CN101441220A - Method for detecting ChIFN-gamma double-monoclonal antibody sandwich ELISA - Google Patents
Method for detecting ChIFN-gamma double-monoclonal antibody sandwich ELISA Download PDFInfo
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- CN101441220A CN101441220A CNA2008102354382A CN200810235438A CN101441220A CN 101441220 A CN101441220 A CN 101441220A CN A2008102354382 A CNA2008102354382 A CN A2008102354382A CN 200810235438 A CN200810235438 A CN 200810235438A CN 101441220 A CN101441220 A CN 101441220A
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Abstract
The invention discloses a double monoclonal-antibodies sandwich ELISA method for detecting ChIFN-gamma, wherein the specific monoclonal antibodies (mAbs) against ChIFN-gamma are used to build the double monoclonal-antibodies sandwich ELISA method for detecting ChIFN-gamma. The double monoclonal-antibodies sandwich ELISA method for detecting ChIFN-gamma, with mAb3E5 as capture antibody and biotin-labeled mAb 3E3 as detection antibody, can quickly detect the trace ChIFN-gamma (125-500pg/ml). The method is rapid, useful, sensitive, and effective, can be used to detect the ChIFN-gamma and recombinant ChIFN-gamma in biological sample, and provides a sensitive detection tool for detecting and evaluating the cell immune response of poultry, and is useful for the research on the function of the ChIFN-gamma in various immune response mechanisms of poultry, and efficiently makes up for the prior ChIFN-gamma detection.
Description
Technical field
The invention belongs to the immunological detection method field, be specifically related to detect the double-monoclonal antibody sandwich ELISA method of poultry cell factor.
Background technology
Gamma interferon (IFN-γ is called immune interferon or II type interferon again) is the important media of cellullar immunologic response, has critical function aspect the immune response adjusting, especially has vital role to inducing and keeping the Th1 cellullar immunologic response.Th1 replys the infection of cause of disease in the opposing born of the same parents extremely important, and IFN-γ secretion property T lymphocytic appearance is the sign that Th1 replys.The appearance of the level of IFN-γ and IFN-γ secretory cell is usually as estimating the sign that the Th1 immune response takes place in the biological sample.
The method of the current detecting cell factor is a lot, mainly contains biological activity determination method, immunological detection, utilizes nucleic acid marking technology for detection cell factor etc.But in the biological sample of complexity, to not reflecting the function that it is actual under a lot of situations of the functional analysis of set cell factor.As; aspect the effect of protection cell resistance virus infections; I, II IFN all have this function; although the structure of this two class IFN is not very closely-related; and it is secreted by different cells; if but only analyze from function, can not accurately reflect the actual conditions of the specific cells factor.And, for specific cell factor, its active half life period is very short, cause no longer carrying out to the analysis of its function, although when sample collection, these cell factors exist, and still can analyze on protein level, but, cause the analysis on the function no longer can carry out because its active half life period is very short.Northern blot, genetic analysis methods such as RNase protection and PCR are all extremely sensitive, and mate fully with genes of interest, but it can only rest on the analysis on the gene transcription level, and a lot of information defy capture.
Therefore, be badly in need of a kind of can be qualitative and the detection by quantitative biological sample in the detection method of IFN-γ.Especially at infecting in the born of the same parents that bacterium, parasite, virus and other cause of disease cause in the born of the same parents, cytokines measurement instrument and detection method are highly profitable to the detection and the diagnosis of this class disease fast and effectively.At present, owing to lack effective testing tool and detection method, very difficult to the analysis and the evaluation of immune state under the disease conditions, in order to improve this present situation, people have carried out many effort, and as producing recombinant cytokine, development specificity antibacterial agent mAb improves detection method.
Along with the development of monoclonal antibody technique, the enzyme linked immunological absorption catching method (ELISA) that detects cell factor has obtained development energetically.In other species, ELISA has obtained very big development, and be proved to be a kind of cytokines measurement method of highly stable, credible, simple, easy row, can be used for detecting the cell factor of activated release, thereby, it is believed that this method is to estimate the good indicating means of cellullar immunologic response (CMI).And this detection method is multiduty, and not only can be used to detect the perhaps many cause of diseases of mitogen stimulates the CMI that induces once more, and can be used for detecting the CMI that excites after the immunity.
