CN107271685A - A kind of method and its dedicated kit for detecting the content of fowl leukocyte interleukin 2 - Google Patents
A kind of method and its dedicated kit for detecting the content of fowl leukocyte interleukin 2 Download PDFInfo
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- CN107271685A CN107271685A CN201710522323.0A CN201710522323A CN107271685A CN 107271685 A CN107271685 A CN 107271685A CN 201710522323 A CN201710522323 A CN 201710522323A CN 107271685 A CN107271685 A CN 107271685A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6863—Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors
- G01N33/6869—Interleukin
Abstract
The invention discloses a kind of method and its dedicated kit for detecting the content of fowl leukocyte interleukin 2.The present invention establishes the double sandwich-ELISA detection method for chIL 2 on protein level using the monoclonal antibodies of chIL 2.It is experimentally confirmed：The monoclonal antibodies of anti-chIL 2 of the present invention have good specificity and affinity, the content of chIL 2 in serum or cell conditioned medium can be reflected exactly, and the detection method of the present invention is quick, it is efficiently and accurate, solve in the prior art using fluorescent quantitative PCR detection method bring it is cumbersome, take time and effort, easily by operating and the problems such as other external conditions are influenceed, not only facilitate the dynamic change level for evaluating chIL 2 in body, prevention and improvement for disease provide reference well, and help to understand the generation of birds communicable disease, development, lapse to the dynamic of situation and body protective immune response.
Description
Technical field
The invention belongs to biological technical field, and in particular to the method for (IL-2) content of one kind detection fowl leukocyte interleukin 2
And its dedicated kit.
Background technology
Interleukin 2 (IL-2) is cell factor necessary to immune response, is grown and differentiation, B cell hair in T cell
Educate and NK cell-stimulatings aspect plays an important role.But because cytokine content is low, had difficulties always in context of detection.
With the popularization of monoclonal antibody technique, the immunoassay technology set up using monoclonal antibody technique very can be detected promptly
To cell factor, the flow of research of cell factor has been greatly accelerated.
Monoclonal antibody technique is to cultivate a large amount of infinite multiplications of energy in vitro using myeloma cell but can not secrete special
Property antibody, and the bone-marrow-derived lymphocyte of antigen immune can produce specific antibody but be unable to the characteristic of infinite multiplication in vitro, will be immune
Splenocyte forms hybridoma after being merged with myeloma cell, makes it have the characteristic that myeloma cell can unrestrictedly breed,
Again there are immunized B cells to synthesize the ability with secreting specificity antibody.The selection of chosen culture medium, the hybridoma only merged
Cell can survive and breed, again by the screening of three subclones and indirect ELISA method, and final obtain can secrete specificity
The hybridoma of monoclonal antibody, through being cultivated in mouse peritoneal, you can unrestrictedly a large amount of to obtain monoclonal antibody.
EUSA (ELISA) is that soluble antigen or antibody are adsorbed on solid phase carrier, utilizes enzyme mark
Antibody or antigen binding form the immune complex of enzyme mark, add corresponding chromogenic enzyme substrate, judge anti-according to reaction solution color
Antigen-antibody reaction situation, this method is the experiment skill for combining antigen-antibody reaction specificity with the high efficiency of enzymatic reaction
Art, available for the haptens, antigen and antibody qualitatively or quantitatively detected in liquid, is widely used in research and production.
No matter all also without the kit of chicken IL-2 gene albumen can be detected on domestic at present or overseas market, but it is similar
It can detect that the kit of IL-2 albumen in people or mice serum or cell conditioned medium has been widely used.In addition with rat,
The IL-2 detection kits of the animals such as monkey have also been applied.The research for IL-2 focuses primarily upon mammal especially at present
Human IL-2, and the research to chicken IL-2 gene is also extremely limited, chicken IL-2 gene has similar biological activity to mammal IL-2.But because
There is species specificity for IL-2, chicken IL-2 gene differs larger with mammal IL-2 homologys, no cross reaction, therefore at present very
The experimental techniques that can be used for mammal and the research that chicken IL-2 gene is cannot be used for from the detection kit of commercialization on the market, pole more
The big further investigation limited to chicken IL-2 gene.
The experimental technique that the change of detection recombinant chIL-2 (IL-2) is mainly applied in the prior art is fluorescent quantitation
PCR, fluorogenic quantitative detection be mRNA level in-site change, but because mRNA level in-site and protein level are in absolute linear pass
System, so the real standard of chicken IL-2 gene albumen can not be reflected using quantitative fluorescent PCR, therefore it can not be used as a detection
The experimental technique of chicken IL-2 gene protein content and apply, while fluorescent quantitative PCR technique is cumbersome, professional skill is required it is higher and
It is expensive, it should not be used in produce reality.
The content of the invention
First purpose of the present invention is to provide anti-chIL-2 monoclonal antibody.
The monoclonal antibody for the anti-chIL-2 that the present invention is provided is thin for CGMCC No.13832 hybridoma by preserving number
What born of the same parents strain FM-1 and preserving number were secreted for CGMCC No.13833 hybridoma cell strain FM-2.It is CGMCC by preserving number
The anti-chIL-2 of No.13832 hybridoma cell strain FM-1 secretions monoclonal antibody is named as FM-1 antibody, is by preserving number
The anti-chIL-2 of CGMCC No.13833 hybridoma cell strain FM-2 secretions monoclonal antibody is named as FM-2 antibody.
Second object of the present invention is to provide the hybridoma cell strain for the monoclonal antibody for secreting anti-chIL-2.
The hybridoma cell strain of the monoclonal antibody for the anti-chIL-2 of secretion that the present invention is provided is FM-1 and FM-2.Hybridoma
Cell line FM-1 Classification And Nomenclature is hybridoma cell line (mouse source), and the hybridoma cell strain is on April 24th, 2017
It is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (abbreviation CGMCC, address：Chaoyang District, Beijing City
The institute 3 of North Star West Road 1, Institute of Microorganism, Academia Sinica, postcode 100101), preserving number is CGMCC No.13832.
Hybridoma cell strain FM-2 Classification And Nomenclature be hybridoma cell line (mouse source), the hybridoma cell strain in
On April 24th, 2017 is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (abbreviation CGMCC, address：
Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Institute of Microorganism, Academia Sinica, postcode 100101), preserving number is CGMCC
No.13833。
Third object of the present invention be to provide it is a kind of detect or auxiliary detection testing sample in chIL-2 contents it is enzyme-linked
Immune reagent kit.
It is upper that the detection or auxiliary that the present invention is provided detect that the enzyme linked immunological kit of chIL-2 contents in testing sample includes
State FM-1 antibody and/or FM-2 antibody.
Above-mentioned enzyme linked immunological kit also includes chIL-2 standard items；The chIL-2 standard items are by by nucleotides sequence
The chIL-2 genes as shown in sequence 1 in sequence table are arranged to carry out in prokaryotic expressing acquisition.
Also include in the enzyme linked immunological kit at least one of following：ELISA Plate, coating buffer solution, washing buffer
Liquid, antibody diluent, confining liquid, the Streptavidin (Streptavidin of HRP marks) of horseradish peroxidase-labeled, colour developing
Liquid and terminate liquid.
