CN102965343A - Hybridoma cell strain secreting bovine gamma-interferon monoclonal antibody, monoclonal antibody thereof and application of monoclonal antibody - Google Patents
Hybridoma cell strain secreting bovine gamma-interferon monoclonal antibody, monoclonal antibody thereof and application of monoclonal antibody Download PDFInfo
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Abstract
The invention provides a hybridoma cell strain secreting a bovine gamma-interferon monoclonal antibody, the monoclonal antibody thereof and the application of the monoclonal antibody. The preservation number of the hybridoma cell strain secreting the bovine gamma-interferon monoclonal antibody provided by the invention is CCTCC NO: C2012107. The monoclonal antibody secreted by the hybridoma cell strain provided by the invention has the advantages of being high in titer, good in specificity and strong in affinity with a natural antigen; and a BoIFN-gamma ELISPOT detection kit established based on the monoclonal antibody is good in sensitivity and specificity, and can be used for the bovine body immune state evaluation and related researches of infectious disease diagnosis.
Description
Technical field
The present invention relates to secrete the moggy gamma interferon monoclonal antibody hybridoma cell strain, its secretion monoclonal antibody and in the application of immunodetection and diagnostic field.
Background technology
IFN-γ (IFN-γ) mainly is by being stimulated and the CD4 of activation by mitogen or specific antigens
+Th1 cell, CD8
+The cytokine that T cell and NK cell etc. produce has extensively antiviral, anti-tumor activity and immunoloregulation function, is the important component part of body defending system.The height that studies show that endogenous IFN-γ level can reflect the cellular immunity of body to a great extent, and the IFN-gamma reaction of antigen-specific then can be used as body for the index of the cellular immunity of certain specific exotic antigen.
IFN-γ and monoclonal antibody thereof (McAb) have a wide range of applications in the animal and veterinary field, gene recombination ox IFN-γ (rBoIFN-γ) and McAb thereof are used for the research of cattle disease, the BoIFN-γ test of antigen-specific has been used to the detection of multiple rinderpest disease in a lot of developed countries, especially with being most widely used in diagnosing bovine tuberculosis, relevant research is also deep.
The immunology detection of IFN-γ is the experimental technique that is used for the IFN-γ of sample is carried out qualitative and quantitative analysis of setting up on the basis of the anti-IFN-γ of specificity McAb.Use different monoclonal antibody marker and detection technique, developed several different methods, such as reversed passive hemagglutination test (RPH), enzyme immunoassay (EIA), enzyme-linked immunosorbent assay (ELISA), enzyme linked immunological spotting method (ELISPOT), flow cytometry (FCM) analytical method etc.
Enzyme linked immunological Spot Jest (the Enzyme-linked Immunospot that nineteen eighty-three sets up, abbreviation ELISPOT) technology has susceptibility and the widespread popularity of height, become in recent years one of the most attracting immunologic detection method, be used widely at the aspects such as research and development, clinical diagnosis and fundamental research of vaccine.Compare with traditional antiviral activity analytical procedure (AVA) that is used for IFN-γ detection, the sensitivity and the specificity that are based upon the IFN-γ detection method on the immune response basis are higher, as the important indicator of cellular immunity, the ELISPOT detection method of IFN-γ has been widely used in the diagnosis of pathogen infection in Research foundation and clinical immunology, immune effect of vaccine assessment, organ transplantation, anaphylaxis and the multiple born of the same parents etc. at present.
Summary of the invention
The purpose of this invention is to provide a kind of hybridoma cell strain of secreting the moggy gamma interferon monoclonal antibody, the monoclonal antibody of this hybridoma cell strain secretion, and the monoclonal antibody of this hybridoma cell strain and secretion thereof is in the application of immunodetection and diagnostic field.
One aspect of the present invention provides a kind of hybridoma cell strain of secreting the moggy gamma interferon monoclonal antibody, and the preserving number of described hybridoma cell strain is CCTCC NO:C2012107.
(address: register preservation Wuhan City Wuhan University), preserving number was CCTCC NO:C2012107 to this bacterial strain, and Classification And Nomenclature is: hybridoma cell line 2G5 at Chinese Typical Representative culture collection center on September 4th, 2012.
Second aspect present invention provides a kind of moggy gamma interferon monoclonal antibody, is hybridoma cell strain or its passage cell strain secretion generation of CCTCC NO:C2012107 by preserving number.
