CN103361317B - Macaque IFN-gamma (interferon-gamma) monoclonal antibody hybridoma as well as preparation method and application thereof - Google Patents

Abstract

The invention discloses a macaque IFN-gamma (interferon-gamma) monoclonal antibody hybridoma as well as a preparation method and application thereof. According to the invention, the preparation method comprises the following steps: performing RNA extract on Con A irritated macaque whole blood hemocyte, carrying out RT-PCR (reverse transcription-polymerase chain reaction), thus obtaining total cDNA, and amplifying total cDNA to obtain macaque IFN-gamma genes; obtaining IFN-gamma through vector in vitro expression and purification; after the purified macaque IFN-gamma is used for immunizing a mouse, separating the immunized mouse spleen B cell and using a hybridoma technique to obtain a hybridoma which effectively expresses IFN-gamma monoclonal antibody, MmIFN-gamma2F6 with CCTCC NO:C201235. The macaque IFN-gamma monoclonal antibody can be specifically bonded with the macaque IFN-gamma so as to be used for detecting macaque IFN-gamma in supernatant after macaque whole blood antigenic stimulation. The macaque IFN-gamma monoclonal antibody can be specifically bonded with the natural macaque IFN-gamma, the sensitiveness can be 125pg/ml when the macaque IFN-gamma monoclonal antibody is used for specific detection of macaque IFN-gamma sensitiveness, so that the sensitiveness required in the tuberculosis detection can be achieved.

Description

The monoclonal antibody hybridoma cell of a kind of macaque IFN-γ and preparation method and application

Technical field

The present invention relates to the detection field of macaque IFN-γ, more specifically relate to a kind of hybridoma of expressing macaque IFN-γ monoclonal antibody (abbreviation monoclonal antibody), also relate to the preparation method of the monoclonal antibody hybridoma cell of a kind of macaque IFN-γ simultaneously, also relate to a kind of macaque IFN-γ Monoclonal Antibody Cell and detecting the application in macaque tuberculosis.

Background technology

Tuberculosis is a kind of chronic debilitating transmissible disease of Zoonosis, is the transmissible disease that current mortality ratio is the highest, is also one of serious infectious diseases of whole world key monitoring.Tuberculosis is caused by mycobacterium tuberculosis complex, mainly propagates through respiratory system, also can propagate through Digestive tract.Mycobacterium tuberculosis and Mycobacterium bovis are topmost pathogenic bacterium, wherein bovine mycobacterium infection spectrum and extensively, except ox, can infect people and Yue more than 50 kind of Mammals and 25 kinds of birds; And people and and human contact's species many infection mycobacterium tuberculosis frequently.Owing to there is the danger of mutually propagating between humans and animals, animal control lungy not only has the meaning of the welfare that watches for animals, and has more sanitarian meaning.

Macaque is that II grade of China lays special stress on protecting wildlife, is also put in endangered species of wild fauna and flora international trade pact (CITES) appendix II.According to Chinese Pacific Ocean environmental organization statistics, within 1998, macaque about has 254000 in China, only has an appointment 77913 to estimative figure in 2002, and within short 4 years, quantity decreases 2/3; Non-human primates is applied to medical research very long history, because it is closest with people in sibship, the homology of 75% ~ 85% is had with the genetic material of the mankind, highly similar to the mankind in physiological structure and immune metabolism, be the laboratory animal in medical research with high value.Investigation display, the whole world every year for the non-human primates of scientific research between 10 ~ 200,000, wherein the usage quantity of the non-human primates of the U.S. is maximum, within 2002, be 52279, and usage quantity annual in recent years all remains on about 54000; The usage quantity of European Union's non-human primates of 2002 is 10362, and along with constantly setting up of non-human primates research institution, the usage quantity of the non-human primates of China also has the gesture of quick growth.Non-human primates is sensitive species lungy.It is generally acknowledged, old world monkey class (oldworldmonkeys mainly comprises the monkey class in continent, Europe, Asia and Africa) is most susceptible, member's moderate susceptible of pongidae, New World monkey class (New World monkeys, in, four section's monkey classes of South America) in then rare generation lungy.Wherein, macaque is most susceptible.Usually, once infection has tuberculosis in a monkey group, full group will be involved very soon, cause outburst lungy.

Because the prevention and control lungy of current animal do not have reliable vaccine, the major measure that the world controls macaque and other animal tuberculosis is " quarantine-eliminate ", therefore, how diagnosing out animal whether to have tuberculosis definitely, being necessary for taking this measure.Tuberculin skin test (tuberculin skin test, TST) is set up in 1890 by tuberculin inventor Robert Koch, is to measure macaque standard method lungy, is also the designation method of international trade.The TST method that CDC of the U.S. (CDC) formulates uses 0.1mL Mammals ot (MOT) (Synbiotics, San Diego, Calif) upper eyelid intradermal injection or the intradermal injection of belly specific region, and 24h, 48h, 72h observe respectively or measure skin depth difference after injection.It is also TST that the national standard of China detects macaque method lungy, and difference is to use ox type PPD(2000U, 0.1mL physiological saline solution) method of upper eyelid intradermal injection.24h, 48h, 72h observe the change in upper eyelid respectively after injection.This law technology and economic requirement are not high, have very high susceptibility and enough specificitys simultaneously.But there is a lot of defect in the method.The most significant defect is false negative result., in a series of tuerculoderma, there is negative findings in positive macaque sometimes to have ample evidence to show; Infected the macaque of mycobacterium tuberculosis at some, when disease is to late period, or infected Other diseases, or there is the reason of immunosuppression and other the unknown, TST there will be negative findings.The significant defect of another one is, no matter be MOT or ox type PPD, be all mixed protein, the common composition containing multiple non-tuberculous mycobacteria, thus make TST cannot distinguish the infection of pathogenic mycobacterium tuberculosis and the infection of non-tuberculous mycobacteria, cause false positive results.For the case needing to make a definite diagnosis further, then need to carry out Chest X-rays, mycobacterium tuberculosis specific nucleic acid detects (PCR), lavage of trachea thing acid-fast stain microscopy, and " gold standard " point bacterium is cultivated.For macaque, upper eyelid skin test also can cause serious discomfort to animal.Meanwhile, operating time of TST long (72h), labour intensity is large, the defects such as reading subjectivity is strong, also have impact on its application clinically.