Being an important animal model aspect chicken catches in a large amount of poultry born of the same parents, up to the present, selecting chicken to be subjected to many restrictions as the animal model of poultry disease, mainly is in default of suitable testing tool and detection system.Nineteen ninety-five Digby etc. successfully clones, gives expression to ChIFN-γ gene, and to the research mushroom development of ChIFN-γ, its functional study work is also in the middle of carrying out.2000, Cheol H. etc. utilized ChIFN-γ natural or reorganization to develop anti-ChIFN-γ mAb, and has set up the change of serum C hIFN-γ level that direct ELISA method detects the eimeria tenella infected chicken; Subsequently, B.Lambrecht etc. utilize the particle gun immunization to prepare anti-ChIFN-γ mAb, and how anti-as capture antibody the anti-ChIFN-γ of utilization purifying is, and biotin labeled mAb has set up the double-antibodies sandwich ELISA that detects ChIFN-γ as detecting antibody.It is immunizing antigen and detection antigen that the present invention adopts prokaryotic expression recombinant ChIFN-γ, prepare anti-ChIFN-γ mAbs, the anti-ChIFN-γ mAb of utilization purifying is as capture antibody, biotin labeled mAb is as detecting antibody, set up the two monoclonal antibody sandwich ELISA methods of ChIFN-γ in the detection of biological sample, this method has avoided utilization how anti-as capture antibody, the stability and the homogeneity of coated antibody have been guaranteed, because may there be very big difference in resist that different batches is prepared more, more difficult standardization, and the superiority of mAb cell line is to produce the mAb with homogeneous characteristic endlessly.
ChIFN-γ antigen capture ELISA can estimate ChIFN-γ on protein level, and is more easy, practical, sensitive than biological method, is convenient to operation and large tracts of land and uses.Being established as of this method detects the testing tool that chicken CMI provides sensitivity.And this method will help studying the effect of ChIFN-γ in the different immune response mechanism of poultry.
Summary of the invention
The objective of the invention is to set up a kind of simple and direct, practical, sensitive, detect ChIFN-γ double-monoclonal antibody sandwich ELISA method efficiently.
Technical scheme of the present invention is: detect the double-monoclonal antibody sandwich ELISA method of ChIFN-γ, be: adopt anti-ChIFN-γ specificity mAb reagent, set up and detect the two mAb sandwich ELISA methods of ChIFN-γ specificity.This method as capture antigen, as detecting antibody, is set up the two mAb sandwich ELISA methods that detect ChIFN-γ with biotin labeled mAb3E3 with mAb3E5.This method can detect the ChIFN-γ of trace delicately.
The concrete step of said method is:
1. capture antibodies is fixed on the solid support, wherein capture antibodies is the ChIFN-γ monoclonal antibody 3E5 of purifying;
2. with biological sample and the insulation of immobilised capture antibodies, ChIFN-γ is combined with capture antibodies;
The ChIFN-γ that is attached on the immobilization capture antibodies is contacted with biotin labeled anti-ChIFN-γ monoclonal antibody 3E3, detect ChIFN-γ.
Said biological sample is separable from chicken serum, chicken plasma or cells and supernatant.
In the said method, the bag of mAb 3E5 is 85 μ g/ml by concentration, and the working concentration of mAb 3E3 is 0.67 μ g/ml.This method can the sensitive ChIFN-γ that detects 125-500pg/ml.
Use the inventive method and carry out the detection of natural ChIFN-γ: sterile preparation chicken spleen lymphocyte, with companion's canavaline (ConA, Sigma company) stimulates the chicken spleen lymphocyte, get the cells and supernatant of cultivating 48 hours, obtain natural ChIFN-γ, measure natural ChIFN-γ with two monoclonal antibody sandwich ELISA methods of setting up, detect natural ChIFN-γ very significantly.