Third aspect present invention provides the hybridoma cell strain of aforementioned secretion moggy gamma interferon monoclonal antibody and aforementioned moggy gamma interferon monoclonal antibody in the detection reagent of preparation rinderpest disease or the application in the diagnostic reagent.
Fourth aspect present invention provides a kind of ELISPOT detection kit of moggy gamma interferon, and described test kit comprises:
1. be selected from following arbitrary:
1) millipore filtration plate and capture antibody;
2) be coated with the millipore filtration plate of capture antibody;
Described capture antibody is aforementioned moggy gamma interferon monoclonal antibody;
2. biotin labeling moggy gamma interferon monoclonal antibody;
In the described test kit, the millipore filtration plate can be coated with capture antibody in advance, also can only provide blank millipore filtration plate and capture antibody, adopts voluntarily ordinary method at the coated capture antibody of millipore filtration plate by the operator before detection.
Described millipore filtration plate can be the millipore filtration plate of various common specifications, such as 96 hole filter membrane plates.
Moggy gamma interferon monoclonal antibody in the described biotin labeling moggy gamma interferon monoclonal antibody is different from aforementioned capture antibody.The method of biotin labeling moggy gamma interferon monoclonal antibody adopts conventional.
Further preferred, the moggy gamma interferon monoclonal antibody in the described biotin labeling moggy gamma interferon monoclonal antibody is hybridoma cell strain or its passage cell strain secretion generation of CCTCC NO:C2012108 by preserving number.
Preserving number be CCTCC NO:C2012108 hybridoma cell strain on September 4th, 2012 at Chinese Typical Representative culture collection center (address: Wuhan City Wuhan University) registration preservation, Classification And Nomenclature is: hybridoma cell line 5E11.
Further, also comprise in the following reagent one or more in the described test kit:
1) avidin-alkaline phosphatase enzyme conjugates;
2) substrate solution;
3) washings.
Mentioned reagent is the common reagent in the ELISOPT detection, is not subjected to the restriction of concrete test item, therefore can add selectively as required test kit, also can be disposed voluntarily or be bought separately by the operator.Be the handled easily person, optimum selection is to comprise simultaneously avidin-alkaline phosphatase enzyme conjugates, substrate solution and washings in the test kit.
Described substrate solution can be universal substrate liquid commonly used in the ELISPOT detection kit, such as liquid nitrogen blue tetrazolium/5-bromo-4-chloro-3-indoles phosphoric acid (NBT/BCIP) substrate solution.
Described washings can be in the ELISPOT detection kit washings commonly used, such as phosphate buffered saline buffer etc.Can select as required concentrated or unconcentrated washings.
Further, can also comprise selectively in the described test kit that other ELISPOT detect required common reagent, such as confining liquid, cell culture fluid, phosphate buffered saline buffer, phosphoric acid salt tween damping fluid etc.
Described confining liquid can be coated millipore filtration plate confining liquid commonly used, such as separated milk, FBS, BSA or casein etc.
Generally, in the test kit of the present invention, each reagent is isolated respectively storage.
The present invention is further based on above-mentioned BoIFN-γ ELISPOT detection kit, set up the ELISPOT detection method of the moggy gamma interferon with better specificity and sensitivity, for detection of the T lymphocyte of secretion moggy gamma interferon, thereby carry out the research of immune status evaluation and medical diagnosis on disease aspect.
Test kit of the present invention both can be used for analyzing ox peripheral blood mononuclear cell sample, also can be used for analyzing the ox whole blood sample.
The detection method of utilizing test kit of the present invention to detect ox peripheral blood mononuclear cell sample comprises the following steps:
1, the coated millipore filtration plate of capture antibody;
2, preparation ox peripheral blood mononuclear cell suspension;
3, cell is hatched and is detected
1. add following reagent in the coated millipore filtration plate of above-mentioned capture antibody: 50 μ L cell culture fluids are to each control wells, and the dyers' grapes (PWM) of 50 μ L, 5 μ g/mL are to each positive hole.Every hole adds 50 μ L cell suspensions, and 96 hole filter membrane plates are placed 37 ℃, 5%CO
2Incubator was cultivated 24-48 hour;
2. abandon culture supernatant, wash plate after, add the biotin labeling moggy gamma interferon monoclonal antibody of 1 μ g/mL, 100 μ L/ holes place 37 ℃ to hatch 1 hour;
3. after washing plate, the avidin of adding dilution-alkaline phosphatase enzyme conjugates, 100 μ L/ holes place 37 ℃ to hatch 1 hour;
4. every hole adds 100 μ L substrate solutions, is positioned over the colour developing of room temperature lucifuge.In 96 hole filter membrane plates, add the pure water termination reaction, remove liquid, spend the night under the room temperature or 37 ℃ of baking boxs in oven dry in 2-3 hour;
5. use inverted microscope to count the spot of purple in each reacting hole, each point represents the T cell of a secretion moggy gamma interferon; Or the millipore filtration plate put into the ELISPOT instrument, experimental result is carried out scanning and counting and analysis.
