CN102876635A - Haemophilus parasuis outer membrane protein P5 (OMP5) resistant monoclonal antibody, hybridoma cell strain and application - Google Patents
Haemophilus parasuis outer membrane protein P5 (OMP5) resistant monoclonal antibody, hybridoma cell strain and application Download PDFInfo
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Abstract
The invention discloses a haemophilus parasuis outer membrane protein P5 (OMP5) resistant monoclonal antibody, a hybridoma cell strain and an application. The hybridoma cell strain is preserved in the China center for type culture collection (CCTCC), and the preservation serial number is CCTCCC2012135. The monoclonal antibody prepared by the hybridoma cell strain is good in specificity, high in valence, high in generality, free from cross reaction with swine Escherichia coli, swine pasteurella, swine pleuropneumonia actinobacillus, streptococcus suis and swine bordetella bacilli, capable of detecting haemophilus parasuis with different serotypes and widely applicable to etiology diagnosis, serology detection and immunology detection and prevention of haemophilus parasuis diseases, and the enzyme-linked immuno sorbent assay (ELISA) antibody valence can reach 1:204800 after purification.
Description
Technical field
The invention belongs to field of immunology, be specifically related to monoclonal antibody, hybridoma cell strain and the application thereof of a kind of anti-haemophilus parasuis outer membrane protein OMP5.
Background technology
Haemophilus parasuis (
Haemophilus Parasuis, HPS) belonging to Pasteurellaceae, hemophilus is a kind ofly not have that mobility, small-sized, NAD rely on, polymorphic gram negative bacillus.HPS is a kind of bacterium of being everlasting, and usually field planting is at the upper respiratory tract of pig, and usually can be in nasal cavity, tonsilla and the tracheae leading portion be separated to, cause any clinical symptom and lose it.HPS also is the cause of disease of pig Ge Lazeshi sick (Glasser ' s diseas).Under certain condition, HPS can cause the systemic infection take fiber disposition polyserositis, sacroiliitis, meningitis as feature.The Ge Lazeshi disease becomes the predominantly bacteria disease that affects the pig industry in the world, brings massive losses to pig industry.The HPS that has found so far has 15 kinds of different serotypes at least, lacks effective cross immunity protectiveness between the different serotypes bacterial strain, and this has increased the difficulty of prevention Ge Lazeshi disease.Therefore, in time, accurately, fast and easily the etiological diagnosis method is significant for the control of pig Ge Lazeshi disease.
At present domestic diagnosis to haemophilus parasuis mainly contains traditional pathogen separation, the methods such as biochemical identification and serology, and the detection method of PCR-based etc.Immunological method such as ELISA, colloidal gold strip etc. have high specificity, highly sensitive, simple to operate, quick, to characteristics such as the instrument dependency are low, thereby often be used to that disease pathogen detects and the fields such as medicine trace analysis.But the at present domestic commercialization diagnostic method that not yet has based on the HPS detection of antibody.
The outer membrane protein of HPS (Outer member protein, OMP) is one of its Major Virulence Factors, and immunogenicity is strong.OMP5 is wherein main a kind of, and each serotype HPS bacterial strain has OMP5.Therefore OMP5 can be used as the specific diagnosis target position of HPS.
Summary of the invention
The object of the present invention is to provide a kind of hybridoma cell strain HPS 8D9 that secretes anti-haemophilus parasuis outer membrane protein OMP5 monoclonal antibody, be preserved in Chinese Typical Representative culture collection center (CHINA CENTER FOR TYPE CULTURE COLLECTION) on September 25th, 2012, deposit number is CCTCC C2012135.
It is the monoclonal antibody of the hybridoma cell strain preparation of CCTCC C2012135 that the present invention also provides by above-mentioned deposit number.
The present invention also provides said monoclonal antibody in the application that detects on the haemophilus parasuis outer membrane protein OMP5.