The bag of antibody is cushioned the PBS that liquid is pH7.2 in the above-mentioned sandwich ELISA method, detecting antibody diluent is the PBST that contains 0.05% Tween 20 and 1%BSA, the dilution of avidin-HRP is PBST (0.05%Tween-20,1%BSA and 10% calf serum), TMB colour developing, 2M H
2SO
4Cessation reaction.
The ChIFN-γ specificity that the present invention set up is caught the natural ChIFN-γ that exists that detects that ELISA can be responsive in Con A stimulated cells culture supernatant.The ELISA method of being set up provides good testing tool for estimating the Th1 cellullar immunologic response.
Description of drawings
Fig. 1 is that (concentration of His-ChIFN-γ is 1000ng/ml for the susceptibility experiment of the two monoclonal antibody sandwich ELISA methods of chicken interferon-γ, the series doubling dilution carries out): monoclonal antibody 3E5 is as capture antibody, biotin labeled monoclonal antibody 3E3 can detect 125-500pg/ml recombination chicken gamma-interferon (His-ChIFN-γ) as detecting antibody
The specificity experiment of the two monoclonal antibody sandwich ELISAs of Fig. 2: two monoclonal antibody sandwich ELISAs detect recombination chicken gamma-interferon (His-ChIFN-γ of serial doubling dilution, 20ng/ml) and the specificity of natural chicken interferon-gamma (ConA stimulate chicken spleen lymphocyte produce) chicken interferon-gamma double-monoclonal antibody sandwich ELISA test (His-ChIFN-γ 20ng/ml, serial doubling dilution carries out; Con A stimulates the chicken spleen lymphocyte culture supernatant of 72h, and doubling dilution carries out, and X-axis is unit with Log2)
The two monoclonal antibody sandwich ELISAs of Fig. 3 detect varying number chicken spleen lymphocyte (1 * 10
6Individual cell/ml, 2.5 * 10
6Individual cell/ml, 5 * 10
6Individual cell/ml) stimulate chicken interferon-gamma in different incubation time sections (24 hours, 48 hours, 72 hours, the 96 hours) cell conditioned medium through ConA (12ug/ml).The two monoclonal antibody sandwich ELISAs of chicken IFN-detect varying number chicken spleen lymphocyte (1 * 10
6Individual cell/ml, 2.5 * 10
6Individual cell/ml, 5 * 10
6Chicken interferon-gamma (Con A:12 μ g/ml) in the culture supernatant of individual cell/ml)
Embodiment:
Employed in the present invention term unless other explanation is arranged, generally has the implication of those of ordinary skills' common sense.
Below in conjunction with specific embodiment, and comparable data is described the present invention in further detail.Should be understood that these embodiment just in order to demonstrate the invention, but not limit the scope of the invention by any way.
In following embodiment, various processes of Xiang Ximiaoshuing and method are not conventional methods as known in the art.The source of agents useful for same, trade name and be necessary to list its constituent person indicate when occurring first that all used thereafter identical reagent if no special instructions, and is all identical with the content of indicating first.
Anti-ChIFN-γ monoclonal antibody specific 3E3,3E5,1G10,2C3 are respectively by hybridoma cell strain 3E3,3E5,1G10,2C3 secretion among the present invention, wherein: hybridoma cell strain 3E3,3E5,1G10,2C3 (anti-chicken interferon-gamma Study of Monoclonal Antibodies and evaluation, Yangzhou University's magazine (agricultural and life science version), 2007,28 (4): 6-9.) Amphixenosis by Yangzhou University learns key lab's structure preservation, provides to the public in 20 years applyings date.
Embodiment 1: the present invention detects the establishment of the double-monoclonal antibody sandwich ELISA method of ChIFN-γ
1. the anti-ChIFN-γ of purifying and mark monoclonal antibody specific: 3E3,3E5,1G10,2C3 (mAbs), adopt Proein G purifying mAb, use biotin labeling mAb, concrete operations are carried out according to standard method.