Fourth object of the present invention be to provide above-mentioned anti-chIL-2 monoclonal antibody or above-mentioned hybridoma cell strain or
The new application of above-mentioned enzyme linked immunological kit.
The invention provides above-mentioned anti-chIL-2 monoclonal antibody or above-mentioned hybridoma cell strain or above-mentioned enzyme linked immunological
Application of the kit in chIL-2 contents in detecting or aiding in detection testing sample.
Above-mentioned anti-chIL-2 monoclonal antibody or above-mentioned hybridoma cell strain is preparing detection or is aiding in detection to treat test sample
Application in product in the reagent or colloidal gold strip or kit of chIL-2 contents falls within protection scope of the present invention.
The 5th purpose of the present invention is to provide a kind of method for detecting or aiding in chIL-2 contents in detection testing sample.
The detection or auxiliary that the present invention is provided detect that the method for chIL-2 contents in testing sample is using FM-1 antibody as bag
By the double-antibody sandwich elisa detection method of antibody, biotinylated FM-2 antibody for detection antibody.
The above method is detected or auxiliary detects that the method for chIL-2 contents in testing sample specifically includes following steps：
(1) by FM-1 antibody coating to ELISA Plate, wash；
(2) ELISA Plate that closing (1) is obtained, washing；
(3) chIL-2 standard items or testing sample are added in the ELISA Plate obtained to (2), be incubated, washing；
(4) biotinylated FM-2 antibody is added in the ELISA Plate obtained to (3), is incubated, washing；
(5) Streptavidin of HRP marks is added in the ELISA Plate obtained to (4), is incubated, washing；
(6) substrate colour developing is added in the ELISA Plate obtained to (5), terminate liquid terminating reaction is added after colour developing；
(7) with ELIASA detect add chIL-2 standard items each hole absorbance, using chIL-2 standard concentrations as
Abscissa, the drafting standard curve by ordinate of the reading of ELIASA；
(8) absorbance in the hole for adding testing sample is detected with ELIASA, absorbance is substituted into the standard curve,
Produce the concentration of chIL-2 in testing sample.
In the above method,
In the step (1), it is coated with after with carbonate buffer solution, FM-1 antibody is diluted on ELISA Plate；
In the step (2), the ELISA Plate that (1) is obtained is closed with defatted milk solution；
In the step (3), added after with BSA solution, chIL-2 standard items are diluted in ELISA Plate；
In the step (4), ELISA Plate is added after biotinylated FM-2 antibody is diluted into 1 μ g/mL with BSA solution
In.The preparation method reference antibody biotin labeling reagent box of the biotinylated FM-2 antibody (is purchased from Thermo
Scientific, Prod#21440) in method in specification.
In the above method,
In the step (1), the concentration dilution of the antibody is 5 μ g/mL；The coated condition is 4 DEG C of coating 12h；
The concentration of the carbonate buffer solution is 0.05M, and pH is 9.6；
In the step (2), the mass fraction of the defatted milk solution (solvent is lavation buffer solution) is 5%；The envelope
The condition closed is 4 DEG C of closing 12h；
The mass fraction of the BSA solution (solvent is lavation buffer solution) is 1%；
In the step (3), the incubation time is 1h；
In the step (4), the incubation time is 2h；
In the step (5), the incubation time is 15min；
Wavelength during the detection absorbance with ELIASA is 450nm；
The number of times of the washing is 3 times；
The substrate is TMB；The terminate liquid is 2M H2SO4。
The present invention sets up double-antibody sandwich elisa on the basis of enzyme linked immunological with reference to biotin-Streptavidin system,
This method utilizes the high-affinity and high specific knot between the Streptavidin for biotin and the HRP mark that marked antibody
Close, substantial amounts of enzyme molecule is gathered in around antigen antibody complex, produce multistage amplification, the enzyme for carrying each antibody
Molecule is dramatically increased, so as to be greatly enhanced sensitivity.
Compared with quantitative fluorescent PCR, the double-antibody sandwich elisa detection method that the present invention is set up has the advantage that：(1)
Sample treatment is simple, it is not necessary to takes the tissue of animal to carry out RNA extraction and reverse transcription, only takes the blood of a small amount of animal, point
Carried out from serum after suitably diluting, you can detected；(2) simple, quick, operating method is simple, and whole detection process is only
Need 3-4 hour；(3) accuracy is high, is detected directly on protein level for chIL-2, is difficult to be operated or other
External condition influences；(4) cost is low, not high to instrument requirements.
The present invention establishes the double sandwich-ELISA detection for IL-2 on protein level using fowl IL-2 monoclonal antibodies
Method, can be used for IL-2 contents in detection serum and cell conditioned medium.It is experimentally confirmed：The fowl IL-2 monoclonals of the present invention resist
Body has good specificity and affinity, and the content of fowl IL-2 in serum or cell conditioned medium, and this can be reflected exactly
The detection method of invention is quick, efficient and accurate, solve in the prior art using PCR detection method bring it is cumbersome,
Take time and effort, easily by operating and the problems such as other external conditions are influenceed, not only facilitate the dynamic change for evaluating IL-2 in body
Change level, prevention and improvement for disease provide reference well, and help to understand the generation of birds communicable disease, hair
Open up, lapse to the dynamic of situation and body protective immune response.
Brief description of the drawings
Fig. 1 is the expression that SDS-PAGE analyzes recombinant protein.Figure 1A is His-chIL-2 SDS-PAGE testing results, its
In, M is protein standard, and 1 is His-chIL-2 protein liquids to be purified, and 2 is penetrate liquid, and 3 be cleaning solution, and 4 be after purification
His-chIL-2；Figure 1B is GST-chIL-2 SDS-PAGE testing results, wherein, M is protein standard, and 1 is pGEX-6P-1 empty
Before carrier induction, 2 be that after pGEX-6P-1 empty carriers are induced, 3 be that before recombinant vector chIL-2-pGEX-6P is induced, 4 be restructuring load
After body chIL-2-pGEX-6P inductions.
Fig. 2 is the expression that Western blotting verify recombinant protein.Fig. 2A is His-chIL-2 Western
Blotting testing results, wherein, 1 is that after pGEX-6P-1 empty carriers are induced, 2 be the His-chIL-2 after induction；Fig. 2 B are
GST-chIL-2 Western blotting testing results, wherein, 1 is that Escherichia coli are split after pGEX-6P-1 empty carriers are induced
Liquid is solved, 2 be E. coli lysate after chIL-2-pGEX-6P inductions.
Fig. 3 is monoclonal antibody specific detection.
Fig. 4 is fowl IL-2 protein truncation schematic diagrames.
Fig. 5 is that Western Blot verify antibody identification region.
Fig. 6 is that monoclonal antibody recognizes epitope schematic diagram.
Fig. 7 is the foundation of standard curve.
Embodiment
Experimental method used in following embodiments is conventional method unless otherwise specified.
Material, reagent used etc., unless otherwise specified, are commercially obtained in following embodiments.