Interferon-γ (IFN-γ) is CD ₄ ⁺th1 cell, CD ₈ ⁺a kind of cytokine that cytotoxic T cell and NK cell etc. produce under antigen (bacterium, virus etc.) and mitogen (ConA and PHA etc.) stimulate.IFN-γ has anti-infective, anti-tumor activity and immunoregulation effect, as activated macrophage, improve MHC I class and class Ⅱmolecule expression, promote angtigen presentation etc., body anti-infective with immunity in play a significant role.Based on IFN-γ and the close ties between infecting, medical science and veterinarily all utilize pathogen specific antigen protein stimulated in vitro peripheral blood lymphocytes to produce IFN-γ to carry out Diagnosis of Infectious Diseases.The method sensitivity and specificity are all high, demonstrate very large application prospect.Tuberculosis detects IFN-γ release in vitro detection method (interferon-gamma in vitro release assay, IGRA) be by inspection animal's whole blood or peripheral blood lymphocytes (PBMC) with the external stimulus of mycobacteria specific antigen, a small amount of T cell is made fully to accept stimulation, thus secrete a large amount of IFN-γ, then detect the Laboratory blood detection method of IFN-γ concentration with ELISA, the early diagnosis of this method to tuberculosis infection is significant.IFN-γ method for releasing and TST are the diagnosis of tuberculosis methods based on cell immune response, and the advantage compared to TST, IGRA is obvious: in susceptibility, IGRA for the detection sensitivity of active tuberculosis higher than TST; In specificity, because IGRA can use the distinctive antigen of mycobacterium tuberculosis complex as stimulator antigen, thus can get rid of nonpathogenic mycobacteria infect and BCG inoculate the false positive produced; IGRA can use different antigen to stimulate simultaneously, can determine the classification of the infection mycobacterium of body more accurately; In clinical manipulation and context of detection, IGRA does not need testing staff to contact body, and result judges objective and accurate, and detected result has very high repeatability; And stimulation fluid can cryopreservation, is conducive to the backtracking of detected result.

In the diagnosis of tuberculosis of macaque, also there is commercial IFN-γ method for releasing detection kit abroad, namely (Prionics AG, Switzerland).This test kit is mainly used in macaque detection lungy, also may be used for the detection lungy of other non-human primate, as orangutan, and gibbon, Squirrel monkey, baboon etc.In susceptibility, be 68%, TST be 84%; Specificity aspect, can reach 97%, TST is 87%.2007 by USDA approval as cynomolgus monkey and macaque detection method lungy.But this test kit is too expensive, every increment detection originally about needs Renminbi 200 yuan, and is difficult to buy.Therefore develop domestic macaque IGRA test kit and have a very big significance.

Summary of the invention

The object of the invention is the monoclonal antibody hybridoma cell that there are provided a kind of macaque IFN-γ, macaque IFN-γ monoclonal antibody expressed by it can with macaque IFN-γ specific binding, can be used for the release detecting macaque IFN-γ.This macaque monoclonal antibody can with macaque IFN-γ specific binding.This hybridoma is by merging through the mouse spleen lymphocyte of macaque IFN-γ recombinant protein immunity and murine myeloma cell, the feature not only remaining splenic lymphocyte antibody-secreting function and can grow in Selective agar medium, and there is murine myeloma cell then can the characteristic of divide without limit, propagation under culture conditions.

Another object of the present invention is the preparation method of the monoclonal antibody hybridoma cell that there are provided a kind of macaque IFN-γ, easy to implement the method, easy and simple to handle, the method can prepare the monoclonal antibody of anti-macaque IFN-γ, can be applied to the monoclonal antibody of other similar cytokine of preparation.

Another object of the present invention is the application of monoclonal antibody hybridoma cell in the ELISA detection method detecting macaque IFN-γ that there are provided a kind of macaque IFN-γ, and the monoclonal antibody of macaque IFN-γ expressed by macaque IFN-γ monoclonal antibody hybridoma cell, foundation detect the ELISA of macaque IFN-γ.The ELISA detection method set up with this monoclonal antibody can the content of specific detection macaque IFN-γ, and can reduce costs, therefore the ELISA set up based on it is at detection macaque IFN-γ, and the aspect evaluating the cell immune response of macaque has good using value.

Another object of the present invention there are provided a kind of macaque IFN-γ Monoclonal Antibody Cell to detect the application in macaque tuberculosis, the monoclonal antibody of macaque IFN-γ expressed by macaque IFN-γ monoclonal antibody hybridoma cell, foundation detect the ELISA of macaque IFN-γ, and detection method is applied to macaque diagnosis of tuberculosis.The ELISA detection method set up with this monoclonal antibody can specific detection whole blood stimulate after the burst size of macaque IFN-γ in supernatant, and can reduce costs, the IGRA therefore set up based on it has good using value in the macaque diagnosis of China.

To achieve the above object, the present invention is according to macaque IFN-γ mRNA sequence (GenBank accessionNo:AY376145.1) that GenBank issues, with the hemocyte total serum IgE of the local macaque of China for template carries out RT-PCR, amplify the cDNA of macaque IFN-γ further, and obtain macaque IFN-γ recombinant protein by vector in vitro prokaryotic expression and purifying, after the macaque restructuring IFN-γ immune mouse of purifying, be separated by the mouse spleen B cell of immunity and adopt hybridoma technology to obtain hybridoma, restructuring IFN-γ is utilized to filter out the hybridoma cell strain of energy high expression macaque IFN-γ monoclonal antibody.Set up macaque IFN-γ by this monoclonal antibody further and detect double-antibody sandwich elisa test kit, for macaque detection lungy.

The present invention adopts following technical measures:

RNA is extracted by the macaque whole blood hemocyte stimulated ConA, carry out RT-PCR, after obtaining total cDNA, amplification is carried out to it and obtain macaque IFN-γ gene, and obtain macaque IFN-γ by vector in vitro expression and purification, after the macaque IFN-γ immune mouse of purifying, be separated by the mouse spleen B cell of immunity and adopt hybridoma technology to obtain the hybridoma of high expression macaque IFN-γ monoclonal antibody, MmIFN-γ 2F6, CCTCC NO:C201235, this macaque IFN-γ monoclonal antibody can with macaque IFN-γ specific binding, for detecting the macaque IFN-γ after antigenic stimulation macaque whole blood in supernatant.This macaque IFN-γ monoclonal antibody can with natural macaque IFN-γ specific binding, can 125pg/ml be reached for susceptibility during specific detection macaque IFN-γ, reach and be applied to susceptibility required in diagnosis.

A preparation method for the monoclonal antibody hybridoma cell of macaque IFN-γ, the steps include:

1, the prokaryotic expression plasmid pET-30a-MmIFN-γ of macaque IFN-γ is built:

Asepticly take local macaque whole blood, anticoagulant heparin, get 10mL whole blood, add ConA 60 μ g/mL in 37 DEG C and 5%CO ₂16h is cultivated under environment.Extract the template of peripheral blood leucocyte total serum IgE as RT-PCR of Con A stimulation, according to macaque IFN-γ (MmIFN-γ) the mRNA sequence (GenBank accession No:AY376145.1) that GenBank issues, the sequences Design pair of primers for removing after signal peptide: upstream primer (P1): 5 ' CGC gAATTCtGTTACTGCCAGGACCCATAT-3 ' (underscore is EcoR I restriction enzyme site), downstream primer (P2): 5 ' GCG aAGCTTcTACTGGGATGCTCTTCGACCTCG-3 ' (underscore is Hind III site); One step amplification total length MmIFN-γ mature peptide sequence 438bp.With endonuclease EcoR I and Hind III respectively enzyme cut MmIFN-γ amplified production and carrier pET30a(novagen company), with ligase enzyme, carrier pET30a is entered in the MmIFN-γ gene clone of amplification, through transform competent E. coli BL21 (DE3) (invitrogen company), obtain the plasmid pET-30a-MmIFN-γ expressing macaque IFN-γ albumen.