Aforesaid method is by density gradient centrifugation Separation of Bovine peripheral blood mononuclear cell, with the positive sample of ox peripheral blood mononuclear cell that stimulates through dyers' grapes, take without the ox peripheral blood mononuclear cell that stimulates as control sample, and the monoclonal antibody of the secretion of the hybridoma cell strain take the preserving number of purifying as CCTCC NO:C2012017 is as capture antibody, take biotin labeled another moggy gamma interferon monoclonal antibody as detecting antibody, the result shows that the method can effectively detect the T lymphocyte of secretion moggy gamma interferon.
The detection method of utilizing test kit of the present invention to detect the ox whole blood sample comprises the following steps:
1, the coated millipore filtration plate of capture antibody;
2, whole blood is hatched and is detected
1. the aseptic 1mL of taking ox blood adds the heparin tube that contains heparin sodium, puts upside down mixing and obtain anticoagulation after gathering blood;
2. add following reagent in the coated millipore filtration plate of above-mentioned capture antibody: 50 μ L nutrient solutions are to each control wells, and the PWM of 50 μ L5 μ g/mL is to each positive hole.Every hole adds 50 μ L ox whole bloods (with sterilization PBS 1:1 dilution), and 96 hole filter membrane plates are placed 37 ℃, 5%CO
2Incubator was cultivated 24-48 hour;
3. abandon culture supernatant, wash plate after, add the biotin labeling moggy gamma interferon monoclonal antibody of 1 μ g/mL, 100 μ L/ holes place 37 ℃ to hatch 1 hour;
4. wash plate, the avidin of adding 1:1000 dilution-alkaline phosphatase enzyme conjugates, 100 μ L/ holes place 37 ℃ to hatch 1 hour;
5. every hole adds 100 μ L substrate solutions, is positioned over the colour developing of room temperature lucifuge.In 96 hole filter membrane plates, add the pure water termination reaction, remove liquid, spend the night under the room temperature or 37 ℃ of baking boxs in oven dry in 2-3 hour;
6. use inverted microscope to count the spot of purple in each reacting hole, each point represents the T cell of a secretion moggy gamma interferon; Or 96 hole filter membrane plates are put into the ELISPOT instrument, experimental result is carried out scanning and counting and analysis.
Technique effect of the present invention:
The monoclonal antibody of hybridoma cell strain of the present invention secretion has the height of tiring, advantage that specificity is good, strong with natural antigen avidity, BoIFN-γ ELISPOT detection kit based on this foundation, when detecting ox peripheral blood lymphocytes sample, the specificity spot digital display work that occurs in the positive hole is more than the control sample hole, show that this test kit can effectively detect the T lymphocyte that PWM stimulates secretion moggy gamma interferon in the ox peripheral blood, has preferably sensitivity and specificity.In addition, this test kit is by carrying out direct-detection to the ox whole blood, and the result shows the T lymphocyte that can detect equally secretion moggy gamma interferon in the positive hole.Adopt test kit of the present invention easy and simple to handle, greatly shortened detection time, can be widely used in immunology research, as evaluation and the relevant research of infectious diseases diagnosis of ox immune status.
Description of drawings
Fig. 1. ox peripheral blood mononuclear cell sample detection is figure as a result
A.PWM stimulates ox peripheral blood mononuclear cell detected result; B. do not stimulate ox peripheral blood mononuclear cell detected result Fig. 2. ox whole blood sample detected result figure
A.PWM stimulates ox whole blood test result; B. do not stimulate ox whole blood test result
Embodiment
Below by specific specific examples explanation embodiments of the present invention, those skilled in the art can understand other advantages of the present invention and effect easily by the disclosed content of this specification sheets.The present invention can also be implemented or be used by other different embodiment, and the every details in this specification sheets also can be based on different viewpoints and application, carries out various modifications or change under the spirit of the present invention not deviating from.