The technical solution adopted in the present invention is:
A kind of hybridoma cell strain HPS 8D9 that secretes anti-haemophilus parasuis outer membrane protein OMP5 monoclonal antibody is preserved in Chinese Typical Representative culture collection center on September 25th, 2012, and deposit number is CCTCC C2012135.
The monoclonal antibody of a kind of anti-haemophilus parasuis outer membrane protein OMP5 is the hybridoma cell strain generation of CCTCC C2012135 by deposit number.
Described monoclonal antibody is the IgG2b subclass.
A kind of test kit that detects haemophilus parasuis antibody, containing deposit number is the monoclonal antibody of the hybridoma cell strain generation of CCTCC C2012135.
A kind of test kit that detects haemophilus parasuis, containing deposit number is the monoclonal antibody of the hybridoma cell strain generation of CCTCC C2012135.
Beneficial effect of the present invention is:
The monoclonal antibody that the present invention prepares has the following advantages:
(1) specificity is good, with swine escherichia coli, pig pasteurellosis bacillus, actinobacillus pleuropneumoniae, swine streptococcus and the equal no cross reaction of pig bordetella bacilli;
(2) height of tiring, the ELISA antibody titer behind the purifying can reach 1:204800;
(3) highly versatile can both detect the different serotypes haemophilus parasuis;
(4) can produce in a large number;
(5) can be widely used in etiological diagnosis, serology detection, immunology detection and the control etc. of Haemophilus parasuis.
Description of drawings
Fig. 1 is techniqueflow chart of the present invention;
Fig. 2 is the specific detection result (M: albumen Marke of monoclonal antibody; P5:OMP5; 1: swine escherichia coli; 2: the streptococcus suis 2-type bacterial strain; 3: pig Pasteurella Multocida Strains A; 4: pig Pasteurella Multocida Strains B; 5: the actinobacillus pleuropneumoniae bacterial strain; 6: pig bordetella bacilli bacterial strain);
Fig. 3 is the versatility detected result (M: albumen Marker of monoclonal antibody; P5:OMP
51:1 type HPS; 2:3 type HPS; 3:4 type HPS; 4:5 type HPS; 5:10 type HPS; 6:11 type HPS; 7:12 type HPS; 8:14 type HPS; 9:15 type HPS; 10:: uncertain serotype HPS);
Fig. 4 is that monoclonal antibody is for detection of adhesion (the confocal laser scanning microscope) (A: monoclonal antibody of HPS on the PK15 cell
HPS8D9; B: anti-OMP5 positive serum contrast; C: the full bacterium positive serum contrast of anti-5 type HPS; D: the negative serum contrast).
Embodiment
Following examples are used for explanation the present invention, but are not used for limiting the scope of the invention.Without departing from the spirit and substance of the case in the present invention, modification or replacement to the inventive method, step or condition are done all belong to scope of the present invention.
If do not specialize, used chemical reagent is conventional commercial reagent among the embodiment, the conventional means that used technique means is well known to those skilled in the art among the embodiment.
Main experiment material and source:
Actinobacillus pleuropneumoniae 1 type (APP259 is available from China Veterinary Drugs Supervisory Inst.); HPS 3 type bacterial strains (SW114, Pat doctor Blackall of Queensland, Australia animal health institute is so kind as to give); HPS 11 types (H456, Pat doctor Blackall of Queensland, Australia animal health institute is so kind as to give); Swine escherichia coli, pig pasteurella multocida 2 strain bacterial strains, streptococcus suis 2-type bacterial strain, pig bordetella bacilli, HPS 1 type bacterial strain, HPS 4 type bacterial strains, HPS 5 type bacterial strains, HPS 10 type bacterial strains, HPS 12 type bacterial strains, HPS 14 type bacterial strains, HPS 15 type bacterial strains and 1 strain prepattern HPS bacterial strain separate, identify and preservation from the sick pig of In Guangdong Province by this laboratory.