2, the expression and purification of macaque IFN-γ and mouse immune:

E. coli bl21 (DE3) (invitrogen company) containing pET-30a-MmIFN-γ expression plasmid is seeded in the LB nutrient solution containing kantlex, 37 DEG C of overnight incubation.Get the LB nutrient solution of 50 μ L overnight culture access 5mL containing kantlex, 37 DEG C of more than shaking culture 2h, to logarithmic phase, add IPTG to final concentration 0.8mmol/L, 37 DEG C of inducing culture 3h.Respectively get 1mL bacterium liquid collected by centrifugation bacterium before and after induction and carry out SDS-PAGE electrophoresis detection.

4 DEG C of centrifugal 10min of 8000rpm/min collect and express thalline, and wash thalline once with the PBS of precooling, 4 DEG C, the centrifugal 10min of 8000rpm/min, with the resuspended thalline of the resuspended damping fluid of the albumen of 15mL.After thalline is fully resuspended, ultrasonic wave or the broken thalline (ice bath) of high pressure cracker, 4 DEG C, the centrifugal 20min of 12000g removes insolubles, collects supernatant.Supernatant 2mL nickel chelating His-albumen affinity chromatography resin purification, obtains the macaque IFN-γ albumen of purifying.

Mouse immune: antigen adds Freund's complete adjuvant neck dorsal sc multi-point injection immunity many BALB/C mice, adds Freund's incomplete adjuvant respectively carry out twice booster immunization behind two weeks and surrounding with antigen.After each immune 7 days, cut the blood sampling of tail point, separation of serum, surveys it with indirect ELISA and tires, and gets the high person that tires and does fusion use.

3, hybridoma and monoclonal antibody preparation:

Prepared by hybridoma: get spleen first three day booster immunization, after three days, mouse draws neck to put to death, asepticly gets spleen and grinds, and leaves standstill 3min absorption upper strata enchylema.Myeloma cell is mixed with the quantitative proportion of splenocyte by 1:10, with 50%(v/v) PEG 4000(SIGMA company) mediates fusion, progressively add incomplete nutrient solution dilution PEG, the termination that is used for eliminating PEG is merged.Culture plate fused cell being added to existing feeder layer is cultivated, cell cultures is to when covering 10 ~ 20% hole floorage, draw culture supernatant ELISA and detect anti-body contg, the secretion situation according to antibody filters out high antibody secretory pit, by the row clone again of cell in hole.

Hybridoma cell clone: adopt limiting dilution assay.Choose Positive hybridoma clones to transfer in 24 orifice plate enlarged culturing, and do the screening of cloning again, after double cloning efficiency reaches 100%, cloning cell is proceeded to culturing bottle enlarged culturing.

Prepared by monoclonal antibody (monoclonal antibody): select BALB/c mouse, first carry out mouse peritoneal injection with Freund's incomplete adjuvant, the hybridoma cell clone abdominal injection (1 × 10 will obtained after a week ⁶individual hybridoma) be inoculated in Mice Body, preparation ascites or serum, can produce ascites after 10 days, and when ascites is many as far as possible, put to death mouse, suck in test tube with dropper by ascites, a general mouse can obtain 5 ~ 10mL ascites.

4, monoclonal antibody Purification and Characterization:

Monoclonal antibody purifying: ascites caprylic acid-ammonium is purified, and with as far as possible few and can the PBS (pH7.4) of dissolution precipitation completely resuspended, put into PBS (pH7.4) dialysed overnight in threading dialysis tubing, be the IgG of extraction.-20 DEG C save backup.Concentration, purity and determination of activity is carried out after dialysis desalination.

The Ig class of monoclonal antibody and the qualification of subclass: the monoclonal antibody the most often obtained is IgM and IgG, adopt Clonotyping System-HRP test kit (Southern Biotech) to identify monoclonal antibody Ig class and subclass.Gained monoclonal antibody of the present invention is accredited as IgG2a subclass.

Hybridoma cell strain is obtained by above-mentioned step, this hybridoma cell strain on February 29th, 2012 at Wuhan University's this cell of China typical culture collection center preservation, preserving number CCTCC NO:C201235, Classification And Nomenclature: hybridoma cell strain MmIFN-γ 2F6.

This hybridoma cell strain MmIFN-γ 2F6 can containing 20% foetal calf serum RPMI-1640 substratum in half adherent growth, require that environment is 37 DEG C, 5%CO2, saturated humidity.This cell strain growth form is perfectly round bright, grows vigorous and becomes tufted, and the monoclonal antibody of secretion anti-macaque IFN-γ that can be stable.It is 91 that chromosome counting shows this cell strain karyomit(e), between the chromosome number (70) and the chromosome number (40) of BALB/c mouse splenocyte of myeloma cell SP2/0, shows that this cell strain is hybridoma cell strain really.

The application of macaque IFN-γ Monoclonal Antibody Cell in the ELISA detection method of macaque IFN-γ, and detect the application in macaque tuberculosis infection medicine, the steps include:

Prepared by A, polyclonal antibody (resisting) more: the macaque IFN-γ proteantigen of purifying does immunogen, and immunizing rabbit 3, gets the highest rabbit anteserum of tiring, purify with ammonium sulfate graded precipitation, obtains anti-macaque IFN-γ rabbit and resists more.

B, ELISA set up: adopt carbonation by macaque IFN-γ monoclonal antibody bag by 96 hole enzyme plates, with containing 5%(m/v) the PBST solution 37 DEG C of skim-milk closes 1 hour; Add sample to be detected, 37 DEG C were rinsed 3 times with PBST after 30 minutes; Anti-add enzyme plate by many for 350 times of macaque IFN-γ diluted, 37 DEG C were rinsed 3 times with PBST after 30 minutes; The goat-anti rabbit two adding HRP mark resists, and 37 DEG C were rinsed 4 times with PBST after 30 minutes, adds substrate solution and TMB 10 minutes, in 630nm densitometric value (OD value) after hydrofluoric acid termination reaction.Determine ELISA sensitivity with Endpoint Dilution Method: negative sample is PBST, detection sensitivity with sample value OD630 value for feminine gender is worth the IFN-γ concentration of 2 times.Table 1 is different dilution macaque IFN-γ sample ELISA detected results.

The sensitivity that table 1 detects IFN-γ based on the sandwich ELISA of monoclonal antibody

C, tuberculosis infection are examined then: gather 7 parts from certain test monkey field and be detected as the negative macaque whole blood of negative tuberculosis through TST eye drip, with ox PPD(25 μ g/mL) and each 100 μ L of PBS stimulate 1mL whole blood respectively, the ELISA that sets up with monoclonal antibody of the present invention detects stimulates supernatant.Detected result is in table 2.Negative stimulation mean value (mean) is 0.11, and standard deviation (SD) is 0.02, when threshold value is set to mean+4SD(0.19) time, PPD stimulating group all values is all in detected value+2SD scope.The ELISA sensitivity of now setting up is decided to be 125pg/mL, and corresponding OD value is 0.18.Therefore the present invention can be used in the detection (example 2 is shown in foundation and reliability thereof that monoclonal antibody is detecting the embody rule method in macaque IFN-γ, and table 2 is detected values of the negative macaque sample of 7 parts of tuberculosis infections) of macaque tuberculosis infection.