Unless otherwise indicated, disclosed experimental technique, detection method, preparation method all adopt the routine techniques of molecular biology, biological chemistry, chromatin Structure and analysis, analytical chemistry, cell cultures, recombinant DNA technology and the association area of the art routine among the present invention.These technology are existing perfect explanation in existing document, specifically can be referring to MOLECULAR CLONING:A LABORATORY MANUAL such as Sambrook, Second edition, Cold Spring Harbor Laboratory Press, 1989 and Third edition, 2001; Ausubel etc., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley﹠amp; Sons, New York, 1987and periodic updates; The series METHODS IN ENZYMOLOGY, Academic Press, San Diego; Wolffe, CHROMATIN STRUCTURE AND FUNCTION, Third edition, Academic Press, San Diego, 1998; METHODS IN ENZYMOLOGY, Vol.304, Chromatin (P.M.Wassarman and A.P.Wolffe, eds.), Academic Press, San Diego, 1999; With METHODS IN MOLECULAR BIOLOGY, Vol.119, Chromatin Protocols (P.B.Becker, ed.) Humana Press, Totowa, 1999 etc.
The acquisition of embodiment 1 hybridoma cell strain
Preserving number is that hybridoma cell strain and the preserving number of CCTCC NO:C2012107 is the acquisition of the hybridoma cell strain of CCTCC NO:C2012108.
1. animal immune
Concrete immune programme for children is as follows: first immunisation, subcutaneous abdomen multi-point injection 100 μ g are through the adequately emulsified recombinant bovine IFN-γ of Freund's complete adjuvant purifying protein, the subcutaneous multi-point injection 100 μ g of 2 all postabdomens carry out second immunisation through the adequately emulsified purifying protein of Freund's incomplete adjuvant, interval 2 all pneumoretroperitoneums are injected the purifying protein that 100 μ g do not add adjuvant and are carried out for the third time immunity, serum antibody titer is measured in posterior orbit blood sampling in 7 days, chooses the higher mouse of tiring and carries out the purifying protein that tail vein booster immunization 100 μ g do not add adjuvant.
2. cytogamy
Concrete steps are as follows: behind the tail vein booster immunization 3d, gather a small amount of blood, separation of serum-20 is ℃ frozen, the positive control during as screening.Put to death immunized mice by the Biosafety method, 75% alcohol-pickled sterilization 5min, aseptic extracting spleen cell be in the myeloma cell SP2/0 of logarithmic phase at PEG(MW4000) the lower fusion of effect, with the ICR Turnover of Mouse Peritoneal Macrophages as feeder cell, merging good cell and feeder cell suspends with the HAT substratum, packing 96 orifice plates are put 37 ℃, 6%CO
2Cultivate in the incubator.Add fresh HAT substratum behind the 5d, use the HT substratum behind the 10d instead and cultivate, routine observation changes liquid and detection.
3. the foundation of indirect ELISA detection method
Adopt indirect ELISA method screening positive cell clone.The coated concentration of detectable antigens is determined in the square formation test.
Detectable antigens dilutes with coated damping fluid transverse gradients, and every hole 50 μ l are coated with elisa plate, and 4 ℃ are spent the night; PBST washing 3 times, every hole adds 200 μ l confining liquids, and 4 ℃ are spent the night; The vertical doubling dilution of immune mouse serum, every hole 50 μ l, the same multiple dilution of normal mouse serum is hatched 2h for 37 ℃ as negative control; With PBST washing 3 times, add the ELIAS secondary antibody of working concentration, every hole 50 μ l are hatched 1.5h for 37 ℃; After the PBST washing, the OPD colour developing, enzyme connection detector is measured OD
490Value, judge the coated concentration of the best of detectable antigens.
The coated concentration of the detectable antigens that test is determined according to square formation, detectable antigens 50 μ l/ holes after the dilution are added in the enzyme plate, 4 ℃ are spent the night, PBST washing lotion washing 3 times, 5min/ time, spend the night washing with the 4 ℃ of sealings of PBST washing lotion that contain 10% calf serum,-20 ℃ of preservations are used for the screening positive cell clone.
