF3 of tubercle bacillus-resistant ESAT-6 and application |
CN102253204A (en) * | 2004-06-30 | 2011-11-23 | 法国巴斯德研究所 | Detection of tuberculosis and infection by mycobacterium yuberculosis using HBHA |
CN102426232A (en) * | 2011-09-06 | 2012-04-25 | 厦门大学 | Kit for detecting tubercle bacillus protein antigens, and preparation method thereof |
CN102608333A (en) * | 2012-03-30 | 2012-07-25 | 中国科学院微生物研究所 | Tuberculosis diagnostic composition and application thereof |
CN102621318A (en) * | 2012-02-27 | 2012-08-01 | 浙江工业大学 | Immune chromatography test paper for detecting mycobacterium tuberculosis secretory proteins and manufacture method thereof |
CN102702360A (en) * | 2012-07-05 | 2012-10-03 | 中国人民解放军第三〇九医院 | Novel mycobacterium tuberculosis specific fusion protein as well as preparation and application thereof |
CN102757971A (en) * | 2012-08-07 | 2012-10-31 | 中国人民解放军第三O九医院 | Mycobacterium tuberculosis recombinant protein and preparation method thereof |
CN102854308A (en) * | 2012-09-20 | 2013-01-02 | 陕西师范大学 | Preparation method and application of double-antibody sandwich ELISA detection kit for alicyclobacillus acidoterrestris in fruit juice |
CN102981002A (en) * | 2012-11-29 | 2013-03-20 | 同昕生物技术(北京)有限公司 | Indirect immunoassay method adopting tag protein humanized chimeric antibody as positive contract and kit |
CN102993283A (en) * | 2010-07-15 | 2013-03-27 | 华中农业大学 | Antigen protein for mycobacterium tuberculosis and application |
CN103814139A (en) * | 2011-04-01 | 2014-05-21 | 澳康姆生物实验室公司 | Methods and kits for detecting cell-free pathogen-specific nucleic acids |
CN103833849A (en) * | 2014-03-07 | 2014-06-04 | 中国人民解放军第四军医大学 | Preparation method and application of ESAT6-CFP10 (Early Secreted Antigen Target 6-Culture Filtrate Protein 10) antibody |
CN110596382A (en) * | 2019-09-16 | 2019-12-20 | 南京拜睿生物科技有限公司 | Tuberculosis diagnostic kit and preparation method thereof |
RU2794855C1 (en) * | 2022-01-24 | 2023-04-25 | Федеральное государственное бюджетное научное учреждение "Центральный научно-исследовательский институт туберкулеза" | Method for diagnosing tuberculosis |
-
2002
- 2002-06-17 CN CN 02113890 patent/CN1388378A/en active Pending
Cited By (26)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN1305900C (en) * | 2004-03-16 | 2007-03-21 | 中国药品生物制品检定所 | Use of Esat-6 protein in diagnosis of tuberculosis |
CN102253204A (en) * | 2004-06-30 | 2011-11-23 | 法国巴斯德研究所 | Detection of tuberculosis and infection by mycobacterium yuberculosis using HBHA |
EP1674868A1 (en) * | 2004-12-21 | 2006-06-28 | Chang Gung University (a university of Taiwan) | Method and device for detection of mycobacterium tuberculosis antigens in biological fluids |
CN100368437C (en) * | 2005-01-28 | 2008-02-13 | 中国农业科学院哈尔滨兽医研究所 | Recombinant antigen protein for diagnosing ox tuberculosis and its prepn process |
CN1866023B (en) * | 2006-05-25 | 2010-11-03 | 上海市肺科医院 | Method for synchronous detection of multiple tubercle bacillus specific secretion antigens |
CN102993283A (en) * | 2010-07-15 | 2013-03-27 | 华中农业大学 | Antigen protein for mycobacterium tuberculosis and application |
CN102993283B (en) * | 2010-07-15 | 2014-11-12 | 华中农业大学 | Antigen protein for mycobacterium tuberculosis and application |
CN101985474A (en) * | 2010-10-12 | 2011-03-16 | 浙江大学 | Monoclonal antibody TBCA6 resistant to mycobacterium tuberculosis CFP-10 and application |
CN101987872A (en) * | 2010-10-12 | 2011-03-23 | 浙江大学 | Monoclonal antibody TBEF3 of tubercle bacillus-resistant ESAT-6 and application |
CN103814139A (en) * | 2011-04-01 | 2014-05-21 | 澳康姆生物实验室公司 | Methods and kits for detecting cell-free pathogen-specific nucleic acids |
CN103814139B (en) * | 2011-04-01 | 2018-11-13 | 澳康姆生物实验室公司 | Method and kit for detecting acellular pathogen specific nucleic acid |
CN102426232A (en) * | 2011-09-06 | 2012-04-25 | 厦门大学 | Kit for detecting tubercle bacillus protein antigens, and preparation method thereof |
CN102426232B (en) * | 2011-09-06 | 2014-02-26 | 厦门大学 | Kit for detecting tubercle bacillus protein antigens, and preparation method thereof |
CN102621318A (en) * | 2012-02-27 | 2012-08-01 | 浙江工业大学 | Immune chromatography test paper for detecting mycobacterium tuberculosis secretory proteins and manufacture method thereof |
CN102608333A (en) * | 2012-03-30 | 2012-07-25 | 中国科学院微生物研究所 | Tuberculosis diagnostic composition and application thereof |
CN102608333B (en) * | 2012-03-30 | 2013-06-05 | 中国科学院微生物研究所 | Tuberculosis diagnostic composition and application thereof |
CN102702360B (en) * | 2012-07-05 | 2014-02-19 | 中国人民解放军第三〇九医院 | Novel mycobacterium tuberculosis specific fusion protein as well as preparation and application thereof |