After table 2 tuberculosis negative macaque whole blood PPD stimulates, IFN-γ detects (representing with OD630 value)

The present invention compared with prior art, has the following advantages and effect:

In susceptibility, IGRA for the detection sensitivity of active tuberculosis higher than TST; In specificity, because IGRA can use the distinctive antigen of mycobacterium tuberculosis complex as stimulator antigen, thus can get rid of nonpathogenic mycobacteria infect and BCG inoculate the false positive produced; IGRA can use different antigen to stimulate simultaneously, can determine the classification of the infection mycobacterium of body more accurately; In clinical manipulation and context of detection, IGRA does not need testing staff to contact body, and result judges objective and accurate, and detected result has very high repeatability; And stimulation fluid can cryopreservation, is conducive to the backtracking of detected result.Compare with external like product, susceptibility of the present invention enough detects the IFN-γ that antigenic stimulation macaque whole blood produces, and low price, easily obtain, be well suited for promotion and application at home.

Accompanying drawing explanation

Fig. 1 is a kind of schema can expressing the hybridoma of macaque IFN-γ monoclonal antibody.

Fig. 2 is the SDS-PAGE electrophoretic analysis figure of the macaque restructuring IFN-γ albumen that a kind of expression plasmid pET-30a-MmIFN-γ expresses.

Before 1:pET-30a/BL21 induction; After 2:pET-30a/BL21 induction; M:protein marker; Before 3:pET-30a-MmIFN-γ/BL21 induces; Full bacterium after 4:pET-30a-MmIFN-γ/BL21 induces; Supernatant after 5:pET-30a-MmIFN-γ/BL21 induces; 6:pET-30a-MmIFN-γ/BL21 induces postprecipitation; 7: MmIFN-γ after purifying.

Fig. 3 is the reading that a kind of ELISA method detects that ConA (100 μ g/mL) stimulates macaque whole blood supernatant, demonstrates this ELISA method and can detect natural macaque IFN-γ.

Table 3 is the readings using Clonotyping System-HRP test kit qualification monoclonal antibody subclass.

Embodiment

Embodiment 1: the foundation of macaque IFN-γ monoclonal antibody.

One, the expression plasmid pET-30a-MmIFN-γ of macaque IFN-γ is built:

1, the acquisition of macaque IFN-γ cDNA and amplification:

Asepticly take local macaque whole blood, anticoagulant heparin, get 10mL whole blood, add Con A 60 μ g/mL in 37 DEG C and 5%CO ₂16h is cultivated under environment.The centrifugal 5min of 3000g, outwells supernatant, adds erythrocyte cracked liquid by the volume of 1:3, and the centrifugal 5min of 5000g, can repeat 2 ~ 3 times, finally washes a white corpuscle with PBS again.

The method provided by RNA-SoIV Reagent extracts Rhesus macaque peripheral blood leukocytes total serum IgE, as the template of RT-PCR.According to macaque IFN-γ (MmIFN-γ) the mRNA sequence (GenBank accession No:AY376145.1) that GenBank issues, the sequences Design pair of primers for removing after signal peptide: upstream primer (P1): 5 '-CGC gAATTCtGTTACTGCCAGGACCCATAT-3 ' (underscore is EcoR I restriction enzyme site), downstream primer (P2): 5 ' GCG aAGCTTcTACTGGGATGCTCTTCGACCTCG-3 ' (underscore is Hind III site); Illustrate according to reversed transcriptive enzyme ReverTra Ace test kit and be RT-PCR, obtain total cDNA.Above downstream primer amplification MmIFN-γ gene.

PCR system:

PCR loop parameter: 95 DEG C of 5min, after [94 DEG C of 30s, 58 DEG C of 30s, 72 DEG C of 45s] 30 circulation, 72 DEG C of 10min, 4 DEG C of preservations.1%(m/v) agarose gel electrophoresis identification reaction product.

2, agarose gel electrophoresis and recovery PCR primer.

By 1% sepharose (containing 10ng/mL EB) electrophoretic separation PCR primer, electrophoresis terminates, gel is placed in uv analyzer, cut the blob of viscose containing PCR primer, reclaim test kit (Beijing Tian Gen company) with sepharose DNA to reclaim, PCR primer is finally dissolved in the Elution Buffer of 35 μ L.

3, restriction enzyme respectively enzyme cut carrier pET30a and MmIFN-γ pcr amplification product

By plasmid pET30a and MmIFN-γ pcr amplification product, with EcoR I and Hind III, (restriction endonuclease is purchased from the precious biotech firm in Dalian respectively, lower same) carry out double digestion, endonuclease reaction, with reference to the operation of restriction enzyme specification sheets, after reaction terminates, is separated digestion products by agarose gel electrophoresis.

4, DNA connection, transformation of E. coli:

In aseptic EP pipe, add the digestion products of 14 μ L MmIFN-γ, the digestion products of 3 μ L plasmid pET30a, 1 μ LT4 ligase enzyme, 2 μ LT4 ligase enzyme damping fluids, add ddH ₂o to 20 μ L, mixing, 16 DEG C of connections are spent the night, connection product is joined 100 μ L competence bacillus coli DH 5 alphas (invitrogen company), mixing, in placing 30min on ice, 42 DEG C of water-bath thermal shock 90s, ice bath is cold immediately hits 2-3min, add 400 μ L LB substratum, 37 DEG C of shaking culture 1h, 45min cultivated by 37 DEG C of 180r/m shaking tables, the centrifugal 5min of 4000r/m discards 300 μ L supernatants, with remaining liquid by resuspended for thalline and to be coated with kalamycin resistance LB dull and stereotyped, just standing on 37 DEG C of incubator 30min to be absorbed to liquid, then be inverted plate and cultivate 12-16h, bacterium colony can be there is.

5, the qualification of positive colony:

From pET-30a-MmIFN-γ/DH5 α flat board that step 4 transforms, picking 6 colony inoculations contain the LB of kantlex to 5ml, and 37 DEG C of shaking culture are spent the night.Extract plasmid with the little extraction reagent kit of ordinary plasmids (Beijing Tian Gen company), reference reagent box specification sheets operates, and obtains plasmid pET30a-MmIFN-γ.With EcoRI and Hind III double digestion plasmid; And be template with plasmid, utilize the PCR amplification system of the MmIFN-γ in step 1 and condition to carry out pcr amplification.

6, determined dna sequence:

Hua Da genome company is sent to check order plasmid pET-30a-MmIFN-γ.The macaque IFN-γ mRNA sequence alignment that sequencing result and GenBank issue, homology is 100%.

7, positive colony transforms expression with e. coli bl21 (DE3) (invitrogen company):

Get positive plasmid 1 μ L by the method for transformation in step 4 and be converted into BL21 (DE3) competent cell, wait to grow single bacterium colony, the single bacterium colony of picking 1 is preserved, this single bacterium colony is the BL21 intestinal bacteria containing expression plasmid pET-30a-MmIFN-γ, can be used for external great expression and produces MmIFN-γ fusion rotein.This bacterial classification for express MmIFN-γ fusion rotein and immune mouse describe as after.