4. screening positive clone
The indirect ELISA method that employing establishes detects the situation of hybridoma secretory antibody.Concrete grammar is as follows: the Hybridoma Cell Culture supernatant added in advance in the coated good elisa plate, and 50 μ l/ holes, as negative control, the immunized mice polyvalent antibody is as positive control with the SP2/0 cell conditioned medium, 37 ℃ of water-bath 2h; PBST washing 3 times; The sheep anti-mouse igg and the IgM antibody that add the HRP mark of working concentration, 50 μ l/ holes, 37 ℃ of water-bath 1.5h; After the washing, the OPD 10 ~ 15min that develops the color, microplate reader was measured OD after colour developing stopped
490Reading.Measured hole OD
490Reading is greater than being judged to be the positive more than the negative control twice.With two strain positive colonies difference called after 2G5 and the 5E11 that screens.
5. the cloning of positive hybridoma cell
Adopt the subclone that limiting dilution assay carries out 2 ~ 3 times to the positive cell clone 2G5 that screens and 5E11 and carry out preservation.The corresponding preserving number of positive cell clone 2G5 is the hybridoma cell strain of CCTCC NO:C2012107, and the corresponding preserving number of positive cell clone 5E11 is the hybridoma cell strain of CCTCC NO:C2012108.
The preparation of embodiment 2 moggy gamma interferon monoclonal antibodies
1. ascites preparation
Adopt in the body and induce the ascites method, carry out according to a conventional method.10 ~ 12 age in week healthy BALB/c mouse abdominal injection whiteruss 0.3 ~ 0.5ml/ only, behind 7 ~ 10d, intraperitoneal inoculation is through the hybridoma 2G5 and the 5E11 that are cultured to logarithmic phase of PBS dilution, 5 * 10 respectively
5Individual cell/only; Collect ascites behind the 7d, centrifugal removal precipitation is collected supernatant, and indirect elisa method is measured antibody titer, packing ,-70 ℃ of preservations.The corresponding hybridoma 2G5 of hybridoma cell strain CCTCC NO:C2012107() monoclonal antibody of secretion is designated as 2G5, the corresponding hybridoma 5E11 of hybridoma cell strain CCTCC NO:C2012108() monoclonal antibody of secretion is designated as 5E11.
2. antibody purification mark
Use Protein G affinity chromatography method to carry out purifying 2G5 and the 5E11 ascites of preparation, and monoclonal antibody 5E11 is carried out biotin labeling.
Adopt standard biological element labelling method to carry out mark purifying 5E11 monoclonal antibody.Dissolving 2 ~ 10mg monoclonal antibody 5E11 albumen is in the phosphate buffered saline buffer of 1ml, and the mmole number of calculating dissolving; The balance vitamin H adds 2mg Sulfo-NHS-Biotin in the 100ul ultrapure water to room temperature, adds certain density vitamin H; Room temperature 30 minutes, or placed on ice 2 hours; With 30ml PBS prewashing purification column, loading, add the damping fluid identical with wanting collecting amount, collect 0.5ml or 1ml in independent pipe, measure the monoclonal antibody protein content with the absorption value of 280nm.
Embodiment 3 monoclonal antibody Characteristics Detection
1. the evaluation of monoclonal antibody subclass
Undertaken by monoclonal antibody subclass test kit specification sheets, adopt the ELISA method of antigen mediation.In coated enzyme plate, add respectively cells and supernatant 50 μ l/ holes, 37 ℃ of 1h, PBST washing 3 times, each 5min; The sheep anti-mouse igg that adds respectively the 1:1000 dilution
1, IgG
2a, IgG
2b, IgG
3, IgM Subclass Antibodies 50 μ l/ holes, 37 ℃ of 0.5h, every strain monoclonal antibody adds every kind of subclass two holes, PBST washs 3 each 5min; The anti-sheep ELIAS secondary antibody of the rabbit 50 μ l/ holes that add the 1:5000 dilution, 37 ℃ of 15min, PBST washing 3 times; Add nitrite ion O-Phenylene Diamine (OPD) solution 50 μ l/ holes, 37 ℃ of lucifuge colour developing 10 ~ 15min, 2M H
2SO
450 μ l/ hole termination reactions are take the visual inspection color apparently higher than other hole Subclass Antibodies that the person is added as the monoclonal antibody subclass.
