CN101985473A - Monoclonal antibody TBEF3 of tubercle bacillus-resistant ESAT-6 and application thereof - Google Patents
Monoclonal antibody TBEF3 of tubercle bacillus-resistant ESAT-6 and application thereof Download PDFInfo
- Publication number
- CN101985473A CN101985473A CN 201010504707 CN201010504707A CN101985473A CN 101985473 A CN101985473 A CN 101985473A CN 201010504707 CN201010504707 CN 201010504707 CN 201010504707 A CN201010504707 A CN 201010504707A CN 101985473 A CN101985473 A CN 101985473A
- Authority
- CN
- China
- Prior art keywords
- monoclonal antibody
- target protein
- mycobacterium tuberculosis
- tbef3
- antigen target
- 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.)
- Granted
Links
- 101710166488 6 kDa early secretory antigenic target Proteins 0.000 title claims abstract description 45
- 241000193830 Bacillus <bacterium> Species 0.000 title abstract description 11
- 210000004027 cell Anatomy 0.000 claims abstract description 43
- 241000187479 Mycobacterium tuberculosis Species 0.000 claims abstract description 38
- 210000004408 hybridoma Anatomy 0.000 claims abstract description 38
- 102000036639 antigens Human genes 0.000 claims abstract description 34
- 108091007433 antigens Proteins 0.000 claims abstract description 34
- 239000000427 antigen Substances 0.000 claims abstract description 33
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 27
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 24
- 108090000765 processed proteins & peptides Proteins 0.000 claims abstract description 16
- 206010035226 Plasma cell myeloma Diseases 0.000 claims abstract description 13
- 201000000050 myeloid neoplasm Diseases 0.000 claims abstract description 13
- 238000012258 culturing Methods 0.000 claims abstract description 7
- 238000001261 affinity purification Methods 0.000 claims abstract description 6
- 108090000790 Enzymes Proteins 0.000 claims abstract description 5
- 102000004190 Enzymes Human genes 0.000 claims abstract description 5
- 238000003119 immunoblot Methods 0.000 claims abstract description 4
- 238000000034 method Methods 0.000 claims description 31
- 230000028327 secretion Effects 0.000 claims description 22
- 241000699666 Mus <mouse, genus> Species 0.000 claims description 20
- 206010003445 Ascites Diseases 0.000 claims description 14
- 238000010790 dilution Methods 0.000 claims description 14
- 239000012895 dilution Substances 0.000 claims description 14
- NHBKXEKEPDILRR-UHFFFAOYSA-N 2,3-bis(butanoylsulfanyl)propyl butanoate Chemical compound CCCC(=O)OCC(SC(=O)CCC)CSC(=O)CCC NHBKXEKEPDILRR-UHFFFAOYSA-N 0.000 claims description 13
- 241000699670 Mus sp. Species 0.000 claims description 13
- 239000012228 culture supernatant Substances 0.000 claims description 13
- 238000002965 ELISA Methods 0.000 claims description 12
- 238000011081 inoculation Methods 0.000 claims description 11
- 238000010367 cloning Methods 0.000 claims description 9
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 claims description 8
- 238000001514 detection method Methods 0.000 claims description 8
- 230000036039 immunity Effects 0.000 claims description 8
- 238000012216 screening Methods 0.000 claims description 8
- 241001529936 Murinae Species 0.000 claims description 7
- 210000001124 body fluid Anatomy 0.000 claims description 7
- 239000010839 body fluid Substances 0.000 claims description 7
- 230000024835 cytogamy Effects 0.000 claims description 7
- 238000002360 preparation method Methods 0.000 claims description 7
- 238000001114 immunoprecipitation Methods 0.000 claims description 6
- 210000003024 peritoneal macrophage Anatomy 0.000 claims description 6
- 239000006228 supernatant Substances 0.000 claims description 6
- 238000011156 evaluation Methods 0.000 claims description 5
- 108010026228 mRNA guanylyltransferase Proteins 0.000 claims description 5
- -1 polyoxyethylene Polymers 0.000 claims description 5
- 229920003171 Poly (ethylene oxide) Polymers 0.000 claims description 4
- 239000001963 growth medium Substances 0.000 claims description 4
- WGCNASOHLSPBMP-UHFFFAOYSA-N hydroxyacetaldehyde Natural products OCC=O WGCNASOHLSPBMP-UHFFFAOYSA-N 0.000 claims description 4
- 238000002347 injection Methods 0.000 claims description 4
- 239000007924 injection Substances 0.000 claims description 4
- 210000004698 lymphocyte Anatomy 0.000 claims description 4
- 238000000746 purification Methods 0.000 claims description 4
- 238000004445 quantitative analysis Methods 0.000 claims description 4
- 150000001413 amino acids Chemical class 0.000 claims description 3
- 238000003556 assay Methods 0.000 claims description 3
- 230000001580 bacterial effect Effects 0.000 claims description 3
- 230000015572 biosynthetic process Effects 0.000 claims description 3
- 230000010261 cell growth Effects 0.000 claims description 3
- 230000012010 growth Effects 0.000 claims description 3
- 238000002649 immunization Methods 0.000 claims description 3
- 210000000952 spleen Anatomy 0.000 claims description 3
- 239000002671 adjuvant Substances 0.000 claims description 2
- 230000001900 immune effect Effects 0.000 claims description 2
- 230000003053 immunization Effects 0.000 claims description 2
- 230000001939 inductive effect Effects 0.000 claims description 2
- 230000001404 mediated effect Effects 0.000 claims description 2
- 239000006152 selective media Substances 0.000 claims description 2
- 238000010521 absorption reaction Methods 0.000 claims 1
- 238000002331 protein detection Methods 0.000 claims 1
- 238000007920 subcutaneous administration Methods 0.000 claims 1
- 230000003248 secreting effect Effects 0.000 abstract description 7
- 238000012360 testing method Methods 0.000 abstract description 7
- 210000003567 ascitic fluid Anatomy 0.000 abstract 1
- 210000003719 b-lymphocyte Anatomy 0.000 abstract 1
- 239000003547 immunosorbent Substances 0.000 abstract 1
- 230000006698 induction Effects 0.000 abstract 1
- 201000008827 tuberculosis Diseases 0.000 description 22
- 239000000523 sample Substances 0.000 description 20
- 238000006243 chemical reaction Methods 0.000 description 19
- 230000004927 fusion Effects 0.000 description 10
- 239000007788 liquid Substances 0.000 description 10
- 101001057048 Mycobacterium tuberculosis (strain ATCC 25618 / H37Rv) ESAT-6-like protein EsxB Proteins 0.000 description 9
- 238000005516 engineering process Methods 0.000 description 9
- 238000005406 washing Methods 0.000 description 9
- 230000008521 reorganization Effects 0.000 description 8
- 239000000243 solution Substances 0.000 description 8
- 208000030507 AIDS Diseases 0.000 description 7
- 238000003745 diagnosis Methods 0.000 description 7
- 238000013399 early diagnosis Methods 0.000 description 6
- 238000001962 electrophoresis Methods 0.000 description 6
- 235000015097 nutrients Nutrition 0.000 description 6
- 239000000126 substance Substances 0.000 description 6
- 238000001262 western blot Methods 0.000 description 6
- 206010048612 Hydrothorax Diseases 0.000 description 5
- 238000004321 preservation Methods 0.000 description 5
- 102000004196 processed proteins & peptides Human genes 0.000 description 5
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 5