Two, the expression and purification of MmIFN-γ and mouse immune:

1, the abduction delivering of MmIFN-γ:

By the above-mentioned strain inoculation containing pET-30a-MmIFN-γ plasmid in the LB nutrient solution containing kantlex, 37 DEG C of overnight incubation.Get the LB nutrient solution of 50 μ L overnight culture access 5mL containing kantlex, 37 DEG C of more than shaking culture 2h, to logarithmic phase, get 1ml bacterium liquid collected by centrifugation bacterium and carry out SDS-PAGE electrophoresis; Add IPTG to final concentration 0.8mmol/L, 37 DEG C are continued to cultivate 3h.Get 1mL bacterium liquid collected by centrifugation bacterium and carry out SDS-PAGE electrophoresis.

2, the purifying of MmIFN-γ fusion rotein

Ni-NTA His- superfow filler loads gravity purification column, carries out purifying.

Ni-NTA His Bind affinitive layer purification concrete steps are as follows:

(1) inducing culture 1L bacterium liquid, by under bacterium liquid 4 DEG C of conditions of induction, the centrifugal 10min of 10000r/m, abandons supernatant.

(2) the appropriate 1 × Binding Buffer of thalline washes once, more ice-cold 1 × BindingBuffer (1/10 volume) is resuspended and be placed on ice, and it is limpid that ultrasonic disruption or high pressure are crushed to liquid-transparent.

(3) 4 DEG C, the centrifugal 10min of 12000r/m, abandons precipitation, supernatant through 0.45 μm of frit, 4 DEG C of placements.

(4) preparation of Ni-NTA His Bind affinitive layer purification pillar:

A.5 times packing volume sterilizing deionized water, washes away envelope post liquid;

B.5 times packing volume 1 × NiSO ₄, ionization filler;

C.3 times packing volume 1 × Binding Buffer, balance pillar;

(5) affinity chromatography

A. treat that 1 × Binding Buffer drops to filling surface, carefully add broken supernatant to be purified, slowly can add around pillar inwall, in order to avoid the depression that causes of the local assault of filling surface and inclined-plane, and coutroi velocity is 4-5 second 1; If flow velocity may cause hanging column incomplete too soon, loss part target protein;

B.10 times volume 1 × Binding Buffer, tentatively washes foreign protein off;

C.6 times volume 1 × Wash Buffer, wash-out foreign protein;

D.6 times volume 1 × Elute Buffer elution of bound albumen.Also can directly with 1 × Strip Buffer by Ni ²⁺, target protein elutes from post simultaneously.

1 × Binding Buffer and 1 × Elute Buffer can mix by different ratios, to obtain different imidazole concentration, and the method determination wash-out foreign protein that can increase progressively by gradient concentration and the best imidazole concentration of wash-out target protein.Purge process is preferably omnidistance in 4 DEG C of environment, if desired can add proteinase inhibitor, prevent proteolytic degradation.SDS-PAGE analyzes purification effect.

3, determination of protein concentration: with Nanodrop 2000(Thermo company) measure protein concentration.

4, mouse immune: antigen immune BALB/C mice, immune programme for children is as follows:

(1) initial immunity, purifying protein 100 μ g/ props up mouse, adds Freund's complete adjuvant neck dorsal sc multi-point injection, 0.5mL/ mouse.

After (2) two weeks, second time immunity, a dosage 100ug/ mouse, adds Freund's incomplete adjuvant neck dorsal sc multi-point injection.

(3), after surrounding, third time immunity, a dosage 100ug/ mouse, adds Freund's incomplete adjuvant neck dorsal sc multi-point injection.

After (4) 7 days, cut the blood sampling of tail point, separation of serum, survey it with indirect ELISA to tire, namely use antigen MmIFN-γ wrapper sheet, add mice serum dilution product 37 DEG C and hatch, wash plate to add HRP and mark anti-37 DEG C of sheep anti mouse two and hatch, wash plate and add tmb substrate colour developing, get the high person that tires and do to merge and use.

Mouse through MmIFN-γ fusion protein immunization will be used to separating Morr. cell to build monoclonal antibody hybridoma, concrete grammar describe as after.

Three, hybridoma builds and monoclonal antibody preparation:

1, hybridoma fusion: adopt PEG mediates fusion

1. cytogamy material:

(1) preparation of myeloma cell line: SP2/0 myeloma cell line is selected in this research.The cultivation RPMI1640 substratum of myeloma cell, calf serum concentration 20%, cell concn is with 10 ⁴~ 5 × 10 ⁵/ ml.When cell is in the mid-term of logarithmic growth, can go down to posterity in the ratio of 1:3.Within every 3 ~ 5 days, go down to posterity once.Cell, in succeeding generations, regularly processes with 8-anaguanine, makes the cell of existence be homogeneous susceptibility to HAT.

(2) feeder cell: Turnover of Mouse Peritoneal Macrophages and splenocyte, the amount of feeder cell is for being generally 2 × 10 ⁴or 10 ⁵cells/well.

2. the step of cytogamy

(1) feeder layer is prepared: with Turnover of Mouse Peritoneal Macrophages and splenocyte.

BALB/C mice, in 6 ~ 10 week age, draws neck to put to death, is immersed in 75%(volume ratio, down together) in alcohol, 3 ~ 5min, cuts off skin by sterile scissors, exposes peritonaeum, inject the nutrient solution (forbidding to puncture intestinal tube) of 5 ~ 6mL precooling with asepsis injector, repeatedly rinse, sucking-off washing fluid; Get spleen simultaneously, resuspended with basis 1640 after mill grinding, leave standstill 3min, draw upper strata enchylema; Above-mentioned two kinds of enchylema washing fluids are put into 50ml centrifuge tube, and 1200rpm/ is separated 10min and abandons supernatant, uses 20%(volume ratio, lower with) the RPMI1640 nutrient solution suspendible of new-born calf serum (NCS) or foetal calf serum (FBS), adjustment cell count to 1 × 10 ⁵/ mL, adds 96 orifice plates, and 100 μ L/ holes, put into 37 DEG C of CO ₂incubator is cultivated.

(2) immune spleen cell is prepared

Last booster immunization after 3 days mouse draw neck to put to death, asepticly get spleen, nutrient solution is washed once, and spleen grinds, resuspended with basis 1640, leaves standstill 3min, draws upper strata enchylema.Centrifugal, cell nutrient solution washes 2 times, and counting gets 10 ⁸splenic lymphocyte suspension is for subsequent use.

3. cytogamy program:

(1) myeloma cell and splenocyte are mixed in 1:10 ratio, in 50ml centrifuge tube, wash 1 time with the incomplete nutrient solution of serum-free, centrifugal, 1200rpm, 8min; Abandon supernatant, thieving paper blots residual liquid.Gently at the bottom of attack centrifuge tube, cell precipitation is slightly loosened.

The 1mL 50%(volume ratio of 37 DEG C of pre-temperature is added in (2) 90 seconds, lower same) PEG(molecular weight 4000) solution, limit edged gentle agitation.37 DEG C of water-bath effects 90 seconds.

(3) the incomplete nutrient solution adding 37 DEG C of pre-temperature, to stop PEG effect, adds 1mL, 2mL, 3mL, 4mL, 5mL and 6mL respectively every 2min.