The result shows that monoclonal antibody 2G5 and 5E11 subclass are IgG
1
2. the odd contradictive hydroperitoneum mensuration of tiring
Use coated damping fluid that detectable antigens is diluted to finite concentration, every hole 50 μ l are coated with elisa plate, and 4 ℃ are spent the night; PBST washing 3 times, every hole adds 200 μ l confining liquids, and 4 ℃ are spent the night; With the odd contradictive hydroperitoneum doubling dilution, every hole 50 μ l, same multiple dilution SP2/0 ascites is hatched 2h for 37 ℃ as negative control; With PBST washing 3 times, add the ELIAS secondary antibody of working concentration, every hole 50 μ l are hatched 1.5h for 37 ℃; After the PBST washing, the OPD colour developing, enzyme connection detector is measured OD
490Value, take P/N value 〉=2.1 as criterion, measure odd contradictive hydroperitoneum and tire.
The result shows that tiring of monoclonal antibody 2G5 and 5E11 reaches respectively 1:2048000 and 1:320000.
3. the specific evaluation of monoclonal antibody
Identify the specificity of monoclonal antibody with Dot-ELISA, concrete steps are as follows: a certain size NC film of clip, dry after soaking in deionized water; Draw respectively rHis-BoIL-4, rGST-BoIL-4, rHis-BoIFN-γ, rGST-BoIFN-γ, rHis-ChIFN-γ, rHis-ChIL-4, the rGST-ChIL-4 of working concentration with pipettor, and BL21 (DE3) (pET) and each 5 μ l point of the ultrasonic degradation liquid supernatant of BL21 (pGEX-6P-1) after IPTG induces on the NC film, after 37 ℃ of 30min dryings, with the PBST sealing that contains 10% calf serum, the room temperature jog spends the night; After the PBST washing, again the NC film is immersed in the ascites of cells and supernatant or dilution, hatch 2h for 37 ℃; PBST washing 3 times, each 10min immerses in sheep anti mouse HRP-Ig (G+M) the enzyme labelled antibody solution of working concentration again, hatches 1h for 37 ℃; PBST washing 3 times, each 10min, last DAB colour developing, distilled water termination reaction.
In Dot-ELISA test, monoclonal antibody 2G5 and 5E11 only with the rBoIFN-gamma reaction, and not with above-mentioned other recombinant cytokine and the reaction of contrast bacterium of prokaryotic expression.
The assembling of embodiment 4 test kits
Step:
1) preparation of biotin labeling moggy gamma interferon monoclonal antibody (being designated as Bio-5E11):
Adopt standard biological element labelling method to carry out mark purifying 5E11 monoclonal antibody.Dissolving 2 ~ 10mg monoclonal antibody 5E11 albumen is in the phosphate buffered saline buffer of 1ml, and the mmole number of calculating dissolving; The balance vitamin H adds 2mg Sulfo-NHS-Biotin in the 100ul ultrapure water to room temperature, adds certain density vitamin H; Room temperature 30 minutes, or placed on ice 2 hours; With 30ml PBS prewashing purification column, loading, add the damping fluid identical with wanting collecting amount, collect 0.5ml or 1ml in independent pipe, measure the monoclonal antibody protein content with the absorption value of 280nm.
2) with 96 hole filter membrane plates, monoclonal antibody 2G5, biotin labeling moggy gamma interferon monoclonal antibody respectively packaging group dress up test kit.
Further, foundation need to be assembled in the test kit: one or more in avidin-alkaline phosphatase enzyme conjugates, NBT/BCIP substrate solution, concentrated PBS damping fluid, confining liquid, cell culture fluid, phosphate buffered saline buffer or the phosphoric acid salt tween damping fluid.
In the test kit, millipore filtration plate, monoclonal antibody 2G5 also can adopt the millipore filtration plate of coated moggy gamma interferon monoclonal antibody to substitute.
The preparation method of the millipore filtration plate of coated moggy gamma interferon monoclonal antibody:
What 1. add 2.5 μ g/mL in the 96 hole filter membrane plates catches moggy gamma interferon antibody 2G5,100 μ L/ holes, and 4 ℃ are spent the night coated;
2. abandon coating buffer, use the sterilization PBST that contains 0.05% tween to wash plate 3 times, 5min/ time;
3. add complete 1640 substratum that contain 10% foetal calf serum, sealing 2h is hatched for 37 ℃ in 200 μ L/ holes;
4. abandon confining liquid, use PBST to wash plate 1 time, 96 hole filter membrane plates are put in the sealing bag, put 4 ℃ of preservations.