One, immunogenic preparation
With HPS 5 type bacterial strains after the TSA flat board is cultivated 18～24h, picking list colony inoculation is 150rpm shaking culture 10～12h in 5mL TSB substratum, then be inoculated in the 100mL TSB substratum according to 1%～4%(v/v) ratio and cultivate 10h, gradient dilution, inspection is polluted in bacterial count simultaneously.6000r/min is behind the centrifugal 10min, with the PBS of pH7.4 washing and make 10
9The suspension of CFU/ml concentration, the formaldehyde of adding 0.1%～0.4%, 37 ℃ of deactivation 24h, for subsequent use.
Two, animal immune
Choose the female Balab/c mouse of body weight about 20g.First immunisation is carried out mixing and emulsifying with HPS bacterium liquid and the Freund's complete adjuvant of deactivation in the ratio of 1:1, dosage be 0.1～0.5mL/ only, injecting method is back intracutaneous multi-point injection.Every 2 ~ 3 all booster immunizations once, adjuvant was used Freund's incomplete adjuvant instead after head exempted from.Three exempt to cut the tail blood sampling about rear 1 week, collect serum; HPS 5 type bacterial strain outer membrane protein OMP5(preparation methods with expression and purification see document for details: Chen Shanzhen, Li Chunling etc., the foundation of haemophilus parasuis OMP5 gene cloning, expression and indirect ELISA detection method, Scientia Agricultura Sinica, 2011,44 (14): 3036-3044) antibody in the serum is carried out ELISA and detect.Immunity proceeds to antibody titer and no longer raises.Select the high mouse of antibody titer, at the cytogamy booster immunization of 0.1～0.5mL/ row of 3 days abdominal injection inactivated bacterial liquid that advances.
Three, the fusion of hybridoma, screening and enlarged culturing
Get splenocyte and the myelomatosis SP2/0 cell of the higher immune mouse of tiring, the PEG fusion method prepares hybridoma routinely; Antibody is with recombinant expressed OMP
5Carry out indirect ELISA and detect screening positive clone and enlarged culturing; Adopt simultaneously limiting dilution assay that positive colony is carried out subclone, carry out at least 3 times, to guarantee to obtain the hybridoma cell strain of stably excreting monoclonal antibody.Filter out the specific hybridoma cell strain HPS of hypersecretion 8D9 and be preserved in Chinese Typical Representative culture collection center, preservation date is on September 25th, 2012, and deposit number is CCTCC C2012135.Monoclonal antibody called after HPS 8D9 with its secretion.
The preparation of the used solution of ELISA
Coated damping fluid: (0.05mol/L carbonate buffer solution pH9.6): 1.59g Na
2CO
3, 2.93g Na
2HCO
3Be dissolved in the 800mL distilled water, regulate pH value to 9.6, and be settled to 1000mL.
Confining liquid: bovine serum albumin (BSA) 1g adds in the coating buffer of 100mL, and mixing is now with the current.
Lavation buffer solution (pH7.4 PBST): 0.2g KH
2PO
4, 2.9g Na
2HPO
412H
2O, 8.0g NaCl, 0.2g KCl, 0.5mL Tween-20(0.05% V/V) after the above accurate weighing of each material, after adding distilled water and dissolving fully, adjust pH to 7.4 is settled to 1000mL.
Antibody diluent: 0.5g BSA is dissolved among the 100mL PBST.
Substrate buffer solution: Na
2HPO
412H
2O 3.8g; Citric acid 0.98g accurately after the weighing, is dissolved in the 80mL distilled water, and adjust pH to 5.0 uses the distilled water constant volume to 100mL.
Tmb substrate nitrite ion: A liquid: TMB 50mg is dissolved among the 10mL DMSO 4 ℃ and keeps in Dark Place.B liquid: (pH5.0): 0.2 mol/L Na
2HPO
4.12H
2O 51.4mL, 0.1 mol/L citric acid 48.6mL, the 100mL ultrapure water adds 30% H again
2O
2112 μ L in brown bottle 4 ℃ keep in Dark Place.Mix in 1 ﹕, 50 ratios with front A liquid and B liquid.