- 241000725303 Human immunodeficiency virus Species 0.000 description 4
- 229920001213 Polysorbate 20 Polymers 0.000 description 4
- 239000012980 RPMI-1640 medium Substances 0.000 description 4
- 239000012148 binding buffer Substances 0.000 description 4
- 239000007853 buffer solution Substances 0.000 description 4
- 238000003114 enzyme-linked immunosorbent spot assay Methods 0.000 description 4
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 4
- 229920001184 polypeptide Polymers 0.000 description 4
- XOJVVFBFDXDTEG-UHFFFAOYSA-N pristane Chemical compound CC(C)CCCC(C)CCCC(C)CCCC(C)C XOJVVFBFDXDTEG-UHFFFAOYSA-N 0.000 description 4
- 235000018102 proteins Nutrition 0.000 description 4
- 238000005215 recombination Methods 0.000 description 4
- 230000006798 recombination Effects 0.000 description 4
- 239000008118 PEG 6000 Substances 0.000 description 3
- 229920002584 Polyethylene Glycol 6000 Polymers 0.000 description 3
- 238000004113 cell culture Methods 0.000 description 3
- 239000011248 coating agent Substances 0.000 description 3
- 238000000576 coating method Methods 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 238000001556 precipitation Methods 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 238000007789 sealing Methods 0.000 description 3
- 239000000758 substrate Substances 0.000 description 3
- 229920000936 Agarose Polymers 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- BVKZGUZCCUSVTD-UHFFFAOYSA-L Carbonate Chemical compound [O-]C([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-L 0.000 description 2
- 108060003951 Immunoglobulin Proteins 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- 241000283973 Oryctolagus cuniculus Species 0.000 description 2
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 2
- 239000006180 TBST buffer Substances 0.000 description 2
- 230000003187 abdominal effect Effects 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 230000000890 antigenic effect Effects 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 238000003759 clinical diagnosis Methods 0.000 description 2
- MTHSVFCYNBDYFN-UHFFFAOYSA-N diethylene glycol Chemical compound OCCOCCO MTHSVFCYNBDYFN-UHFFFAOYSA-N 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 230000005611 electricity Effects 0.000 description 2
- 239000012149 elution buffer Substances 0.000 description 2
- 238000004945 emulsification Methods 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 102000037865 fusion proteins Human genes 0.000 description 2
- 108020001507 fusion proteins Proteins 0.000 description 2
- 239000003292 glue Substances 0.000 description 2
- 238000003384 imaging method Methods 0.000 description 2
- 102000018358 immunoglobulin Human genes 0.000 description 2
- 208000015181 infectious disease Diseases 0.000 description 2
- 230000031700 light absorption Effects 0.000 description 2
- 238000004020 luminiscence type Methods 0.000 description 2
- 229920002981 polyvinylidene fluoride Polymers 0.000 description 2
- 238000004393 prognosis Methods 0.000 description 2
- 239000003531 protein hydrolysate Substances 0.000 description 2
- 238000004451 qualitative analysis Methods 0.000 description 2
- 238000011084 recovery Methods 0.000 description 2
- 210000002966 serum Anatomy 0.000 description 2
- 235000020183 skimmed milk Nutrition 0.000 description 2
- 208000011580 syndromic disease Diseases 0.000 description 2
- 229960005486 vaccine Drugs 0.000 description 2
- 125000003088 (fluoren-9-ylmethoxy)carbonyl group Chemical group 0.000 description 1
- CDGHMJJJHYKMPA-DLOVCJGASA-N Asn-Phe-Ala Chemical compound C[C@@H](C(=O)O)NC(=O)[C@H](CC1=CC=CC=C1)NC(=O)[C@H](CC(=O)N)N CDGHMJJJHYKMPA-DLOVCJGASA-N 0.000 description 1
- 238000011725 BALB/c mouse Methods 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 1
- QFJPFPCSXOXMKI-BPUTZDHNSA-N Gln-Gln-Trp Chemical compound C1=CC=C2C(=C1)C(=CN2)C[C@@H](C(=O)O)NC(=O)[C@H](CCC(=O)N)NC(=O)[C@H](CCC(=O)N)N QFJPFPCSXOXMKI-BPUTZDHNSA-N 0.000 description 1
- HKSNHPVETYYJBK-LAEOZQHASA-N Gly-Ile-Glu Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCC(=O)O)C(=O)O)NC(=O)CN HKSNHPVETYYJBK-LAEOZQHASA-N 0.000 description 1
- 101000911390 Homo sapiens Coagulation factor VIII Proteins 0.000 description 1
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 1
- FXBKQTOGURNXSL-HJGDQZAQSA-N Met-Thr-Glu Chemical compound CSCC[C@H](N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@H](C(O)=O)CCC(O)=O FXBKQTOGURNXSL-HJGDQZAQSA-N 0.000 description 1
- 241000186359 Mycobacterium Species 0.000 description 1
- 241000545499 Mycobacterium avium-intracellulare Species 0.000 description 1
- 241000186363 Mycobacterium kansasii Species 0.000 description 1
- 208000001388 Opportunistic Infections Diseases 0.000 description 1
- 239000002033 PVDF binder Substances 0.000 description 1
- 241001494479 Pecora Species 0.000 description 1
- 102000001708 Protein Isoforms Human genes 0.000 description 1
- 108010029485 Protein Isoforms Proteins 0.000 description 1
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 1
- 229920002684 Sepharose Polymers 0.000 description 1
- 102000005937 Tropomyosin Human genes 0.000 description 1
- 108010030743 Tropomyosin Proteins 0.000 description 1
- 206010053613 Type IV hypersensitivity reaction Diseases 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 108010047495 alanylglycine Proteins 0.000 description 1
- 235000001014 amino acid Nutrition 0.000 description 1
- 230000001355 anti-mycobacterial effect Effects 0.000 description 1
- 238000011091 antibody purification Methods 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- 210000001185 bone marrow Anatomy 0.000 description 1
- UDSAIICHUKSCKT-UHFFFAOYSA-N bromophenol blue Chemical compound C1=C(Br)C(O)=C(Br)C=C1C1(C=2C=C(Br)C(O)=C(Br)C=2)C2=CC=CC=C2S(=O)(=O)O1 UDSAIICHUKSCKT-UHFFFAOYSA-N 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 210000001728 clone cell Anatomy 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 238000005138 cryopreservation Methods 0.000 description 1
- 230000002498 deadly effect Effects 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 230000002950 deficient Effects 0.000 description 1
- 230000008021 deposition Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000002405 diagnostic procedure Methods 0.000 description 1
- 238000000502 dialysis Methods 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 239000000975 dye Substances 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 108010049041 glutamylalanine Proteins 0.000 description 1
- 238000000703 high-speed centrifugation Methods 0.000 description 1
- 102000057593 human F8 Human genes 0.000 description 1
- 238000009396 hybridization Methods 0.000 description 1
- 238000003018 immunoassay Methods 0.000 description 1
- 238000012417 linear regression Methods 0.000 description 1
- 239000007791 liquid phase Substances 0.000 description 1
- 239000012160 loading buffer Substances 0.000 description 1
- 239000006210 lotion Substances 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 238000004949 mass spectrometry Methods 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 229940126619 mouse monoclonal antibody Drugs 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 238000007500 overflow downdraw method Methods 0.000 description 1
