CN101987872B - Monoclonal antibody TBEF3 of tubercle bacillus-resistant ESAT-6 and application - Google Patents
Monoclonal antibody TBEF3 of tubercle bacillus-resistant ESAT-6 and application Download PDFInfo
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Abstract
The invention provides a monoclonal antibody TBEA8 of tubercle bacillus-resistant secretory antigen target protein (ESAT-6) in early stage. The monoclonal antibody TBEA8 is prepared by a method comprising 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 assay and an immunoblotting testing method to establish a hybridoma cell line TBEA8 for secreting monoclonal antibody of tubercle bacillus-resistant secretory ESAT-6 in early stage; carrying out multiplication culturing on the positive cells and injecting the cells to congenic strain mouse enterocoelia for induction to generate ascitic fluid containing antibody; and carrying out collecting and affinity purification of the antibody to obtain the monoclonal antibody of tubercle bacillus-resistant secretory antigen target protein in early stage. The invention also provides the 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 TBEA8 for preparing anti-EAST-6 is accredited as IgG2b, 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: Ser Ile His Ser Leu LeuAsp Glu Gly Lys Gln Ser Leu.This monoclonal antibody hypotype is IgG2b, κ type, called after hybridoma cell strain TBEA8, and energy specific recognition mycobacterium tuberculosis is the aa 24-36SIHSLLDEGKQSL 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 TBEA8 (IgG2b, κ type) in early days, and deposit number is: CCTCCNO:C201081.
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 24-36 amino acids of ESAT-6,13 peptide SIHSLLDEGKQSL) carry out immunity to mouse with antigen peptide.13 peptide SIHSLLDEGKQSL 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.As feeder cell, merging the day before yesterday with the BALB/C mice peritoneal macrophage, inoculation BALB/C mice peritoneal macrophage is in 96 well culture plates, contains the HAT culture medium culturing one day of 20%FCS.Get spleen lymphocyte of mouse in (1) and the myeloma cell of (2) middle mouse, above-mentioned two kinds of cytomixis are centrifugal, merge with polyoxyethylene glycol (PEG 6000) mediated cell then, the cell after the fusion suitably dilutes, be seeded to the feeder cell culture plate, 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 G affinity purification purifying ascites.
(8) the present invention obtains a hybridoma cell line that produces anti-ESAT-6 monoclonal antibody, i.e. TBEA8, TBEA8 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 TBEA8 culture supernatant tires, and ascites is tired and is respectively 1: 16384.Show that through the analysis of monoclonal antibody immunity tropomyosin isoform the antibody type that this hybridoma produces is IgG2b.TBEA8 is in China typical culture collection center (CCTCC) preservation, and preserving number is CCTCC No.C201081.
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 TBEA8, the monoclonal antibody TBEA8 of the anti-ESAT-6 of energy stably excreting by the BALB/c mouse splenocyte of immunity and murine myeloma cell SP2/0.
Another object of the present invention provide this monoclonal antibody TBEA8 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 TBEA8,, 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 TBEA8, identify the expression of ESAT-6 in the 1. described test sample with immunoprecipitation method.
3. with monoclonal antibody TBEA8 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 TBEA8 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 13 peptides (sequence aa24-36SIHSLLDEGKQSL) 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:, give every 0.5ml of BALB/C mice abdominal injection pristane, then every inoculation 5.0 * 10 in inoculation hybridoma the last week
6Positive hybridoma cell is collected ascites and is measured antibody titer after 10 days.
(7) Purification of Monoclonal Antibodies: adopt monoclonal antibody in affinity purification (Sepharose that Protein G 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 G 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 TBEA8 hybridoma excretory monoclonal antibody is IgG2b, κ type, aminoacid sequence with SEQ No.1: Ser Ile His Ser Leu Leu Asp Glu Gly Lys GlnSer Leu, called after hybridoma cell strain TBEA8, energy specific recognition mycobacterium tuberculosis is the aa 24-36 SIHSLLDEGKQSL 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; Deposit number is: CCTCC NO:C201081.
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 TBEA8 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 igg 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 TBEA8 of purifying, and 4 ℃ were slowly shaken 2 hours; Add fully resuspended Protein G 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 G 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.
Embodiment 3, carry out the detection by quantitative of ESAT-6 molecule with this monoclonal antibody
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 pH7.4PBS-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 TBEA8 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 monoclonal antibody TBEA8 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 0.01M pH7.4PBS-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 anti-ESAT-6 monoclonal antibody TBEF3 (the IgM type is at the different target position of EAST-6) or the anti-ESAT-6 of rabbit that add suitably dilution 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 result is referring to 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
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 (3)
1. the Killing Mycobacterium Tuberculosis monoclonal antibody TBEA8 of secretion property antigen target protein in early days, this monoclonal antibody hypotype is IgG2b, 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.C201081 produces by preserving number.
2. Killing Mycobacterium Tuberculosis according to claim 1 in early days the monoclonal antibody TBEA8 of secretion property antigen target protein in the application in the secretion property antigen target protein detection reagent in early days of preparation mycobacterium tuberculosis.
3. Killing Mycobacterium Tuberculosis according to claim 1 is the application of monoclonal antibody TBEA8 in preparation mycobacterium tuberculosis secretion property antigen target protein quantitative analysis in early days reagent of secretion property antigen target protein in early days.
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