Stop buffer: (2 mol/L H
2SO
4): distilled water 178.3mL dropwise adds the vitriol oil (98%) 21.7mL.
The indirect ELISA concrete operations are as follows:
1) coated: with coating buffer dilution restructuring HPS outer membrane protein OMP
5To concentration be 3.475 μ g/mL, 100 μ L/ holes add 96 hole polystyrene enzyme-linked reaction plates, 4 ℃ of coated spending the night; Discard coating buffer, every hole adds 250 μ L PBST washingss, leaves standstill 3min, abandons washings, elisa plate is firmly patted dry triplicate on thieving paper.
2) sealing: add confining liquid, 2h is sealed under 37 ℃ of conditions in 100 μ L/ holes, discards confining liquid, adds 250mL PBST in every hole, leaves standstill 3min, firmly pats dry triplicate on thieving paper.
3) application of sample: every hole add 100 μ L cells and supernatant ascites antibody behind the purifying, dilute the positive mice serum of anti-HPS with PBST by 1:1000, as positive control, get the negative contrast of SP2/0 culture supernatant, 37 ℃ of effect 1h act on and discard sample after complete.Every hole adds 250 μ L PBST and leaves standstill 3min, firmly pats dry triplicate on thieving paper.
4) add ELIAS secondary antibody: every hole adds the horseradish peroxidase mark goat anti-mouse antibody that 50 μ L dilute with PBST 1:4000,37 ℃ of effect 30min act ons and discard two after complete and resist, and every hole adds 250 μ L PBST and leaves standstill 3min, on thieving paper, firmly pat dry triplicate.
5) add nitrite ion: every hole adds 100 μ L nitrite ions, and nitrite ion will be with freshly prepared, lucifuge colour developing 10min under the room temperature condition.
6) add stop buffer: develop the color complete after, add immediately the stop buffer termination reaction, every hole 100 μ L.
7) survey the OD value: measure OD450nm place light absorption value with microplate reader.
Four,
The mensuration that hybridoma cell strain supernatant and ascites monoclonal antibody (HPS 8D9) are tired
With the ascites of cleer and peaceful preparation on the Hybridoma Cell Culture, then doubling dilution is used indirect ELISA (working method is with three) to measure it and is tired, and establish the positive, negative serum and SP2/0 cells and supernatant and make negative control continuously.The result shows that tiring of Hybridoma Cell Culture supernatant is 1:2560, and titer of ascites is 1:51200, and the antibody titer behind the purifying is 1:204800.
Five, a large amount of preparations of monoclonal antibody HPS 8D9 ascites
Get female Sexual health Balb/c mouse in 10 ages in week, 0.5ml/ abdominal injection pristane; After 1 week, 0.2～0.5 ml/ abdominal injection concentration is 5 * 10
7The hybridoma of/ml; Treat that mouse web portion obviously swells, have when touching skin with hand when compacting sense, collect ascites and with the centrifugal 5min of 2000rpm, supernatant liquor is the ascites that contains monoclonal antibody.Ascites is carried out doubling dilution from 1:100, then use indirect ELISA (working method is with four) to measure it and tire, the result shows that tiring of ascites is 1:51200.The ascites that obtains can be preserved in-80 ℃ or liquid nitrogen.
Six, the purifying of monoclonal antibody HPS 8D9
With the protein A affinity chromatography test kit of Roche company with monoclonal antibody from the Hybridoma Cell Culture liquid that screens or ascites purifying out, concrete steps are pressed the specification sheets operation of test kit.
Tiring behind the purifying is 1:204800.
Seven, monoclonal antibody IgG subgroup identification
With reference to the Mouse Monoclonal Antibody Isotyping Reagents operation instruction of Sigma company, use to catch ELISA method mensuration
HPSThe subclass type of the immunoglobulin (Ig) of 8D9 hybridoma secretion, the result shows
HPS8D9 is the IgG2b subclass.