(4) centrifugal, 1000rpm, 10min.

(5) fill with clearly, select nutrient solution resuspended with containing 20% calf serum HAT.

(6) by above-mentioned cell, be added in 96 orifice plates of existing feeder layer, every hole adds 100 μ L.A general immune spleen can inoculate 4 piece of 96 orifice plate.

(7) culture plate is put 37 DEG C, 5%CO ₂cultivate in incubator, every hole can obtain the positive hybridoma cell system that one or more express MmIFN-gamma antibodies.

2, the detection of antibody:

Adopt indirect elisa method:

1) MmIFN-γ is diluted to 0.2 μ g/mL at the carbonate buffer solution of PH9.6, and every hole 100 μ L wraps by 96 orifice plates.

2) wash plate to close, add cells and supernatant 100 μ L to be recorded.Hatch, wash plate for 37 DEG C.

3) add HRP mark sheep anti mouse two to resist, hatch for 37 degree and wash plate.

4) wash plate to add the high hole of the positive colour developing of TMB colour developing choosing and do and clone again.

3, the cloning of hybridoma:

Hybridoma cloning generally refers to carries out cloning by antibody positive wells.Hybridoma clone after HAT screening can not ensure to only have a clone in a hole.These cells are separated from each other and just need cloning.The principle of cloning is, carries out cloning as early as possible for the hybridizing clones detecting antibody positive; Even if the hybridoma of cloning also needs regular cloning again, to prevent sudden change or the chromosome elimination of hybridoma, thus lose the ability producing antibody.

Employing limiting dilution assay is cloned

(1) preparation feeder layer (same to cytogamy) in first 1 day is cloned.

(2) hybridoma will cloned dries up gently in culture hole, counting.

(3) adjusting cell is 3 ~ 10 cell/mL.

(4) get the Tissue Culture Plate of the feeder layer that the day before yesterday prepares, every hole adds diluting cells 100 μ L.Hatch in 37 DEG C, 5%CO ₂in incubator.

(5) change liquid at the 7th day, within later every 2 ~ 3 days, change liquid 1 time.

(6) 8 ~ 9 days visible cell Clone formation, detect antibody activity in time.

(7) cell in positive hole is moved to enlarged culturing in 24 orifice plates.

(8) forward to culturing bottle by cell from 24 holes, each clone should be frozen as early as possible.

(9) double cloning efficiency obtains 100% expression MmIFN-γ protein positive hybridoma.Hybridoma cell strain on February 29th, 2012 at Wuhan University's this cell of China typical culture collection center preservation, preserving number CCTCC NO:C201235, Classification And Nomenclature: hybridoma cell strain MmIFN-γ 2F6.

4, the frozen and recovery of hybridoma:

(1) hybridoma is frozen

(2012/2/29 at Wuhan University's this cell of China typical culture collection center preservation, preserving number CCTCC NO:C201235)

The subcloned cells that each cloning of hybridoma of timely frozen archioporus obtains is very important.The cryopreservation methods of hybridoma is the same with the cryopreservation methods of other clones, and cell should often prop up ampoule containing 1 × 10 in principle ⁶above, but can change because culture environment is different the hybridoma of archioporus.

Cells frozen storing liquid (volume ratio): 90% calf serum; 10%DMSO(dimethyl sulfoxide (DMSO)).

The best precooling of frozen storing liquid, operational motion is soft, rapid.After room temperature can be down to 0 DEG C immediately, put into-70 DEG C of Ultralow Temperature Freezers time frozen, next day proceeds in liquid nitrogen.Also available cells cryopreservation device carries out frozen.Freeze-stored cell will regularly be recovered, and checks the activity of cell and the stability of secretory antibody, and in liquid nitrogen, cell can preserve several years or longer time.

(2) cell recovery method

Glass ampoule is carefully taken out in liquid nitrogen, puts in 37 DEG C of water-baths, in 1min, make frozen cell thawing, cell complete culture solution is washed twice, then move in the culturing bottle of the feeder layer cells prepared the day before yesterday, put 37 DEG C, 5%CO ₂cultivate in incubator, when cell forms colony, detect antibody activity.

5, the expansion preparation of monoclonal antibody:

Inoculation hybridoma, preparation ascites.

The preparation of ascites: first abdominal injection 0.5mL Freund's incomplete adjuvant is in BALB/C mouse, and pneumoretroperitoneum injected 1 × 10 in 1 week ⁶individual hybridoma, inoculating cell can produce ascites after 10 days, the healthy state of close observation animal and ascites sign, treat that ascites is many as far as possible, and mouse is frequency domain before death, puts to death mouse, suck in test tube with dropper by ascites, a general mouse can obtain 5 ~ 10mL ascites.Also usable syringes extracting ascites, can collect for several times repeatedly.Monoclonal Antibodies in Mice Ascites content can reach 5 ~ 10mg/mL, and this is method the most frequently used at present, also cell cryopreservation in ascites can be got up, and after recovery, transferred species mouse peritoneal then produces that ascites is fast, amount is many.The making of typical hybridization oncocyte, cultivate and morphological specificity as follows:

Hybridoma is in preparation monoclonal antibody process, with the cell of myeloma cell and effect B cytogamy.The ultimate principle of hybridoma antibody technology is the principal character simultaneously keeping both by merging two kinds of cells.These two kinds of cells are make murine myeloma cell through the mouse cell of antigen immune respectively.The principal character of splenic lymphocyte is its antibody-secreting function and can grows in Selective agar medium, and murine myeloma cell then can divide without limit, propagation, i.e. so-called immortality under culture conditions.Under the effect of Selective agar medium, the hybridization of B cell and myeloma cell fusion is only had just to have the ability of continuous proliferation, form the cell clone possessing antibody-secreting function simultaneously and keep cell immortality two kinds of features, this hybrid cell is called hybridoma.

(1) oncocyte cultivates (without particular requirement)

Myeloma cell, in suspending or the growth of slight attached form, when the cell as tack growth is many, to blow and beat with suction pipe or knocking culture vessel can separate gently gently.Usually will maintain cell in exponential phase of growth (the cell cultures time is 15 ~ 20 hours), within every 3 ~ 5 days, get cell 0.2 ~ 1mL and be moved in the new nutrient solution of 10mL, its maximum cell density can not more than 5 × 10 ⁵~ 10 ⁶/ ml.

(2) preparation of immune mouse and splenocyte

Immunity: get the female mouse of Balb/C in 6 ~ 8 weeks age, fundamental immunity 2 weeks, vein again booster immunization once, after 3 ~ 5 days for fusion.

Prepared by splenocyte:

1. crane one and put to death mouse, disinfect table body in alcohol.

2. take out spleen under aseptic condition, shave except reticular tissue and fat, put into mill, add 3mL serum-free medium.

3. soft spleen of milling, adds 7mL serum-free medium, leaves standstill 3min.

4. draw 7mL supernatant in 50mL centrifuge tube, add 7mL serum-free medium, leave standstill 3min.

5. repeating step 4, until be finished 50mL serum free medium.

6. centrifugal (1000 ~ 1200 revs/min) counting, for subsequent use.