The formula of solution used in following embodiments is as follows：
1st, coating buffer solution is 0.05M phosphate buffers (pH6.0), is to mix A liquid 438.5mL and B liquid 61.5mL
Obtained solution, the formula of wherein A liquid and B liquid is as follows：
2nd, the formula of lavation buffer solution (PBST, pH7.4) is as follows：
| Reagent | Consumption |
| KH2PO4 | 0.2g |
| Na2HPO4·12H2O | 2.9g |
| NaCl | 8.0g |
| KCl | 0.2g |
| Tween-20 | 0.5mL |
| Distilled water | 1000mL |
3rd, the formula of confining liquid (5% defatted milk) is as follows：
| Reagent | Consumption |
| Defatted milk | 5g |
| Lavation buffer solution | 100mL |
4th, the formula of sample and antibody diluent (1% BSA) is as follows：
| Reagent | Consumption |
| Bovine serum albumin(BSA) (BSA) | 1g |
| Lavation buffer solution | 100mL |
5th, terminate liquid (2M H2SO4) collocation method
| Reagent | Consumption |
| The concentrated sulfuric acid | 22mL |
| Water | 178mL |
6、PBS(pH 7.4)
Weigh 8.0g NaCl, 0.2g KCl, 1.44g Na2HPO4, 0.24g KH2PO4800mL distilled water is dissolved in, it is dense
Salt acid for adjusting pH value is settled to 1L, 121 DEG C of high pressure 20min, 4 DEG C of preservations to 7.4.
" high person of outstanding talent peak etc., chicken interleukin-2 10 (chIL-10) is single in document by the chIL-10 of prokaryotic expression in following embodiments
Mistake disclosed in the preparation and identification of clonal antibody, Chinese Veterinary Journal ", the public can obtain from China Agricultural University.
The preparation method of buffer solution in following embodiments is as follows：
1st, 0.05M glycine-HCIs buffer solution (pH 2.8)
A liquid：Weigh 1.5g glycine to be dissolved in 100mL distilled water, 0.2M glycine is made standby as mother liquor.B liquid：Amount
1.65mL concentrated hydrochloric acids are taken, 100mL is settled to distilled water, 0.2M hydrochloric acid are made standby as mother liquor.Take A liquid 25mL, B liquid
8.4mL, plus distilled water are settled to 100mL.
2nd, 0.05M citric acid-sodium citrate buffer solutions (pH 3.0-5.0)
A liquid：Weigh citric acid 10.5g to be dissolved in 500ml distilled water, 0.05M citric acids are made standby as mother liquor.B liquid：
Weigh sodium citrate 14.7g to be dissolved in 500ml distilled water, 0.05M sodium citrates are made standby as mother liquor.By 93ml A liquid and
7ml B liquid is mixed, and obtains 0.05M citric acid-sodium citrate buffer solutions (pH 3.0).
65.5ml A liquid and 34.5ml B liquid are mixed, 0.05M citric acid-sodium citrate buffer solutions (pH 4.0) are obtained.
41ml A liquid and 59ml B liquid are mixed, 0.05M citric acid-sodium citrate buffer solutions (pH 5.0) are obtained.
3rd, 0.05M phosphate buffers (pH 6.0-8.0)
A liquid：Weigh sodium dihydrogen phosphate dihydrate 3.9g to be dissolved in 500ml distilled water, 0.05M sodium dihydrogen phosphates are made as mother
Liquid is standby.B liquid:Weigh disodium hydrogen phosphate 8.975g to be dissolved in 500ml distilled water, 0.05M disodium hydrogen phosphates work is made
It is standby for mother liquor.87.7ml A liquid and 12.3ml B liquid are mixed, 0.05M phosphate buffers (pH 6.0) are obtained.By 39ml
A liquid and 61ml B liquid are mixed, and obtain 0.05M phosphate buffers (pH 7.0).5.3ml A liquid and 94.7ml B liquid are mixed,
Obtain 0.05M phosphate buffers (pH 8.0).
4th, 0.05M carbonate buffer solutions (pH 9.6)
1.59g sodium carbonate is weighed, 2.93g sodium acid carbonates are dissolved in 1 000mL distilled water.
5th, 0.05M sodium acid carbonates-sodium hydrate buffer solution (pH 10)
A liquid：Weigh 0.21g sodium acid carbonates and be dissolved in 50ml distilled water that 0.05M sodium acid carbonates are made is standby as mother liquor.B
Liquid:Weigh 0.4g sodium hydroxides and be dissolved in 100ml distilled water that 0.1M sodium hydroxides are made is standby as mother liquor.Take A liquid 50mL, B
Liquid 10.7mL, plus distilled water are settled to after 100mL and mixed.
The formula of confining liquid in following embodiments is as follows：
1st, 5% defatted milk：5g defatted milks are weighed, is dissolved in 80mL PBST, is finally settled to 100mL.
2nd, 2% BSA：2g BSA are weighed, is dissolved in 80mL PBST, is finally settled to 100mL.
3rd, 1% gelatin：1g gelatin is weighed, is dissolved in 80mL PBST, stirring and dissolving is finally settled to 100mL.
4th, the casein of 5% defatted milk+1%：0.5g caseins are weighed, 50mL, stirring and dissolving mistake are settled to PBST
Night, then the defatted milk with the 5% of equivalent mix.
Embodiment 1, enzyme-linked immunologic detecting kit and double-antibody sandwich for detecting fowl leukocyte interleukin 2 (IL-2)
The foundation of ELISA detection method and the optimization of condition
First, for the preparation for the enzyme-linked immunologic detecting kit for detecting fowl leukocyte interleukin 2 (IL-2)
The enzyme-linked immunologic detecting kit for being used to detect fowl leukocyte interleukin 2 (IL-2) of the present invention includes polystyrene enzyme
Mark reaction plate, chIL-2 standard items (recombinant protein His-chIL-2, sandwich albumen), anti-chIL-2 monoclonal antibodies FM-1 (bags
By antibody, the antibody of present invention selection hybridoma cell strain FM-1 secretions is used as coated antibody), anti-chIL-2 monoclonal antibodies
FM-2 (detection antibody, the antibody of present invention selection hybridoma cell strain FM-2 secretions is used as detection antibody), 0.05M phosphate delay
Fliud flushing (pH8.0, coating buffer), PBST (pH7.4, lavation buffer solution), 5% defatted milk (confining liquid), 1%BSA (sample and antibody
Dilution), HRP mark Streptavidin, substrate TMB, 2M H2SO4Solution (terminate liquid).
1st, the preparation of immunogene
(1) structure of chicken IL-2 gene prokaryotic expression carrier
Chicken IL-2 gene gene order insertion vector pET-28a shown in sequence 1 (is purchased from EMD Biosciences
(Novagen), catalog number is 69864-3) EcoR I and Hind III digestions site between, and keep carrier pET-28a's
Other sequences are constant, obtain recombinant vector chIL-2-pET-28a.
Chicken IL-2 gene gene order insertion vector pGEX-6p-1 shown in sequence 1 (is purchased from You Bao biotech firms, product mesh
Record number is VT1258) EcoR I and Hind III digestions site between, and keep vector pGEX -6p-1 other sequences constant, obtain
To recombinant vector chIL-2-pGEX-6P.