2.ChIFN-the preliminary establishment of γ antigen capture ELISA: with the rChIFN-γ of prokaryotic expression as capture antigen (anti-chicken interferon-gamma Study of Monoclonal Antibodies and Preliminary Identification, cell and molecular immunology magazine, 2006,22 (4): 510-512), 3E3 with above-mentioned mark, 3E5,1G10,2C3mAbs and commercially available mAbs are (as Abs:Serotec:Rabbit anti chicken interferon gamma (polyclonal antibody, AHP945Z) match experiment between, chessboard method is found out the optimum antibody of setting up ChIFN-γ antigen capture ELISA, and (bag is 3E5 by mAbs, detecting antibody is biotin labeled mAb3E3) and optimum concentration, fixedly the working concentration of avidin-HRP (BD company) is 1:2500 (with reference to an instructions), set up negative control (1%BSA) simultaneously, blank is set up the ELISA method of catching;
3.ChIFN-the mensuration of γ antigen capture ELISA sensitivity: carry out according to the condition that " 2 " are set up: the bag of mAb3E5 is 85 μ g/ml by concentration, the working concentration of bio-m3E3 is 0.67 μ g/ml, the avidin-HRP working concentration is 1:2500, with rChIFN-γ doubling dilution, set up negative control (1%BSA) simultaneously, this method can detect the rChIFN-γ (Fig. 1) of 125-500pg/ml.
Embodiment 2: use the inventive method and detect natural ChIFN-γ
The preparation of natural ChIFN-γ: aseptic win 4 age in week the SPF chicken spleen, grind back 200 order copper mesh and filter; Collect filtrate, 4 ℃ centrifugal, 1000rpm, 5min; Lymphocyte separation medium (Sigma company) isolated lymphocytes, 21 ℃, 2000rpm, 30min is centrifugal; The cloud layer is lymphocyte in the middle of collecting, and the cell centrifugation of collecting is washed 2 times with PBS, use complete RPMI 1640 (10% hyclone, 100U/ml penicillin, 100U/ml streptomysin) washing 1 time again, use complete RPMI 1640 with cell suspension at last, adjust cell concentration to 1 * 10
7Individual/ml, 5 * 10
6Individual/ml, 2 * 10
6Individual/ml; Get 96 porocyte culture plates, add the 100ul cell, and then add the nutrient culture media 100 μ l that contain stimulant ConA (Sigma company), make that the final concentration of ConA is 12 μ g/ml, set up negative control (RPMI 1640 fully) simultaneously; Put 41 ℃, 5%CO
2Cell culture incubator is cultivated, and collects the cells and supernatant of cultivating 72h, obtains natural ChIFN-γ.
Sandwich ELISA method detects natural ChIFN-γ:
MAb3E5 is cushioned liquid (0.01M PBS pH7.2) with bag is diluted to 85 μ g/ml, wrap by elisa plate 100 μ l/ holes, 4 ℃, 24h; PBS-T (0.5%Tween-20) washing 3 times, 5min/ time; With the PBS-T sealing that contains 2%BSA, 200 μ l/ holes, 37 ℃., 2h; The same washing adds detected sample, 100 μ l/ holes, 37 ℃., 2h; The same washing; The bio-3E3 (0.67 μ g/ml) that adds working concentration, 37 ℃., 1h; The same washing back adds avidin-HRP, and (1:2500, dilution are the PBS-T that contains 1%BSA, 10% top grade calf serum, 100 μ l/ holes, 37 ℃, 30min; The same washing 7 times adds TMB, 100ul/ hole, 37 ℃., 15min, 2M H
2SO
4Stop, microplate reader reads the OD450nm value.Establish positive control, negative control and blank simultaneously.
Use this sandwich ELISA method can detect natural ChIFN-γ (Fig. 2,3) very significantly.