- 230000008506 pathogenesis Effects 0.000 description 1
- 239000012071 phase Substances 0.000 description 1
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- 239000008057 potassium phosphate buffer Substances 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 229940047431 recombinate Drugs 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 238000003118 sandwich ELISA Methods 0.000 description 1
- 229920006395 saturated elastomer Polymers 0.000 description 1
- 238000009991 scouring Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000002791 soaking Methods 0.000 description 1
- 238000010532 solid phase synthesis reaction Methods 0.000 description 1
- 230000003393 splenic effect Effects 0.000 description 1
- 210000004988 splenocyte Anatomy 0.000 description 1
- 229910001220 stainless steel Inorganic materials 0.000 description 1
- 239000010935 stainless steel Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 230000001052 transient effect Effects 0.000 description 1
- 229960001005 tuberculin Drugs 0.000 description 1
- 210000005239 tubule Anatomy 0.000 description 1
- 230000005951 type IV hypersensitivity Effects 0.000 description 1
- 208000027930 type IV hypersensitivity disease Diseases 0.000 description 1
- 230000000007 visual effect Effects 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Images
Landscapes
- Peptides Or Proteins (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention provides a monoclonal antibody TBEF3 of tubercle bacillus-resistant secretory antigen target protein in early stage. The monoclonal antibody TBEF3 is prepared by the following steps: selecting a specific antigen peptide mouse; collecting sensitized mouse B lymphocyte and mouse myeloma cell to fuse; judging positive clones by using an enzyme linked immunosorbent assayelisa and an immunoblotting testing method; establishing a hybridoma cell line TBEF3 for secreting monoclonal antibody of tubercle bacillus-resistant secretory ESAT-6 in early stage; carrying out multiplication culturing on the positive cells and injecting to congenic mouse enterocoelia for induction of generation of ascitic fluid containing antibody; and carrying out collection and antibody affinity purification to obtain the monoclonal antibody TBEF3 of tubercle bacillus-resistant secretory antigen target protein in early stage. The invention also provides an application of the monoclonal antibody in testing the secretory antigen target protein of mycobacterium tuberculosis in early stage.
Description
Technical field
The invention belongs to biological technical field, relate to the Killing Mycobacterium Tuberculosis MONOCLONAL ANTIBODIES SPECIFIC FOR and the application of secretion property antigen target protein (ESAT-6) in early days, be to utilize cell engineering, antibody engineering technology, obtain the hybridoma cell line of the monoclonal antibody of the anti-ESAT-6 of secretion, induce ascites by mouse with strain, the monoclonal antibody TBEF3 for preparing anti-EAST-6 is accredited as IgM, κ type, again by the application of technology such as affinity purification, electrophoresis, immunity realization to this antibody.
Background technology
Tuberculosis early diagnosis technology is to perplex an important difficult problem of area of medical diagnostics always.In 20th century, because of the development and the widespread use of microbiotic research, the mankind had once successfully controlled tuberculosis.But because resistance constantly occurs and accumulation, mycobacterium tuberculosis still is a realistic threat for the mankind of 21 century.More seriously, in recent years, global human immunodeficiency virus (HIV) and acquired immune deficiency syndrome (AIDS) (AIDS) popular become the major cause that global incidence of tuberculosis and mortality ratio are gone up for the third time, and tuberculosis also is the patient's AIDS most common opportunistic infection and the deadly cause of disease simultaneously.The trend that tuberculosis and acquired immune deficiency syndrome (AIDS) are concurrent makes that treatment is extremely difficult.Diagnosis is the prerequisite of treatment, if can early diagnosis, harm lungy can reduce significantly.The quick growth area that China is regional as the high infection rate of tubercule bacillus and HIV infects, the early stage accurate diagnostic level that improves tuberculosis and HIV/AIDS merging tuberculosis infection is very urgent.
Traditional early diagnosis means are tuberculin skin tests.This is based on a kind of tuerculoderma of delayed type hypersensitivity principle.But two defectives make that the meaning of this diagnostic method is very limited.One: this method can not be distinguished the healthy person and the tubercule bacillus that once infected tubercule bacillus and just send out the patient at active; Its two: this method can not be distinguished the infected of tubercule bacillus and the inoculator of bacille Calmette-Guerin vaccine.So traditional method of early diagnosis can only provide very limited reference information, make a definite diagnosis and often will wait until that patient occurs after the tuberculose focus.And it is late at this time to do treatment again, and particularly to the resistance tubercule bacillus, patient's prognosis is very poor.
The ELISPOT technology has been brought the revolution of tuberculosis disease early diagnosis.One because the ELISPOT method adopts the specific polypeptide of mycobacterium tuberculosis, can be avoided bacille Calmette-Guerin vaccine inoculator's cross reaction; Its two be patient's cellular immune level at that time because ELISPOT detects, can effectively distinguish the TB activity patient that healthy TB takes bacterium person and morbidity.But ELISPOT detects complex steps, needs special instruments and equipment and reagent costliness, difficult popularization the in the high burden of the tuberculosis countries and regions of most low incomes.
Develop a kind of fast, sensitive tuberculosis method of early diagnosis has been extremely urgent.Based on above background, the selected mycobacterium tuberculosis specific antigen albumen ESAT-6 that generally acknowledges at present of this project is a target antigen, utilize modern information biology and Protocols in Molecular Biology, design and synthesize several sections candidates' the special antigenic peptide sequence of ESAT-6, adopt and merge the hybridoma cell line that hybridoma technology is set up stably excreting Killing Mycobacterium Tuberculosis specific antigen protein monoclonal antibody, and a large amount of preparation, purifying and these monoclonal antibodies of evaluation.The successful acquisition of this monoclonal antibody, novel diagnosis of tuberculosis method---basic substance is established in the diagnosis based on immunological technique in order to set up.Research to aspects such as disease pathogenesis, diagnosis, prognosis and efficacy determinations simultaneously plays an important role.