(2) structure of recombinant bacterium and the induced expression of recombinant protein and purifying
Recombinant vector chIL-2-pET-28a and chIL-2-pGEX-6P are transferred into Rosetta expression bacterium respectively (to be purchased from
Beijing health is that century is biological, and article No. is CW0811A) middle progress induced expression, add IPTG to final concentration of 1mmol/L, 37 DEG C
6h is induced, thalline is collected by centrifugation.Through ultrasonic degradation, (Ultrasound Instrument power ratio 30%, 40 DEG C of bath temperature degree, ultrasonic 3s stops 2s to thalline, surpasses
Sound total time 30min) afterwards 12 000r/min centrifuge 20min, collect supernatant.Supernatant carries out affinitive layer purification, respectively obtains pure
Recombinant protein His-chIL-2 (16kDa) and GST-chIL-2 (40kDa), SDS-PAGE and Western blotting after change
Testing result difference is as depicted in figs. 1 and 2.His-chIL-2 albumen is used to mouse is immunized, and GST-chIL-2 albumen is used to hybridize
Oncocyte is screened.
Above-mentioned purifying is comprised the following steps that：1st, supernatant is crossed into post, flow velocity is 10 times of column volume/hours.2nd, using 15
The Soluble Binding Buffer of times column volume rinse pillar, and collection flows through peak.3rd, using the Soluble of 5 times of column volumes
Elution Buffer are eluted, and collect eluting peak.4th, after eluting, successively using the Soluble Binding of 3 times of column volumes
The deionized water washing pillar of Buffer and 5 times of column volume, then (ethanol is by filler with the 20% ethanol balances of 3 times of column volumes
Submergence), Feng Zhuhou 2-8 DEG C are preserved.
2nd, mouse is immune
His-chIL-2 albumen after purification is immunized to the BALB/c mouse of 6-8 week old as immune protein, 4 are immunized altogether
Secondary, each At intervals of two to three weeks, three immune one week afters, mouse docking takes blood, serum titer is determined using indirect ELISA, if potency
It is qualified, merge first three day and do booster immunization, specific immune programme for children is shown in Table 1.
Table 1, specific immune programme for children
3rd, cell fusion
Mice serum is taken to carry out antibody titer detection using indirect ELISA after mouse three times is immune, potency eligible is carried out
Booster immunization is used for cell fusion.Cell fusion is comprised the following steps that：By ready sp2/0 hybridomas and immune spleen
Cell is according to 1:2-1:5 ratio mixing, is added in a sterile 50mL centrifuge tube, 4 DEG C, 1000rpm centrifugation 10min, abandons
Supernatant, the centrifuge tube for filling cell is inserted in 37 DEG C of warm water, centrifuge tube is gently rocked on one side, is dissolved while drawing 1mL
PEG4000 fusion agents, be drop by drop added in cell mud, complete this operation in 60s, then static 60s, afterwards according to
Following method adds 15mL10%DMEM nutrient solutions：1-2min is slowly added into 1mL10%DMEM nutrient solutions, adds within the 3rd minute
1mL, four minutes add 2mL, and remaining 10%DMEM nutrient solutions are then added in 3min, and 1000rpm centrifugation 10min are abandoned
Supernatant, adds 40mLHAT selection nutrient solution suspension sedimentation cells, suspension cell is added in 96 orifice plates, 100uL/ holes are placed in 37
℃CO2Incubator culture.
Above-mentioned indirect ELISA carries out comprising the following steps that for antibody titer detection：With pH9.6 carbonate buffer solution (bag
By liquid) GST-chIL-2 albumen is diluted to 1ug/ml, 100uL/ holes are coated with CORNING ELISA Plates, and 4 DEG C are overnight；Dry in hole
Residual liquid, 300uL PBST are added per hole, are washed 3 times, 3min/ times；Dry in hole and added after residual liquid per hole
300uL5% skimmed milks, 4 DEG C of closings are stayed overnight；Washing；Docking blood sampling, does 2 doubling dilutions by yin and yang attribute serum, will dilute respectively
Serum add ELISA Plate, 100uL/ holes, each dilution factor does the parallel hole of more than three, and 37 DEG C are incubated 1 hour, cleaning 5 times；
Plus 1：The goat anti-mouse IgG ELIAS secondary antibody 100ul/ holes of the horseradish peroxidase-labeled of 10000 times of dilutions, 37 DEG C are incubated 1
Hour；PBST is washed three times；Dry residual liquid in hole, plus TMB developer 100ul/ holes, color development at room temperature 10-15 minutes, per hole
Plus ELIASA detects D after 50ul terminate liquids450Value.As a result judgement is by detecting OD450>Average+2x the standards of standard female sample
The standard deviation of negative sample carries out positive judgement, and statistically the result of determination can reach 95% confidential interval.
4th, positive hybridoma cell is screened
When cell clone to be fused grows into 1/4-1/3, culture supernatant is detected using indirect ELISA method,
Method is determined with serum antibody titer, is added yin and yang attribute serum control and the control of sp2/0 cell conditioned mediums, is used sp2/0 cell conditioned mediums
Value screens positive hybridoma cell as critical value, and negative hole is examined once again after three days, and it is abandoned if still for feminine gender.Between twice
The cell hole of ELISA test positive is connect, higher being enlarged of the positive value of selection is cultivated and be subcloned.
5th, the subclone of positive hybridoma cell
Positive cell is gently blown afloat, takes a small amount of cell to carry out cell count with Trypan Blue liquid after diluting, uses cell
Nutrient solution draws after 10 times of dilutions of positive hybridoma cell 80-100 hybridoma and is added to 10mL15%DMEM cultures
In liquid, mix, the cell suspension diluted is added in 96 porocyte culture plates, make about thin containing 1 hybridoma in every hole
Born of the same parents, each positive colony spreads one block of plate, have in addition in every block of plate 1 hole add in 10 cells, 1 hole 100 cells of addition with
The loss of positive cell is avoided, cell is put in 37 DEG C of incubator cultures, whne cell length to when covering bottom hole portion 1/4-1/3, is inhaled
Cell conditioned medium is taken to be detected using indirect ELISA, the positive hole of selection only one of which cell clone is cloned again, Ya Ke
It is grand to be 3-4 times altogether.After the hybridoma cell strain of stably excreting antibody is obtained, culture is enlarged.
After being immunized through three times, chIL-2 antibody titers reach 1 in mice serum:128000.Using indirect after cell fusion
ELISA method is detected to chIL-2 antibody in cell conditioned medium after fusion, and 6 plants of positive hybridomas are sieved to altogether, by three Asias
Gram and expand it is numerous it is final obtain 2 plants can stably excreting chIL-2 antibody hybridoma cell strain, respectively name FM-1 and FM-2.