3) preparation of cell is raised: the abdominal cavity cell of conventional mouse boosting cell or mouse

Peritoneal Cells of Mice preparation method:

1. crane one and put to death mouse, disinfect table body in alcohol.

2. the stripping of hara kiri skin is to both sides, exposes stomach wall.

3., with aseptic No. 7 needle applicators, draw 3mL serum-free medium.

4. pick up stomach wall with little tweezer, inject 3mL serum-free medium, repeatedly rub stomach wall gently with hand, more repeatedly aspirate 3-5 time.

5. collect the cell inhaled of last, inject the centrifuge tube of 50mL, then add 20mL serum-free medium, with suction pipe rinsing once.

6. centrifugal, remove supernatant, regulate cell concn to be 2 × 10 with containing blood serum medium ⁵/ ml, inoculates in culture plate, and the 24 every holes of orifice plate add 0.1mL.

4) cytogamy

The preparation of HAT substratum: 50 × HAT salt (SIGMA company) adds in proportion containing in 20% serum free culture system liquid after adding the dissolving of 10mL serum-free medium.

The preparation of HT substratum: 50 × HT salt (SIGMA company) adds in proportion containing in 20% serum free culture system liquid after adding the dissolving of 10mL serum-free medium.

Merge and use PEG4000(50% volume ratio) purchased from SIGMA company.

Cell prepares:

1. collect myeloma cell, wash 3 times (37 DEG C) with serum-free medium, counting vigor cell (being no less than 90%).

2. collect mouse boosting cell, wash 3 times (37 DEG C) with serum-free medium, and count cell count and measure vigor cell.

3. after pressing 1:5 or 1:10 mixing splenocyte and myeloma cell, centrifugal supernatant discarded, and to exhaust unnecessary supernatant with sterilized filter paper.

Merge:

Method one:

1. join in cell mass by one after another drop of for the PEG liquid of 1mL50%, added in 60 seconds, simultaneously and constantly fine rotation centrifuge tube or flick centrifuge tube with finger.

2. add 1mL serum-free medium continuous rotation in centrifuge tube, add in 60 seconds.

3. in 5 minutes, slowly add 20mL serum free medium.Now cell is very responsive to physical abuse.

4. centrifugal (1000 revs/min, 8 minutes), remove supernatant, suspend with complete culture solution 10mL, mix gently.

5. get 96 orifice plates, every hole adds 50 μ L.

6. get equal-volume cell suspension, in another 96 orifice plate, add 50 μ L.

7. send into 5%CO ₂in incubator, 37 DEG C of cultivations, are replaced with HAT selectivity nutrient solution after 24 hours.

Method two:

1. add the PEG of 0.2ml 50% in centrifuge tube.

2. stretch in pipe with the thin glass needle of sterilizing in advance and stir cell mass gently.

3. mid-2 minutes of room temperature.

4. add 10mL complete culture solution.

5.800 revs/min centrifugal 5 minutes.

6. cultivate suspension cell with 2 times of HAT nutrient solutions, get 24 orifice plates, in every hole, add 0.5mL, in the another 96 every holes of orifice plate, add 0.1mL.

7.5%CO ₂incubator, 37 DEG C, first time changes nutrient solution after one week, and in culture plate, inoculation has feeder cell in advance, adds after 2 × HAT selects nutrient solution to mix with former equivalent nutrient solution, namely become 1 × HAT substratum.

Cell cultures after merging

1. within 7 ~ 10 days, change liquid with HAT nutrient solution variable after merging, change liquid once every 2 ~ 3 days half amounts later.

2. a liang Zhou Houke uses HT nutrient solution half amount instead and changes liquid, or still uses HAT nutrient solution.

Occur hybrid cell colony after 3.2 ~ 3 weeks, cell is large, circle and transparent.

4., when colony proliferation grows to 1/3 hole, antibody test should be carried out.

Colonized culture

[soft agar cultivation]

1. claim 1g agar powder, join in 100mL distilled water, autoclaving, 4 DEG C of Refrigerator stores.

2. before using, the agar of 1% and 2 × the respectively temperature bath in 45 DEG C of water-baths of DMEM nutrient solution.

3., after equal-volume mixing both, add 1.5 × 10 immediately ⁷mouse boosting cell, mixing.

4. immediately to entering in plate, every plate (diameter 10cm) adds 10mL, mid-15 minutes of room temperature.

5. separately get hybridoma suspension and 0.5% agar equal-volume mixing (0.25% agar), add 2mL in the bottle ware being covered with bottom-layer agar in advance.

6.CO ₂incubator is cultivated.

After 7.10 ~ 15 days, there is cell colony to be formed, move into enlarged culturing in 24 well culture plates in good time.As with agarose, bottom concentration is 0.6%, and upper strata is 0.3% agar.

[limiting dilution assay]

1. preparation has the Tissue Culture Plate of feeder cell bottom.

2. collecting cell, count positive culturing cell.

3. cell suspension is adjusted to 5 ~ 10 cell/mL.

In 4.96 orifice plates, every hole adds 0.1mL.

5.CO ₂after incubator cultivates one week, half amount changes liquid, continues to cultivate.

6. detect every hole culture supernatant, the cell selecting the culture hole that is positive proceeds limiting dilution, till be sure oing that in hole, cell is mono-clonal in good time.

7. carry out enlarged culturing.

Four, monoclonal antibody Purification and Characterization:

1, monoclonal antibody purifying: adopt the IgG in caprylic acid-ammonium purified mouse ascites.

1) by centrifugal for ascites 12000r/m 10min, to remove the impurity such as lipid.

2) acetate buffer solution (pH4.5) of the 0.06mol/L adding 4mL in purifying ascites is wanted to 1mL.

3), under condition of ice bath, every mL ascites adds sad 33 μ L, and limit edged slowly stirs.

4), under condition of ice bath, continue to stir 30min.

5) the centrifugal 30min of 12000r/m, gets supernatant liquor after filter paper filtering, regulates pH to 7.4.

6) slowly add the saturated ammonium sulphate of pH7.4 under condition of ice bath, ammonium sulfate saturation concentration is no more than 45%, stirs 30min, leaves standstill 2-4h or 4 DEG C and spends the night.

7) 4 DEG C of centrifugal 30min of 12000r/m, collecting precipitation thing, and with the PBS(pH7.4 of proper volume) resuspended, put in dialysis tubing and put into PBS(pH7.4) dialysed overnight, be the IgG of extraction.

2, the Ig class of monoclonal antibody and the qualification of subclass

The monoclonal antibody the most often obtained is IgM and IgG, and the hybridoma of secretion IgE is rarely found.The method of this research qualification monoclonal antibody Ig class and subclass uses Clonotyping System-HRP test kit (SouthernBiotech).

1) MmIFN-γ is diluted to 0.2ug/mL at the carbonate buffer solution of PH9.6, and every hole 100ul wraps by 96 orifice plates.

Wash plate to close.

2) the monoclonal antibody to be measured 100 μ L of dilution is added.Hatch, wash plate for 37 DEG C.

3) HRP added in Clonotyping System-HRP test kit marks sheep anti mouse IgM, IgA, IgG1, IgG2a, IgG2b, IgG3 subclass two and resists, and hatches for 37 DEG C and washes plate.