CN102702360A (en) * | 2012-07-05 | 2012-10-03 | 中国人民解放军第三〇九医院 | Novel mycobacterium tuberculosis specific fusion protein as well as preparation and application thereof |
CN102757971A (en) * | 2012-08-07 | 2012-10-31 | 中国人民解放军第三O九医院 | Mycobacterium tuberculosis recombinant protein and preparation method thereof |
CN102854308A (en) * | 2012-09-20 | 2013-01-02 | 陕西师范大学 | Preparation method and application of double-antibody sandwich ELISA detection kit for alicyclobacillus acidoterrestris in fruit juice |
CN102854308B (en) * | 2012-09-20 | 2015-04-08 | 陕西师范大学 | Double-antibody sandwich ELISA detection kit for alicyclobacillus acidoterrestris in fruit juice |
CN102981002A (en) * | 2012-11-29 | 2013-03-20 | 同昕生物技术(北京)有限公司 | Indirect immunoassay method adopting tag protein humanized chimeric antibody as positive contract and kit |
CN102981002B (en) * | 2012-11-29 | 2014-08-06 | 同昕生物技术(北京)有限公司 | Indirect immunoassay method adopting tag protein humanized chimeric antibody as positive contract and kit |
CN103833849A (en) * | 2014-03-07 | 2014-06-04 | 中国人民解放军第四军医大学 | Preparation method and application of ESAT6-CFP10 (Early Secreted Antigen Target 6-Culture Filtrate Protein 10) antibody |
CN110596382A (en) * | 2019-09-16 | 2019-12-20 | 南京拜睿生物科技有限公司 | Tuberculosis diagnostic kit and preparation method thereof |
RU2794855C1 (en) * | 2022-01-24 | 2023-04-25 | Федеральное государственное бюджетное научное учреждение "Центральный научно-исследовательский институт туберкулеза" | Method for diagnosing tuberculosis |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Samanich et al. | Serodiagnostic potential of culture filtrate antigens of Mycobacterium tuberculosis | |
CN1388378A (en) | Tubercle mycobaterium detecting reagent | |
US8216795B2 (en) | Tuberculosis diagnostic test | |
Pfrepper et al. | Seroreactivity to and avidity for recombinant antigens in toxoplasmosis | |
Samanich et al. | Homogeneity of antibody responses in tuberculosis patients | |
Ivanyi et al. | Immunodiagnostic assays for tuberculosis and leprosy | |
Koo et al. | Use of rMPB70 protein and ESAT-6 peptide as antigens for comparison of the enzyme-linked immunosorbent, immunochromatographic, and latex bead agglutination assays for serodiagnosis of bovine tuberculosis | |
De Kesel et al. | Composition and immunological properties of the protein fraction of A36, a major antigen complex of Mycobacterium paratuberculosis | |
Shin et al. | Improved sensitivity of diagnosis of tuberculosis in patients in Korea via a cocktail enzyme-linked immunosorbent assay containing the abundantly expressed antigens of the K strain of Mycobacterium tuberculosis | |
CN107141341B (en) | Antigen polypeptide pool for detecting mycobacterium tuberculosis infection and application | |
Zhang et al. | Identification of RD5‐encoded Mycobacterium tuberculosis proteins as B‐cell antigens used for serodiagnosis of tuberculosis | |
Ng et al. | Serodiagnosis of pericardial tuberculosis | |
ZA200400843B (en) | Early detection of mycobacterial disease using peptides | |
KR101359801B1 (en) | Composition containing m.tuberculosis antigens, diagnostic kit for diagnosis of tuberculosis comprising thereof, and method of providing information for diagnosis of tuberculosis using thereof | |
CA2276491C (en) | Early detection of mycobacterial disease | |
CN102702360B (en) | Novel mycobacterium tuberculosis specific fusion protein as well as preparation and application thereof | |
WO1998029132A9 (en) | Early detection of mycobacterial disease | |
CN101805397A (en) | Mycobacterium tuberculosis ESAT-6 recombinant dipolymer, preparation method and application thereof | |
Pollock et al. | Peptide mapping of bovine T‐cell epitopes for the 38 kDa tuberculosis antigen | |
Yang et al. | Immune responses to a recombinant Rv0057-Rv1352 fusion protein of mycobacterium tuberculosis | |
CA2698826A1 (en) | New antigens for paratuberculosis diagnosis and vaccination | |
CN100401066C (en) | Gold-labeled diagnosis reagent based on combined protein for tubercle bacillus | |
Renneberg et al. | Western blot analysis of the antibody response in patients with Listeria monocytogenes meningitis and septicemia | |
CN106699853A (en) | Mycobacterium tuberculosis Rv0189c recombinant protein, and preparation method and application thereof in diagnosis of tuberculosis | |
Shende et al. | A low molecular weight ES-20 protein released in vivo and in vitro with diagnostic potential in lymph node tuberculosis |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
C06 | Publication | ||
PB01 | Publication | ||
C10 | Entry into substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
C02 | Deemed withdrawal of patent application after publication (patent law 2001) | ||
WD01 | Invention patent application deemed withdrawn after publication |