The elisa plate that uses in this method is irradiation plate (Xiamen cloud roc development in science and technology company limited); It is PBS that bag is cushioned liquid, pH7.2; The dilution buffer liquid of dilution rChIFN-γ and detection antibody is the PBST that contains 0.05%Tween-20,1%BSA, the dilution of avidin-HRP is 0.05% Tween-20,1% BSA, the PBST that reaches 10% top grade calf serum (Lanzhou people's marine growth engineering corporation), TMB (eBioscience company) colour developing, 2M H
2SO
4Cessation reaction.
To sum up, the ChIFN-γ specificity that the present invention set up is caught the natural ChIFN-γ that exists that detects that ELISA can be responsive in ConA stimulated cells culture supernatant.The ELISA method of being set up provides good testing tool for estimating the Th1 cellullar immunologic response.
Claims (5)
1, a kind of double-monoclonal antibody sandwich ELISA method that is used for detection of biological sample ChIFN-γ, it may further comprise the steps:
1. capture antibodies is fixed on the solid support, wherein capture antibodies is the ChIFN-γ monoclonal antibody 3E5 of purifying;
2. with biological sample and the insulation of immobilised capture antibodies, ChIFN-γ is combined with capture antibodies;
The ChIFN-γ that is attached on the immobilization capture antibodies is contacted with biotin labeled anti-ChIFN-γ monoclonal antibody 3E3, detect ChIFN-γ.
2, method as claimed in claim 1, wherein biological sample separates from chicken serum, chicken plasma or cells and supernatant.
3, the process of claim 1 wherein that step holding temperature 1. is 4 ℃, temperature retention time is 24 hours; Step holding temperature 2. is 37 ℃, and temperature retention time is 2 hours; Step holding temperature 3. is 37 ℃, and temperature retention time is 1 hour
4, the method for one of claim 1-3, wherein capture antibodies is the immobilization monoclonal antibody.
5, the method for claim 4, the amount of immobilised capture antibodies are 85 μ g/ml.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101788563A (en) * | 2010-02-03 | 2010-07-28 | 华中农业大学 | Spotted deer gamma-interferon double-antibody sandwich ELISA detection method, kit thereof and application of kit |
| CN102608328A (en) * | 2012-03-06 | 2012-07-25 | 无锡市普生生物工程技术有限公司 | TRFIA (Time-resolved Fluorescence Immunoassay) kit for mouse interferon alpha and detection method thereof |
| CN103344630A (en) * | 2013-08-09 | 2013-10-09 | 郑州安图生物工程股份有限公司 | Kit for quantitatively detecting mycobacterium tuberculosis gamma interferon |
| CN105759054A (en) * | 2016-03-31 | 2016-07-13 | 扬州大学 | ELISPOT detection kit for detecting bovine brucellosis |
| CN107286241A (en) * | 2017-07-10 | 2017-10-24 | 中国农业大学 | A kind of method and its dedicated kit for detecting fowl interferon content |
-
2008
- 2008-11-25 CN CNA2008102354382A patent/CN101441220A/en active Pending
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101788563A (en) * | 2010-02-03 | 2010-07-28 | 华中农业大学 | Spotted deer gamma-interferon double-antibody sandwich ELISA detection method, kit thereof and application of kit |
| CN101788563B (en) * | 2010-02-03 | 2013-03-06 | 华中农业大学 | Spotted deer gamma-interferon double-antibody sandwich ELISA detection method, kit thereof and application of kit |
| CN102608328A (en) * | 2012-03-06 | 2012-07-25 | 无锡市普生生物工程技术有限公司 | TRFIA (Time-resolved Fluorescence Immunoassay) kit for mouse interferon alpha and detection method thereof |
| CN103344630A (en) * | 2013-08-09 | 2013-10-09 | 郑州安图生物工程股份有限公司 | Kit for quantitatively detecting mycobacterium tuberculosis gamma interferon |
| CN105759054A (en) * | 2016-03-31 | 2016-07-13 | 扬州大学 | ELISPOT detection kit for detecting bovine brucellosis |
| CN107286241A (en) * | 2017-07-10 | 2017-10-24 | 中国农业大学 | A kind of method and its dedicated kit for detecting fowl interferon content |
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Application publication date: 20090527 |