4) wash plate and add TMB colour developing, the HRP that the hole of positive colour developing is used marks the anti-subclass being monoclonal antibody of sheep anti mouse subclass two.

This monoclonal antibody is accredited as IgG class IgG2a subclass.(see table 3)

Table 3 is the readings using Clonotyping System-HRP test kit qualification monoclonal antibody subclass.

HRP marks the anti-subclass of sheep anti mouse two

IgM

IgA

IgG1

IgG2a

IgG2b

IgG3

OD630

0．09

0.08

0.07

0.84

0.10

0.09

The foundation of embodiment 2, double antibody enzyme-linked immunosorbent assays (ELISA) method

An application in the ELISA detection method of macaque IFN-γ, the steps include:

1, the purifying that how anti-MmIFN-γ is:

Do immunogen with the purifying MmIFN-γ antigen obtained, immunizing rabbit 5, gets the highest rabbit anteserum of tiring, and carries out purifying by saturated ammonium sulphate method.

1) by Culling heart blood, collect hyper-immune serum, place 1h in 37 DEG C, deactivation complement.Spend the night in 4 DEG C, allow serum naturally separate out.

2) by sample to be extracted through 4 DEG C or the centrifugal 13000r/min 30min of room temperature, collect supernatant.

3) get honest and upright and thrifty 20mL to mix with isopyknic physiological saline, under agitation slowly drip 40mL saturated ammonium sulphate and act on 3h in 4 DEG C or spend the night, albumen is fully precipitated.

4) the centrifugal 10min of centrifugal 30min or 13000r/min of 3000r/min, 4 DEG C are centrifugal, abandon supernatant, by 12mL physiological saline solution precipitation, with step 2), drip 8mL saturated ammonium sulphate, 4 DEG C of reaction 1h.

5) repeating step 4) 1 time, centrifugal, by 13.3mL physiological saline solution precipitation, drip 6.6mL saturated ammonium sulphate, 4 DEG C of reaction 1h.

6) repeating step 4) 1 time, the centrifuged pellet thing PBS(pH 7.4 of a little 0.01mol/L) dissolve.

7) lysate super filter tube carry out surpassing from, desalination, concentrated, glycerol adding-20 DEG C saves backup.

2, double antibodies sandwich enzyme linked immunosorbent assay (ELISA) method detects MmIFN-γ

1) the carbonate buffer solution dilution Sheet of 0.05mol/L pH9.6 resists to wrapping by concentration 2.5 μ g/mL, adds enzyme plate 100 μ L/ hole, puts 4 DEG C and spend the night, dry;

2) containing PBST (0.02mol/LpH7.4PBS containing 0.05%(volume ratio, the lower with) Tween-20 of 5% skim-milk) close 200 μ L/ holes, 37 DEG C 1 hour, dry;

3) wash plate 3 times with PBST, add prokaryotic expression MmIFN-γ respectively, concentration is followed successively by each 100 μ L/ holes of sample to be tested of 62.5,125,250,500,1000pg/mL; Other hole adds the macaque whole blood supernatant 100 μ L/ hole that Con A (100 μ g/mL) stimulates, and establishes negative control hole, hatches 30 minutes for 37 DEG C;

4) rinse 3 times with PBST, dry; Add enzyme plate (100 μ L/ hole) by how after anti-350 times of dilutions, 37 DEG C 30 minutes;

5) rinse 3 times with PBST, dry; Add anti-(Southern Biotech) each 100 μ L/ holes of goat-anti rabbit two of the commercial HRP mark of 20000 times of dilutions, 37 DEG C 30 minutes;

6) rinse 4 times with PBST, dry; Add substrate solution and TMB (each 50 μ L/ holes), 37 DEG C 10 minutes

7) add 0.25mol/L hydrofluoric acid 50 μ L/ hole termination reaction, survey absorbancy in 630nm.Determine ELISA sensitivity with Endpoint Dilution Method: negative sample is PBST, detection sensitivity with sample value OD630 value for feminine gender is worth the IFN-γ concentration of 2 times.

The susceptibility that this result shows this ELISA method is that 125pg/mL(is in table 1).This ELISA also can detect the natural IFN-γ (see figure 3) in the macaque whole blood supernatant that ConA (100 μ g/mL) stimulates simultaneously.

Macaque IFN-γ Monoclonal Antibody Cell is detecting the application in macaque tubercular drugs, and its operation steps is as follows:

3, double antibodies sandwich enzyme linked immunosorbent assay (ELISA) method detects the whole blood supernatant of different antigenic stimulation.

1) aseptic collection macaque whole blood (at least 2ml), heparin sodium anti-freezing; Every part of blood sample is added in 24 orifice plates, every hole 1mL, point holes; Add 100 μ L ox PPD(final concentration 25 μ g/mL respectively) and 100 μ L PBS, mixing, in 37 DEG C, 5%CO ₂, saturated humidity hatches 16 hours; Centrifugal absorption supernatant;

2) the carbonate buffer solution dilution Sheet of 0.05mol/L pH9.6 resists to wrapping by concentration 2.5 μ g/mL, adds enzyme plate 100 μ L/ hole, puts 4 DEG C and spend the night, dry;

3) containing PBST (0.02mol/LpH7.4PBS containing 0.05%(volume ratio, the lower with) Tween-20 of 5% skim-milk) close 200 μ L/ holes, 37 DEG C 1 hour, dry;

4) wash plate 3 times with PBST, adding whole blood respectively stimulates each 100 μ L/ holes of supernatant, hatches 30 minutes for 37 DEG C;

5) rinse 3 times with PBST, dry; Add enzyme plate (100 μ L/ hole) by how after anti-350 times of dilutions, 37 DEG C 30 minutes;

6) rinse 3 times with PBST, dry; Add anti-(Southern Biotech) each 100 μ L/ holes of goat-anti rabbit two of the commercial HRP mark of 20000 times of dilutions, 37 DEG C 30 minutes;

7) rinse 4 times with PBST, dry; Add substrate solution and TMB (each 50 μ L/ holes), 37 DEG C 10 minutes

Add 0.25mol/L hydrofluoric acid 50 μ L/ hole termination reaction, survey absorbancy in 630nm.Detected result is in table 2.Negative stimulation mean value (mean) is 0.11, and standard deviation (SD) is 0.02, when threshold value is set to mean+4SD(0.19) time, PPD stimulating group all values is all in detected value+2SD scope.The ELISA sensitivity of now setting up is decided to be 125pg/mL, and corresponding OD value is 0.18.Therefore the present invention can be used in the detection of macaque tuberculosis infection.

Claims (3)

1. a hybridoma for the monoclonal antibody of macaque IFN-γ, is characterized in that: hybridoma cell strain MmIFN-γ 2F6, CCTCC NO:C201235.

2. the application of the hybridoma of the monoclonal antibody of a kind of macaque IFN-γ according to claim 1 in the ELISA detection kit of preparation macaque IFN-γ.

3. the hybridoma of the monoclonal antibody of a kind of macaque IFN-γ according to claim 1 detects the application in macaque tubercular drugs in preparation.