Hybridoma cell strain FM-1 Classification And Nomenclature be hybridoma cell line (mouse source), the hybridoma cell strain in
On April 24th, 2017 is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (abbreviation CGMCC, address：
Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Institute of Microorganism, Academia Sinica, postcode 100101), preserving number is CGMCC
No.13832。
Hybridoma cell strain FM-2 Classification And Nomenclature be hybridoma cell line (mouse source), the hybridoma cell strain in
On April 24th, 2017 is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (abbreviation CGMCC, address：
Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Institute of Microorganism, Academia Sinica, postcode 100101), preserving number is CGMCC
No.13833。
6th, the preparation and identification of monoclonal antibody
Select through producing BALB/c mouse, intraperitoneal injection sterilized liquid paraffin 1mL/ only, respectively obtains step 5 screening after 7d
2 strain of hybridoma be injected into mouse peritoneal carry out ascites preparation, hybridoma injection volume be 2.0 × 106Individual/only.Receive
The ascites of collection is purified through collected after centrifugation supernatant using caprylic acid saturated ammonium sulphate method.Wherein, hybridoma cell strain
The chIL-2 monoclonal antibodies of FM-1 secretions are named as anti-chIL-2 monoclonal antibodies FM-1 (FM-1 antibody)；Hybridoma cell strain
The chIL-2 monoclonal antibodies of FM-2 secretions are named as anti-chIL-2 monoclonal antibodies FM-2 (FM-2 antibody), and special to it respectively
Property identified, including hypotype identification, titer of ascites determine, affinity determine, antibody specificity identification and Characterization of antigenic epitopes.
(1) identification of monoclonal antibody Ig subclass
The identification of monoclonal antibody subclass is carried out according to the mouse monoclonal antibody subtype typing reagent identification kit specification of Sigma companies,
Identified two plants of monoclonal antibody hypotypes are IgG1.
(2) measure of monoclonal antibody affinity
Using indirect elisa method, multiple proportions is added with 1 μ g/mL concentration GST-chIL-2 coated elisa plates, after closing dilute
The purified monoclonal antibody released is incubated, and the goat anti-mouse IgG marked using HRP is secondary antibody, and ELIASA reads OD450nm light absorption values.
The OD450nm readings of continuous several dilution factors are considered as antigen-antibody 100% when no longer increasing and combined, with antibody concentration (mol/L)
Be that ordinate does scatter diagram for abscissa, OD450nm light absorption values, using antigen-antibody Percentage bound during reading maximum half as
50%, generate logarithm Trendline and formula.
2 plants of monoclonal antibodies FM-1 and FM-2 affinity dissociation constant (K after measuredd) it is respectively 4.15 × 10-9With 1.36 × 10-10, it is high-affinity antibody.
(3) monoclonal antibody specificity identification
Using Western Blot to chIL-2 monoclonal antibodies to the His-chIL-2 of prokaryotic expression, His-chIL-10,
His-chIFN- γ and His-chIFN- β monoclonal antibody specificity are detected.Different albumen are diluted and carried out after certain multiple
Western Blot, respectively with the monoclonal antibody (1 of dilution:Or His monoclonal antibodies (1 5000):10000) as primary antibody, horseradish peroxidase
The goat anti-mouse IgG of enzyme HRP marks detects gained fowl chIL-2 monoclonal antibodies to other common different fowl as secondary antibody
Cell factor has no cross reaction.
The His-chIFN- γ expression vectors of above-mentioned prokaryotic expression are to insert the chicken IFN-γ gene order shown in sequence 4
Carrier pET-28a (being purchased from EMD Biosciences (Novagen), catalog number is 69864-3) EcoR I and Hind
Between III digestion site, and keep carrier pET-28a other sequences it is constant after obtained carrier.
The His-chIFN- β expression vectors of above-mentioned prokaryotic expression are to insert the chicken IFN-β gene order shown in sequence 5 to carry
Body pET-28a (being purchased from EMD Biosciences (Novagen), catalog number is 69864-3) EcoR I and Hind III
Between restriction enzyme site, and keep carrier pET-28a other sequences it is constant after obtained carrier.
As a result it is as shown in Figure 3.As a result show, 2 plants of monoclonal antibodies only recognize the His-chIL-2 albumen of prokaryotic expression, and fail to see
His-chIL-10, His-chIFN- γ and His-chIFN β of other prokaryotic expression, show that 2 plants of monoclonal antibody specificity are good.
(4) odd contradictive hydroperitoneum titration
Ascites prepares the last week, and paraffin is only injected into mouse peritoneal according to 500 μ L/, and hybridoma expands after culture, every
Mouse is according to 106200 μ L hybridoma suspensions are injected intraperitoneally in individual cell number, and after 6 days, mouse abdominal circumference is significantly increased, and gather abdomen
Water, titer of ascites is determined using indirect elisa method.
2 plants of monoclonal antibodies FM-1 and FM-2 titer of ascites are respectively after measured：1.28×107With 6.4 × 106。
(5) monoclonal antibody identification Characterization of antigenic epitopes
132 amino acid of ChIL-2 total lengths, its N-terminal has signal peptide region of one section of size for 11 amino acid, by ChIL-
2 albumen are truncated (to be truncated in units of 40 amino acid from its C-terminal and N-terminal, builds ChIL-2 truncated protein respectively
Expression vector chIL2- Δs 1 and chIL2- Δs 2 (Fig. 4), then Rosetta expression bacterium in induce each truncated protein respectively
Expression vector, and as antigen, using ChIL-2 monoclonal antibodies as primary antibody, the mountain marked with horseradish peroxidase HRP
Goat anti-mouse antibody is the antigen recognizing district that secondary antibody analyzes 2 plants of monoclonal antibodies by Western blotting.
The expression vector chIL-2- Δs 1 of above-mentioned ChIL-2 truncated protein are to insert the DNA fragmentation shown in sequence 2 to carry
Between body pET-28a EcoR I and Hind III digestions site, and keep carrier pET-28a other sequences it is constant after obtain
Carrier.
The expression vector chIL-2- Δs 2 of above-mentioned ChIL-2 truncated protein are to insert the DNA fragmentation shown in sequence 3 to carry
Between body pET-28a EcoR I and Hind III digestions site, and keep carrier pET-28a other sequences it is constant after obtain
Carrier.
As a result it is as shown in Figure 5.Testing result shows：FM-1 identification fowl IL-2 40-80 amino acids, FM-2 identification fowl
The 81-121 amino acids (Fig. 6) of IL-2 albumen.
2nd, the foundation of double-antibody sandwich elisa detection method and the optimization of condition
The present invention establishes double-antibody sandwich on the basis of enzyme linked immunological with reference to biotin-Streptavidin system
ELISA detection method, this method is using anti-chIL-2 monoclonal antibodies FM-1 as coated antibody, using His-chIL-2 as sandwich egg
In vain, using anti-chIL-2 monoclonal antibodies FM-2 as detection antibody, and the strepto- for biotin and the HRP mark that marked antibody is utilized
High-affinity and high specific between Avidin are combined, and substantial amounts of enzyme molecule is gathered in around antigen antibody complex, are produced
Raw multistage amplification, dramatically increases the enzyme molecule that each antibody is carried, so as to be greatly enhanced sensitivity.
Operated according to conventional double antibody ELISA method, often optimize a condition and then fix other conditions.When optimizing
One condition A, then during another condition B of next suboptimization, then using A condition, other conditions are still fixed, by that analogy, directly
Finished to all condition optimizings.A condition optimizing is often completed, obtained data need to be handled, consider P/N values
(P/N values=positive control OD450 averages/negative control OD450 averages, it is generally maximum with P/N values, and P values close to 1, N values compared with
The small judgment basis as optimum reaction condition) and the good aspect of sandwich protein concentration scope two of linear relationship, it is determined that most
Good reaction condition.