Eight, the stability analysis of hybridoma secretory antibody HPS 8D9
With hybridoma recovery after frozen 1 month in liquid nitrogen, with OMP
5For antigen is measured antibody titer in the cell culture supernatant with indirect elisa method, and then it is frozen to put into liquid nitrogen; Again take out recovery after 1 month, repeat so continuously freeze thawing three times.Comparative analysis is the measured value of the antibody titer of Hybridoma Cell Culture supernatant several times.The result shows that hybridoma is through three multigelations, and its cell culture supernatant antibody titer is constant, illustrates that the monoclonal antibody of secretion has satisfactory stability.
Nine, the specific detection of monoclonal antibody HPS 8D9
With different bacteriums, comprise after swine escherichia coli, pig pasteurella multocida, actinobacillus pleuropneumoniae 1 type, streptococcus suis 2-type and pig bordetella bacilli are cultivated and collect thalline; With 4 ℃ of aseptic distillation water washing thalline 3 times, 4 ℃ of ultrasonic disruptions with the centrifugal 10min of 8000rpm, are got supernatant liquor and are carried out the SDS-PAGE electrophoresis, transfer on the nitrocellulose filter (NC), simultaneously with the positive contrast of restructuring HSP OMP5; Take monoclonal antibody HPS8D9 as primary antibodie, detect take the goat anti-mouse antibody of horseradish peroxidase-labeled as the two anti-Western-blotting that carry out.
Western Blot detects and carries out according to the following steps:
(1) sample preparation: after protein sample and sample-loading buffer volume mixed in the ratio of 1:1, boil 10min.
(2) SDS-PAGE electrophoresis: glue, application of sample carries out the SDS-PAGE electrophoresis, after dying in advance albumen Marker and going to the predetermined position, deenergization.
(3) electrophoresis complete after, unload gel, and electricity consumption transfering buffering liquid balance 3 times, each 5min.
(4) pvdf membrane is processed: cut out in advance pvdf membrane and the filter paper onesize with adhesive tape, pvdf membrane is processed 5s with methyl alcohol, then be tiled in the distilled water face, 10min drains the pure qi (oxygen) bubble after making it lean on wick water in the invasion water, puts into subsequently electricity and turns the damping fluid balance.
(5) get glue: glue after the balance, places on the clean sheet glass in transfering buffering liquid, spreads successively NC film, three metafiltration paper (both sides Dou Pu).
(6) transferring film: add an amount of electricity and turn liquid toward electric turn trough, glue is tiled on the sponge that electricity consumption turns the wetting mistake of damping fluid is fixed (fixedly time notice that film faces toward an anodal side); Put into the electrotransfer groove after fixing, after constant current 100mA shifted (electric turn trough is placed in the mixture of ice and water, prevents that electricity from turning over excess Temperature in the journey) 3h, deenergization was got film and is done immunoblotting.
(7) wash film: film is put into TBST, leave standstill 5min, triplicate.
(8) coated: film is added in the coating buffer, and steady shaking ladle is by 2h under the room temperature condition.
(9) wash film: discard coating buffer, film is put into TBST, leave standstill 5min, triplicate.
(10) add primary antibodie: primary antibodie is monoclonal antibody HPS 8D9, presses the 1:500 dilution proportion with TBST, steadily shakes 1h under the room temperature.
(11) wash film: discard primary antibodie, film is put into TBST, leave standstill 5min, repeat four times.
(12) adding two resists: with the goat anti-mouse antibody of horseradish peroxidase mark, dilute with TBST in the 1:5000 ratio, room temperature is steadily shaken sense and is made 1h.
(13) wash film: react complete after, discard ELIAS secondary antibody, successively wash film 3 times with TBST, each 5min; Then film is changed over to and leave standstill 10min among the TBS, repeat twice.
(14) colour developing: add the DAB nitrite ion, lucifuge colour developing under the room temperature condition, when band occurring, colour developing finishes; Immediately film is put into the ultrapure water termination reaction.