The present invention uses hybridoma cell technology.This technology merges the bone-marrow-derived lymphocyte and the myeloma cell of immune mouse, to set up the hybridoma cell line of secretion homogeneous antibody, is also referred to as monoclonal antibody technique.This technology relates to serial of methods such as animal immune, cell cultures, cytogamy, cell clone cultivation and immunoassay.
Summary of the invention
The purpose of this invention is to provide a kind of Killing Mycobacterium Tuberculosis monoclonal antibody of secretion property antigen target protein (ESAT-6) in early days, have the aminoacid sequence of SEQ No.1: Met Thr Glu Gln GlnTrp Asn Phe Ala Gly Ile Glu Ala Ala.This monoclonal antibody hypotype is IgM, κ type, called after TBEF3, and energy specific recognition mycobacterium tuberculosis is the aa 1-14MTEQQWNFAGIEAA antigenic peptide sequence of secretion property antigen target protein (ESAT-6) in early days.Killing Mycobacterium Tuberculosis in early days the monoclonal antibody hybridoma cell of secretion property antigen target protein (ESAT-6) by China's typical culture collection center preservation; Address: Chinese Wuhan Wuhan University; Preservation date: on August 17th, 2010; Culture title, strain number and symbol (comprising genotype): mycobacterium tuberculosis is secretion property antigen target protein (ESAT-6) monoclonal antibody hybridoma cell TBEF3 (IgM, κ type) in early days, and deposit number is: CCTCC NO:C201080.
Second purpose of the present invention provides anti-ESAT-6 MONOCLONAL ANTIBODIES SPECIFIC FOR method, realizes by following steps and technical scheme:
(1) immunity of animal: select the BALB/C mice in 6 ages in week, (the 1-14 amino acids of ESAT-6,14 peptide MTEQQWNFAGIEAA) carry out immunity to mouse with antigen peptide.14 peptide MTEOQWNFAGIEAA are synthetic by solid phase method, carry out on the Peptide synthesizer (431A) of American AB I company, adopt Fmoc (9-fluorenylmethyloxycarbonyl) scheme, and synthesis step carries out according to the polypeptide synthetic operation handbook of ABI company.Through high-efficient liquid phase chromatogram purification, purity＞95%, Sequence Identification is by mass spectroscopy.
(2) cultivation of murine myeloma cell: cultivation murine myeloma cell SP2/0 and the growth conditions that makes it to keep good are used for cytogamy.
(3) cytogamy: adopt the polyoxyethylene glycol fusion method.With the BALB/C mice peritoneal macrophage as feeder cell, merging the day before yesterday, inoculation BALB/C mice peritoneal macrophage is in 96 well culture plates, the HAT culture medium culturing one day that contains 20%FCS is got the myeloma cell of mouse in the spleen lymphocyte of mouse in (1) and (2), and above-mentioned two kinds of cytomixis are centrifugal, merge with polyoxyethylene glycol (PEG 6000) mediated cell then, cell after the fusion suitably dilutes, and is seeded to the feeder cell culture plate, and felicity condition is cultivated.
(4) hybridoma screening: above-mentioned culture is cultivated in the HAT selective medium.When cell colony length arrives size to fit, draw cell culture supernatant and do antibody evaluation, screening positive clone.
(5) hybridoma cloning: with limiting dilution assay clone hybridization oncocyte, will be diluted to cell inoculation to 96 orifice plate of certain density, make every hole have only a cell growth.The hole of formation cell colony is got culture supernatant and is done enzyme-linked immunosorbent assay (ELISA), identifies positive colony.Repeat the limited dilution cloning several times, reach 100% up to the positive porosity of hybridoma.Hybridoma enlarged culturing after the cloning is done antibody to be identified and the physicochemical character analysis.
(6) inducing of odd contradictive hydroperitoneum:, give every 0.5ml of BALB/C mice abdominal injection pristane, then every inoculation 5 * 10 in inoculation hybridoma the last week
6Positive hybridoma cell, collection ascites is centrifugal after 10 days, measures antibody titer, and monoclonal antibody purification.
(7) Purification of Monoclonal Antibodies: utilize the monoclonal antibody in the Protein L affinity purification purifying ascites.
(8) the present invention obtains a hybridoma cell line that produces anti-ESAT-6 monoclonal antibody, i.e. TBEF3, TBEF3 hybridoma cell line be through 3 time cloningizations, and it is surplus to continue to cultivate June, and secretory antibody is stable.This cell strain is through liquid nitrogen cryopreservation, and recovery is well-grown afterwards, and antibody-secreting is not seen decline.It is 1: 256 that the ELISA indirect method records that the TBEF3 culture supernatant tires, and ascites is tired and is respectively 1: 8192.Show that through the analysis of monoclonal antibody immunity tropomyosin isoform the antibody type that this hybridoma produces is IgM.TBEF3 is in China typical culture collection center (CCTCC) preservation, and preserving number is CCTCC NO:C201080.
The invention provides the hybridoma that produces monoclonal antibody, it is through merging, screen, clone, go down to posterity and mouse hybridoma cell frozen repeatedly, that the recovery back obtains being TBEF3, the monoclonal antibody TBEF3 of the anti-ESAT-6 of energy stably excreting by the BALB/c mouse splenocyte of immunity and murine myeloma cell SP2/0.In China's typical culture collection center preservation, preserving number is this hybridoma cell line: CCTCC No.C201080.
Another object of the present invention provide this monoclonal antibody TBEF3 mycobacterium tuberculosis in early days secretion property antigen target protein detect and sample in the proteic expression level of ESAT-6 carry out application in qualitative and quantitative, by following approach realization:
1. be probe with monoclonal antibody TBEF3,, by western blotting method (Western Blot) method the proteic situation of ESAT-6 in tubercule bacillus and culture, various body fluid sample, gene recombination and the synthetic sample is identified with the SDS-PAGE electrophoresis.
2. be binding antibody with monoclonal antibody TBEF3, identify the expression of ESAT-6 in the 1. described test sample with immunoprecipitation method.
3. with monoclonal antibody TBEF3 as coated antibody or detect antibody, by enzyme linked immunosorbent assay (ELISA) the proteic expression level of ESAT-6 in tubercule bacillus culture, various body fluid sample, gene recombination and the synthetic sample is carried out qualitative and quantitative analysis.
The invention has the advantages that the monoclonal antibody that a kind of Killing Mycobacterium Tuberculosis specific antigen ESAT-6 is provided, do not see bibliographical information as yet.The preparation method is simple, the monoclonal antibody of this method preparation of what is more important can have multiple use, as ESAT-6 developed by molecule situation in the bacterial cultures of clinical and breadboard mycobacterium tuberculosis, mycobacterium tuberculosis, clinical body fluid sample, the gene recombinant protein is carried out qualitative and quantitative analysis, and various research purposes.
Figure of description
Fig. 1 is that the immunoglobulin (Ig) hypotype of monoclonal antibody TBEF3 is analyzed.