1st, it is coated with the optimization of condition
Respectively with 0.05M glycine-HCIs (pH2.8), 0.05M citric acid-sodium citrate buffer solutions (pH2.5), 0.05M
Citric acid-sodium citrate buffer solution (pH4.0), 0.05M citric acid-sodium citrate buffer solutions (pH5.0), 0.05M phosphate delay
Fliud flushing (pH6.0), 0.05M phosphate buffers (pH7.0), 0.05M phosphate buffers (pH8.0), 0.05M carbonate buffers
Liquid (pH9.6) and 0.05M sodium acid carbonates-sodium hydrate buffer solution (pH10) are coating buffer.FM-1 antibody is diluted into concentration is
10 μ g/mL, 4 DEG C of coating 12h, after 1% PBST is washed 3 times, with being washed after 5% 4 DEG C of closing 12h of defatted milk, are added with PBS
The recombinant protein GST-IL-2 of doubling dilution is carried out, and using PBS as negative control, 37 DEG C are incubated washing after 1h.1 is pressed with PBS:1
(the preparation method reference antibody biotin labeling reagent box of biotinylated antibody (is purchased from Thermo to 000 pair of biotinylated antibody
Scientific, Prod#21440) in Thermo Scientific, Prod#21440 specification) be diluted, 37 DEG C incubation
Washed after 1h, 1 is pressed with 1% BSA:10 000 couples of HRP mark Streptavidin (be purchased from Thermo Scientific,
Prod#21130) it is diluted, 37 DEG C are incubated washing after 30min, add 2M H after nitrite ion TMB2SO4Terminate.On ELIASA
OD450 light absorption values are read, optimal coating buffer is determined according to decision condition.
Optimization coating temperature and time when, antibody is diluted with optimal coating buffer, respectively room temperature coating 12h, 4 DEG C
12h and 37 DEG C of coating 2h of coating, other reaction conditions ibid, read OD450 light absorption values on ELIASA, true according to decision condition
Fixed optimal coating temperature and time.
In optimization coating concentration, antibody is diluted to respectively with optimal coating buffer 5 μ g/mL, 10 μ g/mL, 15 μ g/mL and
20 μ g/mL, using optimal coating temperature and time, other reaction conditions ibid, read OD450 light absorption values, root on ELIASA
Optimal coating concentration is determined according to decision condition.
According to above-mentioned optimum results, optimal coating condition is as follows：0.05M carbonate buffer solutions (pH9.6) dilution coating
Antibody is to 5 μ g/mL, and 4 DEG C are coated with 12h.
2nd, the optimization of sealing condition
Using the optimum reaction condition optimized, respectively with 5% defatted milk, 2% BSA, 1% gelatin and 5%
The casein of defatted milk+1% at 4 DEG C, 12h and 37 DEG C, is closed, other reaction conditions as confining liquid under conditions of 2h
Ibid, OD450 light absorption values are read on ELIASA, optimal sealing condition is determined according to decision condition.
According to above-mentioned optimum results, optimal sealing condition is as follows：5% defatted milk, 4 DEG C of closing 12h.
3rd, the optimization of biotinylated antibody dilution ratio
Using the optimum reaction condition optimized, biotinylated antibody is pressed 1 respectively:500、1:1 000、1:2 000 Hes
1:4 000 are diluted, and other reaction conditions ibid, read OD450 light absorption values on ELIASA, are determined most according to decision condition
Good biotinylated antibody dilution ratio.
According to above-mentioned optimum results, optimal biotinylated antibody dilution ratio is 1:500.
4th, the optimization of sandwich albumen and biotinylated antibody dilution
Using the optimum reaction condition optimized, respectively with PBS, 5% defatted milk, 2.5% defatted milk, 1.25%
Defatted milk and 1% BSA are diluted to biotinylated antibody and sandwich albumen, and other reaction conditions are constant, on ELIASA
Read OD450 light absorption values, according to decision condition determine optimal sandwich albumen and biotinylated antibody dilution (it is determined that after, will
Optimal dilution is used as negative control).
According to above-mentioned optimum results, the BSA that optimal sandwich albumen and biotinylated antibody dilution is 1%.
5th, the optimization of sandwich albumen incubation time
Using the optimum condition optimized, sandwich albumen is incubated 0.5h, 1.0h, 1.5h and 2.0h respectively, other reaction bars
Part is constant, and OD450 light absorption values are read on ELIASA, and optimal sandwich albumen incubation time is determined according to decision condition.
According to above-mentioned optimum results, optimal sandwich albumen incubation time 1.0h.
6th, the optimization of biotinylated antibody incubation time
Using the optimum reaction condition optimized, biotinylated antibody is incubated 0.5h, 1.0h, 1.5h and 2.0h respectively, its
His reaction condition is constant, and OD450 light absorption values are read on ELIASA, determines that optimal biotinylated antibody is incubated according to decision condition
Time.
According to above-mentioned optimum results, optimal biotinylated antibody incubation time is 1.0h.
7th, the optimization of board-washing number of times
Using the optimum reaction condition optimized, respectively board-washing 3,4 and 6 times, other reaction conditions are constant, on ELIASA
OD450 light absorption values are read, optimal board-washing number of times is determined according to decision condition.
According to above-mentioned optimum results, optimal board-washing number of times is 3 times.
8th, the optimization of the Streptavidin incubation time of HRP marks
Using the optimum reaction condition optimized, the Streptavidin of HRP marks is incubated 15min, 30min, 45min respectively
And 60min, other reaction conditions are constant, and OD450 light absorption values are read on ELIASA, determine that optimal HRP is marked according to decision condition
The incubation time of the Streptavidin of note.
According to above-mentioned optimum results, the incubation time of the Streptavidin of optimal HRP marks is 30min.
9th, the determination of optimum reaction condition
By the above-mentioned optimization to reaction condition, the final specific steps for determining double-antibody sandwich elisa detection method are such as
Under：Coated antibody is diluted to 5 μ g/mL, 4 DEG C of coating 12h with carbonate buffer solution (pH9.6)；5% defatted milk, 4 DEG C of closings
12h；Sandwich albumen, 37 DEG C of incubation 1h are diluted with 1% BSA；With 1% BSA according to 1:500 ratio is by biotinylated antibody
It is diluted to 1 μ g/mL, 37 DEG C of incubation 1h；With 1% BSA according to 1:The Streptavidin of 10 000 dilution proportion HRP marks,
37 DEG C of incubation 30min；PBST board-washings 3 times.
The sensitivity technique of embodiment 2, enzyme-linked immunologic detecting kit for detecting fowl IL-2
First, the present invention is used for the sensitivity technique for detecting fowl IL-2 enzyme-linked immunologic detecting kit
It is anti-by coating of FM-1 antibody according to the optimal conditions for the double-antibody sandwich elisa detection method for implementing to determine in 1
Body, biotinylated antibody FM-2 are detection antibody, and ELISA experiments will be carried out after sandwich albumen doubling dilution to various concentrations, and
Using sandwich protein concentration as abscissa, with OD450Standard curve is set up for ordinate.Comprise the following steps that：
1st, FM-1 antibody is diluted to after 5 μ g/mL, 100 μ L/ holes, 4 DEG C of coating 12h with carbonate buffer solution (pH9.6),
Lavation buffer solution is washed 3 times, 300 μ L/ holes, dries residual liquid in hole；
2nd, after 5% defatted milk, 300 μ L/ holes, 4 DEG C of closing 12h, washing is ibid；
3rd, sandwich albumen (recombinant protein GST-chIL-2) is diluted to various concentrations (the concentration such as institute of table 2 with 1% BSA
Show), 100 μ L/ holes, 37 DEG C are incubated after 1h, and washing is ibid；
4th, biotinylated antibody FM-2 is diluted with 1% BSA, makes its ultimate density for 1 μ g/mL, 100 μ L/ holes,
37 DEG C are incubated after 2h, and washing is ibid；
5th, 1 is pressed with 1% BSA:10 000 ratio is diluted to the HRP Streptavidins marked, 100 μ L/ holes, 37
DEG C be incubated 15min after, washing ibid；
6th, nitrite ion TMB is added, 100 μ L/ holes when negative control slightly becomes indigo plant, add 2M H2SO4, 50 μ L/ holes, eventually
Only react；
7th, ELIASA reads OD450Light absorption value.