The results are shown in Figure 2.As can be seen from the figure, the tropina of the monoclonal antibody that the present invention obtains and swine escherichia coli, pig pasteurella multocida, actinobacillus pleuropneumoniae, streptococcus suis 2-type bacterial strain and pig bordetella bacilli is all reactionless, (about 57KD) locates to see the specific reaction band at the OMP5 of HPS albumen, illustrate the monoclonal antibody that obtains can with HPS OMP5 specific binding.
Ten, the versatility of monoclonal antibody HPS 8D9 detects
To collect thalline after the haemophilus parasuis bacterial strain of 9 different serotypes such as HPS 1 type bacterial strain, 3 type bacterial strains, 4 type bacterial strains, 5 types, 10 type bacterial strains, 11 type bacterial strains, 12 type bacterial strains, 14 type bacterial strains, 15 type bacterial strains and the 1 strain prepattern strain culturing; With 4 ℃ of aseptic distillation water washing thalline 3 times, 4 ℃ of ultrasonic disruptions with the centrifugal 10min of 8000rpm, are got supernatant liquor and are carried out the SDS-PAGE electrophoresis, transfer on the nitrocellulose filter (NC), do simultaneously restructuring HSP OMP5 positive control; With the monoclonal antibody that obtains
HPS8D9 is primary antibodie, detects take the goat anti-mouse antibody of horseradish peroxidase-labeled as the two anti-Western-blotting that carry out.
The results are shown in Figure 3.As can be seen from the figure, the monoclonal antibody that obtains of the present invention
HPSHaemophilus parasuis tropina and the prepattern bacterial strain of 8D9 and all 9 serotypes all react.At OMP
5Albumen (about 57KD) is located to see the specific reaction band, and the monoclonal antibody that obtains is described
HPSThe 8D9 versatility is good.
11, monoclonal antibody HPS 8D9 is used for the immunofluorescence detection of HPS---laser co-focusing immunofluorescence detection
1. the processing of laser co-focusing special cell culture dish:
(1) pre-equilibration: draw the 3mL cell culture fluid in culture dish, then in cell culture incubator, hatch 15min.
(2) outwell cell culture fluid, add 500 μ L cell suspensions, in cell culture incubator, place 2h, make cell settlement adherent.
(3) carefully add the 3mL cell culture fluid.
2.PK-15 the cultivation of going down to posterity of cell: cultivate the PK-15 cell with the MEM nutrient solution that contains 10% foetal calf serum, wait cell length to begin to go down to posterity to the logarithmic growth after date.Concrete operations are as follows: remove nutrient solution, with the residual nutrient solution of sterilization PBS flush away; Add the 1mL pancreatin, jiggle effect 2min, see and outwell pancreatin when a bottle floor cells is vaporific, with the residual pancreatin of sterilization PBS flush away; , then cell suspension is divided into two cell suspension with the MEM nutrient solution that contains 10% foetal calf serum, minute installs in two Tissue Culture Flasks, put into incubator and cultivate, the rear 1h that goes down to posterity changes liquid will be assembled agglomerating cell and outwell, in order to avoid affect experiment effect.
3. after cell grows to 80% full scale, change liquid to cell, add Serotype 5 HPS bacterium liquid, making its final concentration is 1 * 10
6Cfu/mL puts 37 ℃, and 2h carries out common cultivation; Discard nutrient solution, fully wash with PBS, add the PBS that 2ml contains 15 μ L/mL normal goats serum, place 15min for 37 ℃; Wash with PBS.Add fixedly 2h of 4 ℃ of PBS that 2mL contains 2% glutaraldehyde and 1.5% formaldehyde.