Adopt Fig. 2 immunoprecipitation and western-blot method detect the proteic expression of ESAT-6 in reorganization ESAT-6/CFP-10 fusion rotein, mycobacterium tuberculosis and other mycobacterium and the culture supernatant equal samples thereof.
As coated antibody, reorganization ESAT-6/CFP-10 fusion rotein is as the enzyme linked immunosorbent assay standard reaction curve of standard antigen with monoclonal antibody for Fig. 3.
Embodiment
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.
The MONOCLONAL ANTIBODIES SPECIFIC FOR method of embodiment 1. anti-ESAT-6
(1) immunity of mouse: first immunisation, with 14 peptides (sequence aa1-14MTEQQWNFAGIEAA) of the ESAT-6 of crosslinked KLH with Freund's complete adjuvant emulsification, 2nd, 3 times with Freund emulsification, every BALB/C mice 0.2ml (containing ESAT-6 antigen peptide 200 μ g), back intracutaneous multi-point injection, whenever biweekly.Booster immunization once carried out cytogamy after 6 weeks after 3 days.
(2) cultivation of murine myeloma cell SP2/0: the SP2/0 myeloma cell strain from BALB/C mice is gone down to posterity to contain the 10%FBS-DMEM culture medium culturing, containing 5%CO
2Cultivate in 37 ℃ of incubators of saturated humidity.Merge and to go down to posterity that cell enters logarithmic phase when guaranteeing to merge the day before yesterday.
(3) cytogamy: with the BALB/C mice peritoneal macrophage as feeder cell, merging the day before yesterday, inoculation BALB/C mice peritoneal macrophage is in 96 well culture plates, contain the HAT culture medium culturing one day of 20%FCS, get spleen next day, put in the 200 order stainless steel meshs, reject fat and reticular tissue, tear coating, resuspended behind the separating Morr. cell, centrifuge washing cell 1～2 time with nutrient solution.Collect mouse SP2/0 myeloma cell, centrifugal, wash 2 times after the usefulness nutrient solution resuspended, as SP2/0 cell to be merged.With 1.0 * 10
8Individual immune mouse splenic lymphocyte and 1.0 * 10
7Individual murine myeloma cell SP2/0 mixes, and merges under the 50%PEG6000 effect.Two kinds of cytomixis after scouring once, the centrifugal supernatant of abandoning, flicking the outstanding cell (not adding liquid) of opening of tube wall dropwise added in the cell precipitation with 37 ℃ of pre-warm 50%PEG6000 (pH 8.0) 0.8ml in 60～90 seconds, jolting centrifuge tube gently therebetween, but not piping and druming, left standstill 1 minute, then by elder generation's fast principle in slow back, in the 1st minute, add 1ml serum-free RPMI 1640, in the 2nd minute, add 2ml serum-free RPMI 1640, in the 3rd minute, add 7ml serum-free RPMI 1640, add the serum-free RPMI1640 substratum 30～40ml of 37 ℃ of pre-temperature in later 1 minute gradually.800 rev/mins of low-speed centrifugals 5～10 minutes.Add 20%FCS HAT substratum then, be inoculated into 96 well culture plates that are added with feeder cell respectively, 4 blocks of plates in cell shop of general each fusion are put 37 ℃ of 5%CO
2Cultivate in the incubator.
(4) hybridoma screening: changed 1/2 nutrient solution (HAT) every 4 days once, use instead after 10 days and contain the HT nutrient solution.Hybridoma after the fusion approximately continues to cultivate for two weeks in containing the selectivity nutrient solution of HT.Draw the nutrient solution supernatant during to suitable size (observe, the cell clone size is advisable to take a visual field) in that cell colony is long under 10 times of object lens, be ELISA, screening positive clone after suitably diluting.Adopt ELISA indirect method screening positive hybridoma clone.Key step: 1. 0.01M pH9.6 carbonate buffer solution dilution ESAT-6/CFP-10 recombination fusion protein, and synthetic ESAT-6 specific polypeptides, concentration 200ng/ml adds 0.1ml/ hole wrapper sheet respectively at 96 hole enzyme plates, and 4 ℃ are spent the night; 2. 0.01M pH7.4PBS-Tween 20 washes plate three times; 3. the PBS with 2%BSA-0.01M pH 7.2 sealed 1 hour; 4. the same plate of washing; 5. add the hybridoma culture supernatant of dilution in 1: 5, positive control (serum of immune mouse), negative control (SP2/0 culture supernatant) and blank are established, room temperature reaction 2 hours simultaneously in the 0.1ml/ hole; 6. wash plate; 7. goat-anti mouse Ig (the G+M)-HRP that adds dilution in 1: 5000,0.1ml/ hole, room temperature reaction 1 hour; 8. wash plate; 9. add substrate (TMB) room temperature lucifuge reaction 5～10 minutes; 10. 2M H
2The SO4 termination reaction; 450nm measures its OD value, and is positive with P/N 〉=2.1.
(5) hybridoma cloning: hybridoma cloning is cultivated and is undertaken by limiting dilution assay, after selecting the suitable propagation of hybridoma porocyte work of the antibody test positive (ESAT-6/CFP-10 recombination fusion protein and synthetic ESAT-6 specific polypeptides are all positive), the accurate counting cell.The cell suspension inoculation that is diluted to 10/ml with complete 1640 substratum is in 96 well culture plates of existing feeder cell, every hole 0.1ml, observation of cell growing state after 7-10 days, and antibody horizontal in the detection supernatant liquor, select 5 antibody titerss the highest, the culture hole that is single clone cell growth is done cloning once more and is cultivated.This method can repeat repeatedly, is 100% until mono-clonal hole antibody test positive rate.
(6) induce ascites: in inoculation hybridoma the last week, give every 0.5ml of BALB/C mice abdominal injection pristane, every inoculation 5.0 * 106 positive hybridoma cells are collected ascites and are measured antibody titer after 10 days then.
(7) Purification of Monoclonal Antibodies: adopt monoclonal antibody in affinity purification (Sepharose that Protein L is crosslinked) the purifying ascites.1. after ascites is diluted 3 times with cold Binding Buffer (binding buffer liquid), removed throw out in centrifugal 15 minutes in 4 ℃ of 10000rpm.The affinity purification post that 2. Sepharose-Protein L will be housed in advance with the Binding Buffer of 10 times of column volumes fully stream wash.3. with the ascites upper prop of dilution, control 8～10 droplets/minute of flow velocitys.The ascites of 4. stream being worn repeats upper prop once.5. use the Binding Buffer thorough washing of 20 times of column volumes, wear liquid A280 light absorption value less than 0.01 until stream.6. use the monoclonal antibody of Elution Buffer (elution buffer) elution of bound, 8～10 droplets/minute of control flow velocitys, (PH7.9 is in collection tube 0.5M) in the potassium phosphate buffer that is added with 0.1ml in advance to collect elutriant, every pipe is collected the elutriant that 0.5ml contains antibody, collects altogether more than 20 pipes.7. detect the absorbancy of every pipe elutriant in A280nm, and the collection light absorption value is greater than 0.2 elutriant.8. the elutriant of collecting is placed the dialysis card, and in the PBS of 0.1M PH7.0, dialyse.Changed liquid once every 6 hours, dialysed altogether 24 hours.9. after the antibody-solutions dilution (1: 100) after will dialysing, survey protein content in 280nm.10. antibody purified is sub-packed in the tubule, places cryogenic refrigerator standby.