Standard curve is as shown in Figure 7.Sandwich protein concentration and OD450Linear relationship is as shown in table 2.As a result show：Sandwich egg
White concentration is in 100pg/mL-100ng/mL concentration range, its concentration and OD450Linear relationship is good, can be detected most
Small sandwich protein concentration is 100pg/mL.
Table 2, sandwich protein concentration and OD450 linear relationships
| Sandwich protein concentration (ng/mL) | 1000 | 100 | 10 | 1 | 0.1 | 0.01 |
| OD450nm | 3.1185 | 1.4025 | 0.2555 | 0.1163 | 0.0723 | 0.0820 |
2nd, in documents antibody sensitivity detection
According to the method in step one, respectively with document " preparation and identification of anti-recombinant chIL-2 monoclonal antibody "
In hybridoma cell strain secretion anti-chIL-2 antibody for coated antibody and detection antibody, by sandwich albumen doubling dilution to not
With progress ELISA experiments after concentration.The concentration of coated antibody, detection antibody and sandwich albumen is specifically as shown in table 3.
Different antibodies combine sandwich protein concentration and OD450Linear relationship is as shown in table 3.As can be seen from the table：In document
Using 4B5 as coated antibody, biotinylated antibody 1C2 is best for the combine detection effect of detection antibody, and this is to Antibody Combination through bar
The minimum sandwich protein concentration that can be detected after piece optimization is 15.6ng/mL.Therefore, it is of the invention anti-by coating of FM-1 antibody
Body, biotinylated antibody FM-2 are substantially more preferable for the sensitivity of the Antibody Combination of detection antibody.
Antibody secreted by hybridoma cell strain is used for the detection case of sandwich ELISA in table 3, documents
The application of embodiment 3, enzyme-linked immunologic detecting kit
Using the enzyme-linked immunologic detecting kit for being used to detect fowl IL-2 of the present invention to 10 part of 1 Japanese instar chickling serum and 10
Part detection of Salmonella Positive Sera sample is detected, tentatively judges the detection method Clinical practicability.Comprise the following steps that：
1st, FM-1 antibody is diluted to after 5 μ g/mL, 100 μ L/ holes, 4 DEG C of coating 12h with carbonate buffer solution (pH9.6),
Lavation buffer solution is washed 3 times, 300 μ L/ holes, dries residual liquid in hole；
2nd, after 5% defatted milk, 300 μ L/ holes, 4 DEG C of closing 12h, washing is ibid；
3rd, 1 is pressed with 1% BSA:10 ratio is diluted to blood serum sample to be measured, 100 μ L/ holes, and 37 DEG C are incubated after 1h,
Washing is ibid；
4th, biotinylated antibody FM-2 is diluted with 1% BSA, makes its ultimate density for 1 μ g/mL, 100 μ L/ holes,
37 DEG C are incubated after 2h, and washing is ibid；
5th, 1 is pressed with 1% BSA:10 000 ratio is diluted to the HRP Streptavidins marked, 100 μ L/ holes, 37
DEG C be incubated 15min after, washing ibid；
6th, nitrite ion TMB is added, 100 μ L/ holes when negative control slightly becomes indigo plant, add 2M H2SO4, 50 μ L/ holes, eventually
Only react；
7th, ELIASA reads OD450Light absorption value.By OD450Light absorption value is substituted into the standard curve in embodiment 2, obtains to be measured
ChIL-2 concentration in blood serum sample.
As a result it is as shown in table 4.As a result show：In 10 part of 1 Japanese instar chickling serum, have 1 part be detected chIL-2 concentration compared with
ChIL-2 concentration is relatively low in height, remaining serum, not in the linear concentration range of detection of the enzyme linked immunological kit of the present invention
Concentration represented with 0.In 10 parts of detection of Salmonella positive serums, chIL-2 is detected, and concentrations are far above 1 Japanese instar chickling
The concentrations of serum.
Table 4, serum sample detection
Sequence table
<110>China Agricultural University
<120>A kind of method and its dedicated kit for detecting the content of fowl leukocyte interleukin 2
<160>5
<210>1
<211>363bp
<212>DNA
<213>Artificial sequence
<220>
<223>
<400>1
gcatctctat catcagaaaa aaggaaacct cttcaaacat taataaagga tttagaaata 60
ttggaaaata tcaagaacaa gattcatctc gagctctaca caccaactga gacccaggag 120
tgcacccagc aaactctgca gtgttacctg ggagaagtgg ttactctgaa gaaagaaact 180
gaagatgaca ctgaaattaa agaagaattt gtaactgcta ttcaaaatat cgaaaagaac 240
ctcaagagtc ttacgggtct aaatcacacc ggaagtgaat gcaagatctg tgaagctaac 300
aacaagaaaa aatttcctga ttttctccat gaactgacca actttgtgag atatctgcaa 360
aaa 363
<210>2
<211>240bp
<212>DNA
<213>Artificial sequence
<220>
<223>
<400>2
gcatctctat catcagaaaa aaggaaacct cttcaaacat taataaagga tttagaaata 60
ttggaaaata tcaagaacaa gattcatctc gagctctaca caccaactga gacccaggag 120
tgcacccagc aaactctgca gtgttacctg ggagaagtgg ttactctgaa gaaagaaact 180
gaagatgaca ctgaaattaa agaagaattt gtaactgcta ttcaaaatat cgaaaagaac 240
<210>3
<211>243bp
<212>DNA
<213>Artificial sequence
<220>
<223>
<400>3
tgcacccagc aaactctgca gtgttacctg ggagaagtgg ttactctgaa gaaagaaact 60
gaagatgaca ctgaaattaa agaagaattt gtaactgcta ttcaaaatat cgaaaagaac 120
ctcaagagtc ttacgggtct aaatcacacc ggaagtgaat gcaagatctg tgaagctaac 180
aacaagaaaa aatttcctga ttttctccat gaactgacca actttgtgag atatctgcaa 240
aaa 243
<210>4
<211>435bp
<212>DNA
<213>Artificial sequence
<220>
<223>
<400>4
catactgcaa gtagtctaaa tcttgttcaa cttcaagatg atatagacaa actgaaagct 60
gactttaact caagtcattc agatgtagct gacggtggac ctattattgt agagaaactg 120
aagaactgga cagagagaaa tgagaaaagg atcatactga gccagattgt ttcgatgtac 180
ttggaaatgc ttgaaaacac tgacaagtca aagccgcaca tcaaacacat atctgaggag 240
ctctatactc tgaaaaacaa ccttcctgat ggcgtgaaga aggtgaaaga tatcatggac 300
ctggccaagc tcccgatgaa cgacttgaga atccagcgca aagccgcgaa tgaactcttc 360
agcatcttac agaagctggt ggatcctccg agtttcaaaa ggaaaaggag ccagtctcag 420
aggagatgca attgc 435
<210>5
<211>612bp
<212>DNA
<213>Artificial sequence
<220>
<223>
<400>5
atgactgcaa accatcagtc tccagggatg cacagcatcc tactgctctt gcttctgcca 60
gctctcacca ccaccttctc ctgcaaccat cttcgtcacc aggatgccaa cttctcttgg 120
aaaagcctcc agctccttca gaatacggct ccacctccac cacagccttg cccacaacaa 180
gacgtgactt ttccatttcc agaaaccctt ctgaaaagca aggacaagaa gcaagcagcc 240
atcaccaccc tccgcatcct ccaacacctc ttcaacatgc ttagcagccc acacactcca 300
aaacactgga ttgaccgcac acgccacagc ctcctcaacc agatccagca ttacatccat 360
caccttgagc aatgcttcgt aaaccaaggc acgcgctccc agaggcgagg gcctcgcaac 420
gctcacctca gcatcaacaa atacttcaga tccatccaca acttcctacg gcacaacaac 480
tacagtgctt gtacctggga ccatgtccgc ctccaggctc gtgactgctt ccgacacgtg 540
gacacactca tacaatggat gaaaagtcga gctcctctca cagcctcatc caaacgtctc 600
aacacccagt ga 612
Claims (10)
1. anti-chIL-2 monoclonal antibody, is to be secreted by preserving number for CGMCC No.13832 hybridoma cell strain FM-1
's.