4. every hole adds the PBS that contains 10% foetal calf serum and 1%BSA, every hole 500 μ L, 37 ℃ of sealing 1h; Seal complete after, discard confining liquid, add PBS and fully wash; The ascites that to make of the monoclonal antibody HPS8D9 of anti-OMP5 is done the 1:100 dilution, and (using the PBS that contains 5% foetal calf serum and 0.05%Tween-20 is diluent, as follows), add respectively each culture dish, each 500 μ L, 37 ℃ of effect 2h establish simultaneously how anti-the anti-full bacterium of 5 type HPS is, how anti-and HPS negative serum contrast of anti-OMP5; Act on complete after, discard ascites, wash with PBS.
The anti-full bacterium of 5 type HPS how anti-and anti-OMP5 how anti-preparation methods are: will through 5 type HPS of formalin-inactivated or with the recombination outer membrane protein P5 of purifying respectively with Fu Shi fully or the oil-emulsion subunit vaccine of Freunds incomplete adjuvant preparation, respectively subcutaneous multi-point injection immunity Balb/c mouse, immunity is three times altogether, midfeather 14 days, took a blood sample separation of serum in immune rear 14 days for the third time.
5. take the FITC-sheep anti-mouse antibody as two anti-, dilute in the ratio of 1:1000 with PBST, add each culture dish, each 500 μ L, 37 ℃ of effect 2h; Act on and discard after complete two anti-ly, wash with PBS; Add at last a little PBS, to prevent the cell dehydration.
6. laser co-focusing microscopic examination: culture dish is put laser confocal microscope (laser confocal scanning light microscope, Leica TCS SP), transfer Ar Ion Laser wavelength to 488nm, focus information is collected in focusing.
The results are shown in Figure 4.The result shows among the figure, monoclonal antibody HPS8D9 is the same with the contrast of anti-OMP5 positive serum with the full bacterium positive serum of anti-HPS, can cause that absorption has the PK-15 cell peripheral of HPS to send green fluorescence, illustrates that monoclonal antibody HPS 8D9 and natural antigen can react, and can be used for the clinical detection of HPS.
Claims (5)
1. the hybridoma cell strain HPS 8D9 of the anti-haemophilus parasuis outer membrane protein OMP5 monoclonal antibody of secretion is preserved in Chinese Typical Representative culture collection center on September 25th, 2012, and deposit number is CCTCC C2012135.
2. the monoclonal antibody of an anti-haemophilus parasuis outer membrane protein OMP5 is produced by hybridoma cell strain claimed in claim 1.
3. the monoclonal antibody of anti-haemophilus parasuis outer membrane protein OMP5 according to claim 2 is characterized in that, described monoclonal antibody is the IgG2b subclass.
4. a test kit that detects haemophilus parasuis antibody contains claim 2 or 3 described monoclonal antibodies.
5. a test kit that detects haemophilus parasuis contains claim 2 or 3 described monoclonal antibodies.
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| CN103941001A (en) * | 2013-03-15 | 2014-07-23 | 河南省农业科学院 | Rapid detection test strip for haemophilus parasuis |
| CN103941001B (en) * | 2013-03-15 | 2016-03-16 | 河南省农业科学院 | Haemophilus parasuis infection Rapid detection test strip |
| CN103570832A (en) * | 2013-09-22 | 2014-02-12 | 中国农业科学院哈尔滨兽医研究所 | Anti-Haemophilus parasuis porcine chimeric antibody, and construction method and application thereof |
| CN103570832B (en) * | 2013-09-22 | 2015-08-12 | 中国农业科学院哈尔滨兽医研究所 | A kind of pig source chimeric antibody of anti-haemophilus parasuis and construction process thereof and application |
| CN111793130A (en) * | 2019-03-20 | 2020-10-20 | 华中农业大学 | Haemophilus parasuis CdtB hybridoma cell and application of monoclonal antibody |
| CN111551748A (en) * | 2020-05-29 | 2020-08-18 | 临沂大学 | Enzyme-linked immunosorbent assay kit for trichosanthin and use method thereof |
| CN113563466A (en) * | 2021-06-30 | 2021-10-29 | 洛阳莱普生信息科技有限公司 | Monoclonal antibody preparation method, detection kit, detection method and application of monoclonal antibody of P.multocida antibody |
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