(8) hypotype of monoclonal antibody is identified: adopt the mouse monoclonal antibody immunoglobulin (Ig) parting kit of Serotec company to analyze.The monoclonal antibody of purifying is done suitably to detect after the dilution, and operation is strict to be undertaken by the test kit specification sheets.Test-results is that TBEF3 hybridoma excretory monoclonal antibody is IgM, κ type.
The result is referring to accompanying drawing 1.
The evaluation (qualitative) that this monoclonal antibody of embodiment 2. usefulness is carried out ESAT-6 detects
(qualitative detection) that the anti-ESAT-6 monoclonal antibody of the present invention's preparation can be used for identifying, authentication method can be realized by following two kinds of methods:
1. immunoblotting Western Blotting: reorganization ESAT-6 and ESAT-6/CFP-10 fusion rotein and mycobacterium tuberculosis whole protein lysate carry out Western Blotting detection with this monoclonal antibody, present the purpose band in the corresponding position, show to detect the proteic expression of ESAT-6.Concrete steps:
(1) sds polyacrylamide gel electrophoresis: method is referring to " fine works molecular biology experiment guide " (Science Presses 1998) such as F. Ao Sibai.Adopt 15% separation gel and 5% to concentrate glue, deposition condition is voltage 150V, and tetrabromophenol sulfonphthalein dyestuff band stops electrophoresis for about 1.5 centimetres apart from the glue base.
(2) electrotransfer: method is referring to " fine works molecular biology experiment guide " (Science Presses 1998) such as F. Ao Sibai.The electricity consumption tranfer system is transferred to it on PVDF (0.22 μ m) film.
(3) immunoblotting: after electricity changes the film end, the room temperature sealing is after 1 hour in 5% skim-milk confining liquid for film, and anti-as one with the monoclonal antibody TBEF3 of the present invention's preparation, incubated at room 2 hours or 4 ℃ of reactions are spent the night, with TBST (TBS adds 0.5%Tween-20) washing 3 times, each 10min.Sheep anti mouse IgM with horseradish peroxidase-labeled is anti-as two, and room temperature reaction 2h washs the back with ECL effect 1min, in full-automatic chemical luminescence imaging instrument (Bio-Rad VersaDoc 5000MP) exposure image with aforesaid method.
2. immuno-precipitation (IP): will recombinate ESAT-6 and ESAT-6/CFP-10 fusion rotein, mycobacterium tuberculosis whole protein lysate, mycobacterium tuberculosis culture supernatant, clinical tuberculosis patient tuberculosis hydrothorax and this monoclonal antibody carry out immunoprecipitation, after western-blot detects, present the purpose band in the corresponding position, show to detect the proteic expression of ESAT-6.Concrete steps:
1. get recombinant protein or the 100ug mycobacterium tuberculosis crack protein of 1ug, 500ul mycobacterium tuberculosis culture supernatant or clinical tuberculosis patient tuberculosis hydrothorax add the anti-ESAT-6 monoclonal antibody of the 1ug TBEF3 of purifying, and 4 ℃ were slowly shaken 2 hours; Add fully resuspended Protein L Agarose of 20 microlitres again, 4 ℃ of slow shaken over night.
2. 2500rpm is centrifugal 5 minutes, carefully absorbs supernatant, and attention can not sop up Protein L Agarose.
3. use 0.5-1 milliliter TBST washing precipitation 5 times.
4. after finishing last washing, remove supernatant, add the resuspended precipitation of 40 microlitre 1X SDS-PAGE electrophoresis sample-loading buffers, instantaneous high speed centrifugation is centrifugal to managing the end sample.
5. 100 ℃ or boiling water bath were handled 3-5 minute, got sample segment and were used for the SDS-PAGE electrophoresis, temporary transient no sample-20 ℃ preservation.
6. after electrophoresis finishes, albumen is transferred to pvdf membrane.
7. after changeing the film end, film is placed 5% skim-milk confining liquid, room temperature sealing 1 hour.
8. how anti-add the anti-ESAT-6 of rabbit, room temperature reaction 2 hours.
9. add anti-rabbit igg-HRP, room temperature reaction 1 hour after washing film 4 times.
10. wash ECL colour developing behind the film 4 times, VersaDoc 5000 full-automatic chemical luminescence imaging instrument exposure images.
The result is referring to accompanying drawing 2, M: molecular weight of albumen Marker (15%SDS-PAGE) wherein, 1. reorganization ESAT-6/CFP-10 fusion rotein, 2. reorganization ESAT-6 albumen, 3. mycobacterium tuberculosis culture supernatant, 4. tuberculosis hydrothorax, 5. mycobacterium avium-intracellulare crack protein, 6. mycobacterium kansasii crack protein, 7. mycobacterium tuberculosis crack protein.
The anti-ESAT-6 monoclonal antibody of the present invention preparation can be used for tuberculosis specific antigen ESAT-6 level in detection by quantitative reorganization ESAT-6 and ESAT-6/CFP-10 fusion rotein, mycobacterium tuberculosis culture supernatant, the various clinical body fluid sample, and detection method can be by following two kinds of methods realization:
1. indirect elisa method:
1. will detect sample and suitably be diluted in the 0.01M pH9.6 carbonate buffer solution coating buffer, ESAT-6 and the suitable series concentration of ESAT-6/CFP-10 fusion rotein with reorganization is diluted in the coating buffer simultaneously, contrasts as the quantitative criterion product.Add the standard substance 100ul of above-mentioned each sample to be tested and series concentration in the enzyme plate hole of correspondence, incubated at room 2h or 4 ℃ of bags are spent the night.
2. turn liquid and pat dry residual liquid is washed plate three times with 0.01M pH7.4 PBS-Tween 20 washing lotions.
3. the PBS with 2%BSA-0.01M pH 7.2 sealed 1 hour.
4. the samely wash plate 3 times.
5. add the suitably anti-ESAT-6 monoclonal antibody TBEF3 of dilution, 0.1ml/ hole, room temperature reaction 2 hours.
6. the same wash plate 3 times after, add goat-anti mouse IgM-HRP, 0.1ml/ hole, room temperature reaction 1 hour.
7. after soaking plate 5min with washings, the same wash plate 4 times after, add substrate (TMB) room temperature lucifuge reaction 5～10 minutes.
8. 2M H
2The SO4 termination reaction, 450nm measures its OD value.
9. drawing standard response curve, the expression level of EAST-6 in the sample to be tested of asking according to typical curve.