The detection of 5 Ns of peripheral blood mononuclear cell samples of embodiment
1. antibody sandwich
What 1. add 2.5 μ g/mL in the 96 hole filter membrane plates catches moggy gamma interferon antibody 2G5,100 μ L/ holes, and 4 ℃ are spent the night coated;
2. abandon coating buffer, use the sterilization PBST that contains 0.05% tween to wash plate 3 times, 5min/ time;
3. add complete 1640 substratum that contain 10% foetal calf serum, sealing 2h is hatched for 37 ℃ in 200 μ L/ holes;
4. abandon confining liquid, use PBST to wash plate 1 time, 96 hole filter membrane plates are directly used in detection, or are put in the sealing bag, put 4 ℃ of preservations, use in 1 week.
2. the preparation of ox peripheral blood mononuclear cell
1. the aseptic 5mL of taking ox blood adds the heparin tube that contains heparin sodium, puts upside down mixing and obtain anticoagulation after gathering blood;
2. after anticoagulation being diluted with sterilization PBS 1:1, will dilute ox blood in the ratio of 1:1 again and slowly add in the aseptic centrifuge tube that contains the ox lymphocyte separation medium, form sharp interface, the at room temperature centrifugal 20-30min of 2000rpm;
3. visible peripheral blood mononuclear cell is present in the cloud layer, draws in the clean centrifuge tube of buffy coat to the sterilization dropper, adds sterilization PBS, behind the cell mixing, in the centrifugal 10min of room temperature 2000rpm, repeats twice;
4. the supernatant discarded nutrient solution adds the resuspended sedimentation cell of complete 1640 substratum, gets to add blood counting chamber after 10 μ L cell suspensions add the blue mixing of 10 μ L platform phenol, at the microscopically counting, and uses complete 1640 substratum that cell suspension is diluted to 1 * 10
7Individual cell/mL.
3. cell is hatched and is detected
1. add following reagent in above-mentioned 96 coated hole filter membrane plates: 50 μ L nutrient solutions are to each control wells, and the PWM of 50 μ L, 5 μ g/mL is to each positive hole.Every hole adds 50 μ L cell suspensions, places 37 ℃, 5% CO2 incubator to cultivate 24-48 hour 96 hole filter membrane plates;
2. 96 hole filter membrane plates are taken out, abandon culture supernatant, use PBST to wash plate 5 times, 5min/ time, dry after washing plate.The moggy gamma interferon that adds 1 μ g/mL detects antibody Bio-5E11, and 100 μ L/ holes place 37 ℃ to hatch 1 hour;
3. wash plate 3 times with PBS, 5min/ time, dry after washing plate, add the avidin of 1:1000 dilution-alkaline phosphatase enzyme conjugates, 100 μ L/ holes place 37 ℃ to hatch 1 hour;
4. every hole adds 100 μ L substrate solution nitroblue tetrazolium(NBT)s and 5-bromo-4-chloro-3-indoles phosphoric acid, is positioned over the colour developing of room temperature lucifuge.In 96 hole filter membrane plates, add the pure water termination reaction, remove liquid, spend the night under the room temperature or 37 ℃ of baking boxs in oven dry in 2-3 hour;
5. use inverted microscope to count the spot of purple in each reacting hole, each point represents the T cell of a secretion moggy gamma interferon; Or 96 hole filter membrane plates are put into the ELISPOT instrument, experimental result is carried out scanning and counting and analysis.Test-results is referring to Fig. 1, the result is presented at when detecting ox peripheral blood lymphocytes sample, the specificity spot digital display work that occurs in the positive hole is more than the control sample hole, this shows that this test kit can effectively detect the T lymphocyte that PWM stimulates secretion moggy gamma interferon in the ox peripheral blood, has preferably sensitivity and specificity.
The detection of 6 Ns of whole blood samples of embodiment
1. antibody sandwich
What 1. add 2.5 μ g/mL in the 96 hole filter membrane plates catches moggy gamma interferon antibody 2G5,100 μ L/ holes, and 4 ℃ are spent the night coated;
2. abandon coating buffer, use the sterilization PBST that contains 0.05% tween to wash plate 3 times, 5min/ time;
3. add complete 1640 substratum that contain 10% foetal calf serum, sealing 2h is hatched for 37 ℃ in 200 μ L/ holes;
4. abandon confining liquid, use PBST to wash plate 1 time, 96 hole filter membrane plates are directly used in detection, or are put in the sealing bag, put 4 ℃ of preservations, use in 1 week.