2. anti-chIL-2 monoclonal antibody, is to be secreted by preserving number for CGMCC No.13833 hybridoma cell strain FM-2
's.
3. the hybridoma cell strain FM-1 of one plant of anti-chIL-2 of secretion monoclonal antibody, its preserving number is CGMCC
No.13832。
4. the hybridoma cell strain FM-2 of one plant of anti-chIL-2 of secretion monoclonal antibody, its preserving number is CGMCC
No.13833。
5. a kind of enzyme linked immunological kit for detecting or aiding in detect chIL-2 contents in testing sample, it includes claim 1
Monoclonal antibody described in described monoclonal antibody and/or claim 2.
6. hybridoma cell strain described in monoclonal antibody or claim 3 or 4 or claim 5 described in claim 1 or 2
Application of the described enzyme linked immunological kit in chIL-2 contents in detecting or aiding in detection testing sample；
Or, the hybridoma cell strain described in the monoclonal antibody or claim 3 or 4 described in claim 1 or 2 is preparing detection
Or the application in auxiliary detection testing sample in the reagent or colloidal gold strip or kit of chIL-2 contents.
7. a kind of method for detecting or aiding in chIL-2 contents in detection testing sample, is with the monoclonal described in claim 1
Antibody is that coated antibody, the monoclonal antibody described in claim 2 are to detect the double-antibody sandwich elisa detection method of antibody.
8. method according to claim 7, it is characterised in that：Methods described comprises the following steps：
(1) by the monoclonal antibody coating described in claim 1 to ELISA Plate, wash；
(2) ELISA Plate that closing (1) is obtained, washing；
(3) chIL-2 standard items or testing sample are added in the ELISA Plate obtained to (2), be incubated, washing；
(4) monoclonal antibody described in biotinylated claim 2 is added in the ELISA Plate obtained to (3), is incubated, washing；
(5) Streptavidin of HRP marks is added in the ELISA Plate obtained to (4), is incubated, washing；
(6) substrate colour developing is added in the ELISA Plate obtained to (5), terminate liquid terminating reaction is added after colour developing；
(7) absorbance in each hole for adding chIL-2 standard items is detected with ELIASA, is sat using chIL-2 standard concentrations to be horizontal
Mark, the drafting standard curve by ordinate of the reading of ELIASA；
(8) absorbance in the hole for adding testing sample is detected with ELIASA, absorbance is substituted into the standard curve, produced
ChIL-2 concentration in testing sample.
9. the method according to claim 7 or 8, it is characterised in that：
Or, in the step (1), coating after the monoclonal antibody dilution described in claim 1 is arrived into enzyme with carbonate buffer solution
On target；
Or, in the step (2), the ELISA Plate that (1) is obtained is closed with defatted milk solution；
Or, in the step (3), added after with BSA solution, chIL-2 standard items are diluted in ELISA Plate；
Or, in the step (4), the monoclonal antibody described in biotinylated claim 2 is diluted to 1.0 μ with BSA solution
Added after g/mL in ELISA Plate.
10. according to any described method in claim 7-9, it is characterised in that：
In the step (1), the concentration dilution of the antibody is 5 μ g/mL；The coated condition is 4 DEG C of coating 12h；It is described
The concentration of carbonate buffer solution is 0.05M, and pH is 9.6；
In the step (2), the mass fraction of the defatted milk solution is 5%；The condition of the closing is 4 DEG C of closing 12h；
The mass fraction of the BSA solution is 1%；
In the step (3), the incubation time is 1h；
In the step (4), the incubation time is 2h；
In the step (5), the incubation time is 15min；
Wavelength during the detection absorbance with ELIASA is 450nm；
The number of times of the washing is 3 times.
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| CN110412002A (en) * | 2019-08-08 | 2019-11-05 | 中山大学孙逸仙纪念医院 | A method of identifying human archeocyte and mouse source cell |
| CN111487416A (en) * | 2020-03-16 | 2020-08-04 | 北京维德维康生物技术有限公司 | Optra drug-resistant protein double-antibody sandwich E L ISA detection kit and detection method |
| CN112354570A (en) * | 2019-07-11 | 2021-02-12 | 北京理工大学 | Multidimensional microfluidic electrophoresis chip, detection device and detection method |
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| 付梦姣等: "禽IL-2单克隆抗体的制备与鉴定", 《中国兽医杂志》 * |
| 翁凌等: "抗体夹心酶联免疫吸附法测定鸡白细胞介素-2", 《厦门大学学报》 * |
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| CN107703311B (en) * | 2017-11-01 | 2019-12-03 | 合肥瀚科迈博生物技术有限公司 | Enzyme linked immunological kit and preparation method thereof for detecting IL-2 |
| CN109655619A (en) * | 2019-01-23 | 2019-04-19 | 中国农业大学 | A kind of method and its dedicated kit detecting 12 content of chicken interleukin-2 |
| CN112354570A (en) * | 2019-07-11 | 2021-02-12 | 北京理工大学 | Multidimensional microfluidic electrophoresis chip, detection device and detection method |
| CN110412002A (en) * | 2019-08-08 | 2019-11-05 | 中山大学孙逸仙纪念医院 | A method of identifying human archeocyte and mouse source cell |
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