2. sandwich ELISA method:
1. wrap quilt: be cushioned liquid with bag the monoclonal antibody TBEA8 of another ESAT-6 is diluted to 1 μ g/mL.Add the 0.1mL/ hole in the enzyme plate reacting hole, 4 ℃ are spent the night.Discard solution in the hole next day, wash plate 3 times, each 3min with 0.01MpH7.4 PBS-Tween 20 lavation buffer solutions.
2. sealing: the PBS room temperature with 2%BSA-0.01M pH 7.2 was sealed 1 hour.
3. application of sample: room temperature reaction was put in the wet box 2 hours in the reorganization ESAT-6 standard substance 0.1mL/ hole that adds the sample to be checked of suitable dilution and series concentration dilution in the above-mentioned reacting hole that has wrapped quilt.
4. wash plate: wash plate 3 times with lavation buffer solution, each 3min.
5. adding two resists: the monoclonal antibody TBEF3 (the IgM type is at the different target position of EAST-6) or the anti-ESAT-6 of rabbit that add this anti-ESAT-6 that suitably dilutes resist 0.1ml/ hole, room temperature reaction 2 hours more.
6. enzyme-added thing: the same wash plate 3 times after, add goat-anti mouse IgM-HRP or anti-rabbit igg-HRP, 0.1ml/ hole, room temperature reaction 1 hour.
7. colour developing: the same wash plate 4 times after, add substrate (TMB) room temperature lucifuge reaction 5～10 minutes.
8. stop, read plate: 2M H
2The SO4 termination reaction, 450nm measures its OD value.
9. typical curve and calculating: with standard substance concentration is X-coordinate, and absorbancy is an ordinate zou, generates typical curve and linear regression equation, the expression contents of EAST-6 in the sample to be tested of asking according to the standard reaction curve.
The results are shown in accompanying drawing 3 and table 1
Table 1ELISA method detects positive rate and the application in the tuberculosis diagnosis thereof of ESAT-6 in the clinical body fluid sample
| Sample classification | The example number | The EAST-6 positive detects number |
| The mycobacterium tuberculosis culture supernatant | 38 | 38 |
| The non-tuberculous |
20 | 0 |
| The tuberculosis hydrothorax of clinical diagnosis | 32 | 29 |
| The non-tuberculous hydrothorax of clinical diagnosis | 24 | 0 |
| Healthy |
20 | 0 |
The ELISA method of utilizing the ESAT-6 monoclonal antibody to set up is tested clinical sample, and its susceptibility reaches 92.4% in the tuberculosis diagnosis, and specificity reaches 100%.
Should understand, the present invention is described in conjunction with most preferred embodiment, yet after having read foregoing of the present invention, those skilled in the art can make various changes or modifications the present invention, and these equivalent form of values fall within the application's appended claims institute restricted portion equally.
Claims (6)
1. the Killing Mycobacterium Tuberculosis monoclonal antibody TBEF3 of secretion property antigen target protein in early days, this monoclonal antibody hypotype is IgM, κ type, can with mycobacterium tuberculosis secretion property antigen target protein ESAT-6 specific combination in early days, be that the hybridoma of CCTCC No.C201080 produces by preserving number.
2. Killing Mycobacterium Tuberculosis according to claim 1 is the preparation method of the monoclonal antibody TBEF3 of secretion property antigen target protein in early days, it is characterized in that this monoclonal antibody obtains by following steps:
(1) mouse immune: selecting the BALB/C mice in 6 ages in week, is the mycobacterium tuberculosis 1-14 amino acids of secretion property antigen target protein gene in early days with special antigen peptide, and 14 peptide MTEQQWNFAGIEAA carry out immunity to mouse.Every mouse mixes subcutaneous multi-point injection immunity behind the adjuvant with the antigen peptide of 200ug, per 2 weeks once, totally 3 times; The 4th booster immunization is used for after 3 days merging.
(2) cultivation of murine myeloma cell: cultivate murine myeloma cell SP2/0 and make it keep good growth conditions to be used for hybridoma and merge;
(3) cytogamy: adopt polyoxyethylene glycol cytogamy method, with the BALB/C mice peritoneal macrophage as feeder cell, merging the day before yesterday, inoculation BALB/C mice peritoneal macrophage is in 96 well culture plates, the HAT culture medium culturing one day that contains 20%FCS is mixed the myeloma cell of mouse in the spleen lymphocyte of immune mouse in the step (1) and the step (2) centrifugal, merges with the polyoxyethylene glycol mediated cell then, cell after dilution is merged is seeded to culture plate and cultivates;
(4) hybridoma screening: above-mentioned culture is cultivated in containing the HAT selective medium, when cell colony length arrives size to fit, drawn culture supernatant and do antibody evaluation, screening positive clone with enzyme-linked immuno-sorbent assay;
(5) hybridoma cloning is cultivated: with limiting dilution assay clone male hybridoma, cell inoculation to the 96 porocyte culture plate of certain density will be diluted to, make every hole have only a cell growth, the hole of formation cell colony is got supernatant and is done enzyme linked immunological absorption detection, screening and evaluation positive colony, the repeated cloning several times reach 100% up to the positive porosity of hybridoma, with the hybridoma enlarged culturing after the cloning and do the antibody physicochemical character and analyze and identify;
(6) inducing of mouse ascites: give every BALB/C mice inoculation 5 * 10
6Positive hybridoma cell, collection ascites is centrifugal after 10 days, measures antibody titer, and the monoclonal antibody in the purifying ascites;
(7) Purification of Monoclonal Antibodies: utilize the monoclonal antibody in the Protein L affinity purification purifying ascites.
3. Killing Mycobacterium Tuberculosis according to claim 1 is the application of monoclonal antibody TBEF3 in mycobacterium tuberculosis secretion property antigen target protein detection in early days of secretion property antigen target protein in early days.
4. the Killing Mycobacterium Tuberculosis according to claim 3 monoclonal antibody TBEF3 of secretion property antigen target protein in early days is characterized in that in the application of mycobacterium tuberculosis during secretion property antigen target protein detects in early days: the mycobacterium tuberculosis expression of secretion property antigen target protein in early days in method bacterial detection culture by antibody folder enzyme linked immunosorbent assay, immunoblotting and immunoprecipitation and the various body fluid sample.
5. Killing Mycobacterium Tuberculosis according to claim 3 is the application of monoclonal antibody TBEF3 in mycobacterium tuberculosis secretion property antigen target protein quantitative analysis in early days of secretion property antigen target protein in early days.