2. whole blood is hatched and is detected
1. the aseptic 1mL of taking ox blood adds the heparin tube that contains heparin sodium, puts upside down mixing and obtain anticoagulation after gathering blood;
2. add following reagent in above-mentioned 96 coated hole filter membrane plates: 50 μ L nutrient solutions are to each control wells, and the PWM of 50 μ L, 5 μ g/mL is to each positive hole.Every hole adds 50 μ L ox whole bloods (with sterilization PBS 1:1 dilution), and 96 hole filter membrane plates are placed 37 ℃, 5%CO
2Incubator was cultivated 24-48 hour;
3. 96 hole filter membrane plates are taken out, abandon culture supernatant, use PBST to wash plate 5 times, 5min/ time, dry after washing plate.The moggy gamma interferon that adds 1 μ g/mL detects antibody Bio-5E11, and 100 μ L/ holes place 37 ℃ to hatch 1 hour;
4. wash plate 3 times with PBS, 5min/ time, dry after washing plate, add the avidin of 1:1000 dilution-alkaline phosphatase enzyme conjugates, 100 μ L/ holes place 37 ℃ to hatch 1 hour;
5. every hole adds 100 μ L substrate solution nitroblue tetrazolium(NBT)s and 5-bromo-4-chloro-3-indoles phosphoric acid, is positioned over the colour developing of room temperature lucifuge.In 96 hole filter membrane plates, add the pure water termination reaction, remove liquid, spend the night under the room temperature or 37 ℃ of baking boxs in oven dry in 2-3 hour;
6. use inverted microscope to count the spot of purple in each reacting hole, each point represents the T cell of a secretion moggy gamma interferon; Or 96 hole filter membrane plates are put into the ELISPOT instrument, experimental result is carried out scanning and counting and analysis.The result as shown in Figure 2.The result shows that test kit of the present invention also can be used for the ox whole blood is carried out direct-detection, and the result can detect the T lymphocyte of secretion moggy gamma interferon in the positive hole equally.
Above-described embodiment is illustrative principle of the present invention and effect thereof only, but not is used for restriction the present invention.Any person skilled in the art scholar all can be under spirit of the present invention and category, and above-described embodiment is modified or changed.Therefore, have in the technical field under such as and know that usually the knowledgeable modifies or changes not breaking away from all equivalences of finishing under disclosed spirit and the technological thought, must be contained by claim of the present invention.
Claims (8)
1. hybridoma cell strain of secreting the moggy gamma interferon monoclonal antibody, the preserving number of described hybridoma cell strain is CCTCC NO:C2012107.
2. a moggy gamma interferon monoclonal antibody is hybridoma cell strain or its passage cell strain secretion generation of CCTCC NO:C2012107 by preserving number.
3. secrete as claimed in claim 1 the hybridoma cell strain of moggy gamma interferon monoclonal antibody or the described moggy gamma interferon monoclonal antibody of claim 2 in the detection reagent of preparation rinderpest disease or the application in the diagnostic reagent.
4. the ELISPOT detection kit of a moggy gamma interferon, described test kit comprises:
A. be selected from following arbitrary:
1) the described moggy gamma interferon monoclonal antibody of millipore filtration plate, and claim 2;
2) be coated with the millipore filtration plate of the described moggy gamma interferon monoclonal antibody of claim 2;
B. biotin labeling moggy gamma interferon monoclonal antibody.
5. the ELISPOT detection kit of moggy gamma interferon as claimed in claim 4 is characterized in that the moggy gamma interferon monoclonal antibody in the described biotin labeling moggy gamma interferon monoclonal antibody is different from the described moggy gamma interferon monoclonal antibody of claim 2.
6. the ELISPOT detection kit of moggy gamma interferon as claimed in claim 4, it is characterized in that the moggy gamma interferon monoclonal antibody in the described biotin labeling moggy gamma interferon monoclonal antibody is hybridoma cell strain or its passage cell strain secretion generation of CCTCC NO:C2012108 by preserving number.
7. the ELISPOT detection kit of moggy gamma interferon is characterized in that as described in arbitrary such as claim 4-6, also comprises in the following reagent one or more in the described test kit:
1) avidin-alkaline phosphatase enzyme conjugates;
2) substrate solution;
3) washings.
8. the ELISPOT detection kit of moggy gamma interferon as claimed in claim 7 is characterized in that described substrate solution is NBT/BCIP.
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