6. Killing Mycobacterium Tuberculosis according to claim 3 is the application of monoclonal antibody TBEF3 in mycobacterium tuberculosis secretion property antigen target protein quantitative analysis in early days of secretion property antigen target protein in early days, it is characterized in that: the method by enzyme linked immunosorbent assay to the mycobacterium tuberculosis in bacterial cultures and body fluid sample in early days secretion property antigen target protein expression level carry out qualitative and quantitative.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2010105047078A CN101985473B (en) | 2010-10-12 | 2010-10-12 | Monoclonal antibody TBEF3 of tubercle bacillus-resistant ESAT-6 and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2010105047078A CN101985473B (en) | 2010-10-12 | 2010-10-12 | Monoclonal antibody TBEF3 of tubercle bacillus-resistant ESAT-6 and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN101985473A true CN101985473A (en) | 2011-03-16 |
| CN101985473B CN101985473B (en) | 2011-11-09 |
Family
ID=43709873
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2010105047078A Expired - Fee Related CN101985473B (en) | 2010-10-12 | 2010-10-12 | Monoclonal antibody TBEF3 of tubercle bacillus-resistant ESAT-6 and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101985473B (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102426244A (en) * | 2011-11-21 | 2012-04-25 | 浙江工业大学 | Immunity-chromatography test paper for rapid detection of mycobacterium tuberculosis secretory protein and preparation method thereof |
| CN105732808A (en) * | 2016-03-20 | 2016-07-06 | 张福华 | Preparation method of mycobacterium tuberculosis monoclonal antibody |
| CN110878309A (en) * | 2019-12-05 | 2020-03-13 | 中国人民解放军第四军医大学 | Heavy chain and light chain variable region gene of mycobacterium tuberculosis secretory protein ESAT-6 monoclonal antibody, encoded polypeptide and application thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1350546A (en) * | 1998-11-04 | 2002-05-22 | Isis创新有限公司 | Tuberculosis diagnostic test |
| CN1866023A (en) * | 2006-05-25 | 2006-11-22 | 上海市肺科医院 | Method for synchronous detection of multiple tubercle bacillus specific secretion antigens |
| CN1936582A (en) * | 2005-09-21 | 2007-03-28 | 台塑生医科技股份有限公司 | Method for detecting tubercle bacillus antigen in body fluid |
-
2010
- 2010-10-12 CN CN2010105047078A patent/CN101985473B/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1350546A (en) * | 1998-11-04 | 2002-05-22 | Isis创新有限公司 | Tuberculosis diagnostic test |
| CN1936582A (en) * | 2005-09-21 | 2007-03-28 | 台塑生医科技股份有限公司 | Method for detecting tubercle bacillus antigen in body fluid |
| CN1866023A (en) * | 2006-05-25 | 2006-11-22 | 上海市肺科医院 | Method for synchronous detection of multiple tubercle bacillus specific secretion antigens |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102426244A (en) * | 2011-11-21 | 2012-04-25 | 浙江工业大学 | Immunity-chromatography test paper for rapid detection of mycobacterium tuberculosis secretory protein and preparation method thereof |
| CN102426244B (en) * | 2011-11-21 | 2014-04-09 | 浙江工业大学 | Immunity-chromatography test paper for rapid detection of mycobacterium tuberculosis secretory protein and preparation method thereof |
| CN105732808A (en) * | 2016-03-20 | 2016-07-06 | 张福华 | Preparation method of mycobacterium tuberculosis monoclonal antibody |
| CN110878309A (en) * | 2019-12-05 | 2020-03-13 | 中国人民解放军第四军医大学 | Heavy chain and light chain variable region gene of mycobacterium tuberculosis secretory protein ESAT-6 monoclonal antibody, encoded polypeptide and application thereof |
| CN110878309B (en) * | 2019-12-05 | 2023-09-26 | 中国人民解放军第四军医大学 | Mycobacterium tuberculosis secretory protein ESAT-6 monoclonal antibody heavy chain and light chain variable region gene, coded polypeptide and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN101985473B (en) | 2011-11-09 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN101985472B (en) | Monoclonal antibody TBCD6 resistant to mycobacterium tuberculosis CFP-10 and application | |
| CN105087497B (en) | Hybridoma cell strain ZJEB8-01, anti-Ebola virus GP protein monoclonal antibody, and preparation and application thereof | |
| US8546093B2 (en) | Detection method for methicillin resistant staphylococcus aureus | |
| CN105112375B (en) | Hybridoma cell strain ZJED0-02, anti-Ebola virus GP protein monoclonal antibody, and preparation and application thereof | |
| CN101560255B (en) | Anti-rabies virus monoclonal antibody and application | |
| CN103173420B (en) | Hybridoma cell capable of secreting anti-cardiac troponin I monoclonal antibodies and applications thereof | |
| CN104862283B (en) | The monoclonal antibody of a pair of of high specific high-affinity combination human muscle hemoglobin and its application | |
| CN111484552A (en) | Monoclonal antibody against SpA5 protein, application thereof and kit containing monoclonal antibody | |
| CN103163299A (en) | Avian leukosis double-antibody sandwich enzyme-linked immuno sorbent assay (ELISA) antigen detection kit | |
| CN104745534B (en) | A kind of Procalcitonin monoclonal antibody hybridoma 2H4 and monoclonal antibody | |
| CN101985473B (en) | Monoclonal antibody TBEF3 of tubercle bacillus-resistant ESAT-6 and application thereof | |
| CN109517802A (en) | Secrete hybridoma cell strain, its monoclonal antibody and its application of Salmonella Pullorm IpaJ monoclonal antibody | |
| CN101987872B (en) | Monoclonal antibody TBEF3 of tubercle bacillus-resistant ESAT-6 and application | |
| CN101985474B (en) | Monoclonal antibody TBCA6 resistant to mycobacterium tuberculosis CFP-10 and application | |
| CN109374886A (en) | Infectious bovine rhinotrachetis virus antibody assay kit and its application | |
| CN102539778A (en) | Enzyme linked immunosorbent assay kit for detecting recombinant human tumor necrosis factor-alpha | |
| CN101921337B (en) | Antibody against lactate dehydrogenase of plasmodium vivax, related preparation method, hybridoma cell strain and application | |
| CN101570742A (en) | Monoclonal antibody of human myxovirus resistance A (A-hMxA), preparation and application thereof | |
| CN114456262B (en) | Anti-H1N 1 influenza virus nucleoprotein monoclonal antibody ZJU-NP-A1 and application thereof in detection | |
| KR20120132227A (en) | Monoclonal antibody for detecting multiple type Foot and Mouth Disease Virus and method for detecting Foot and Mouth Disease Virus using the same | |
| CN105315367B (en) | For detecting the enzyme linked immunological kit of methicillin-resistant staphylococcus aureus | |
| CN109796527A (en) | A kind of bluish dogbane mitochondrial protein COX3 antigen polypeptide and the methods and applications for preparing polyclonal antibody | |
| CN106442983B (en) | The application of antigen of mycobacterium tuberculosis albumen Rv3793 and its t cell epitope peptide | |
| CN109655619A (en) | A kind of method and its dedicated kit detecting 12 content of chicken interleukin-2 | |
| CN109652382A (en) | A kind of application of Procalcitonin monoclonal antibody hybridoma cell strain and its monoclonal antibody |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20111109 Termination date: 20151012 |
|
| EXPY | Termination of patent right or utility model |