CN101671654B - Monoclonal antibody resisting anaplasma marginale MSP5 protein and application thereof - Google Patents
Monoclonal antibody resisting anaplasma marginale MSP5 protein and application thereof Download PDFInfo
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- CN101671654B CN101671654B CN2009101784814A CN200910178481A CN101671654B CN 101671654 B CN101671654 B CN 101671654B CN 2009101784814 A CN2009101784814 A CN 2009101784814A CN 200910178481 A CN200910178481 A CN 200910178481A CN 101671654 B CN101671654 B CN 101671654B
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Abstract
The invention discloses a monoclonal antibody resisting anaplasma marginale MSP5 protein and an application thereof. The invention adopts the purified anaplasma marginale MSP5 protein expressed by escherichia coli as the immunogen to immune mice; a hybridoma cell line which can steadily secrete monoclonal antibody resisting anaplasma marginale MSP5 protein is obtained by cell fusion and screening; and the micromechanism collection number is CGMCC No.3205. The monoclonal antibody secreted by the hybridoma cell line can react with the anaplasma marginale MSP5, simultaneously can carry out idiosyncratic reaction with the cattle anaplasma marginale and the sheep anaplasma marginale, and can not react with mycoplasmas, neospora canimum, eperyt hrozoon and the like. The monoclonal antibody in the invention can be used for differentiating anaplasma marginale and other related pathogenies and provides material basis for the building of a rapid, convenient and accurate diagnostic method and for the immune mechanism research, immune function detection, detection method establishment and the like.
Description
Technical field
The present invention relates to monoclonal antibody, relate in particular to a kind of monoclonal antibody of resisting anaplasma marginale MSP 5 protein, the invention still further relates to the hybridoma cell strain of this anti-MSP5 protein monoclonal antibody of secretion, belong to the cell engineering field.
Background technology
No slurry is sick; Be once called as anaplasmosis; Be not have slurry (Anaplasma marginale), central authorities' no slurry (A.centrale) and sheep by the edge that Rickettsiales (Rickettsiales), no slurry section (Anaplasmataceae), no slurry belong to not have slurry kinds such as (A.bovis) and parasitize caused blood property protozoal disease in the red corpuscle; Mainly propagating with type of sucking blood arthropods (tick class, the gadfly, stable fly, mosquito etc.), is distribute during tick spreads disease the widest a kind of (Ristic M.Bovine anaplasmosis.ParasiticProtozoa, edited by Kreier J P; 1977, (IV): 235-249).Found that the edge did not have slurry and the no slurry of central authorities in 1910 and 1911 first in north African.After this find that no slurry disease is widely current all over the world, wherein the edge does not have the harm maximum of slurry, and it often causes that in the acute course of disease the serious anaemia of animal, weight loss, high fever, miscarriage, milk yield descend, even dead.China part provinces and cities also carried out should disease epidemiology survey, confirmed to exist in various degree infection (Li Haihui what moral wantonly; Ou Yangxuxiang etc. some areas, Hunan Province ox does not have the sick infection conditions investigation of slurry [J]; The China phytophagous animal, 2009,29 (1): 41-43).
The edge does not have slurry and has a complete annular genome, and about 1.2-1.6Mb utilizes SIGNALP (version3; Ref18) software prediction goes out 163 CDSs that contain signal peptide, infers that by TMPRED (http://ch.embnet.org/software/TMPRED) software per three CDSs just comprises one and stride the film district.Because PSORT (http://psort.nibb.ac.jp/form.htmL) and PSORTB (http://psort.org/psortb/index) only can dope 43 and 13 outer membrane proteins (OMPs) separately; Lack many known no slurry outer membrane proteins; So (Brayton K A also can't be inferred in each albumen position; Kappmeyer L S; Herndon D R; Et al.Complete genome sequencingof Anaplasma marginale reveals that the surface is kewed to twosuperfamilies of outer membrane proteins [J] .Proc Natl Acad Sci U S A.2005,102 (3): 844-849).The edge does not have slurry MSP1 superfamily and mainly is made up of MSP1.MSP1 is the heteropleural property mixture that the surface exposes; Polypeptide by 105ku and 100ku is polymerized; Be named as MSP1a and MSP1b (Barbet A F separately; Palmer G H; Myler P L et al.Characterizationof an immunoprotective protein complex of Anaplasma marginale by cloningand expression of the gene coding for polypeptide Am105L [J] .Infect Immun.1987,55:2428-2435).The edge does not have slurry MSP2 superfamily and mainly comprises MSP2, MSP3 and MSP4; Be positioned at adventitia with surperficial exposure zone; MSP2 and MSP3 are that immundominance albumen and antigenicity are variable; No slurry can produce persistent infection in the ox body mainly be because MSP2 and MSP3 morph easily and escape host immune response (Simpson C F.Morphologic alterations of Anaplasmamarginale in calves after treatment with oxytetracycline [J] .Am J VetRes; 1975, (36): 1443-1446; Tettelin H, Saunders N J, Heidelberg J, etal.Complete genome sequence of Neisseria meningitidis serogroup B strainMC58 [J] .Science.2000,287 (5459): 1809-1815).Anaplasma marginale MSP 5 is the albumen by 633bp single copy gene coding 19ku, and is quite conservative between having or not slurry (comprise that the edge does not have slurry, sheep does not have the no slurry of slurry and central authorities) not of the same race; Be suitable as diagnostic antigen (Duzgun A; Schuntner CA, Wright I G, et al.A sensitive ELISA technique for the diagnosisof Anaplasma marginale infections [J] .Vet Parasitol; 1988,29:127.).
The routine inspection method of using clinically is to carry out comprehensive diagnos according to means such as this sick epidemiology, clinical symptom, nectropsy and blood smear inspections.Because this disease popular has three kinds of basic factors, i.e. cause of disease, hard tick and susceptible animal, the three forms a popular chain link, and lacking wherein, any one factor all can not make this disease generation and popular.So, when this disease of diagnosis, see whether this locality once had this sick popular history, whether susceptible animal was arranged from Pest-or disease-free area, whether the activity of the media tick of propagating cause of disease is arranged.This disease is mainly in the summer, autumn under field conditions (factors).Usually working as every milliliter of blood has greater than 10
9Will produce tangible clinical symptom when individual red corpuscle is infected, can detect this moment through Ji's nurse Sa dyeing microscopic examination method, but in every milliliter of blood infected RBC number less than 10
6Just can not detect (Aguirre D H when individual through the method for Ji's nurse Sa dyeing microscopic examination; Bermudez A C; Mangold A J, et al.Natural infection withAnaplasma marginale in cattle of the Hereford, Criolla and Nelore breedsin Tucuman; Argentina.Rev.Latinoam.Microbiol.1988,30:37-41).Ristic (1962) report can shorten this time greatly with SP 15 Lemon Yellow dyeing; But this method is to painted bad (the Ristic M.A capillary tube-agglutination tests foranaplasmosis-A preliminary report.Am J Vet Med Ass of immature erythrocytic nucleic acid; 1962, (141): 588-594).Because routine inspection is difficult to detect early this disease, also be unfavorable for large-scale epidemiology survey, in order to solve these contradiction, many serological methods just be applied to should the inspection of disease on.Serological method commonly used at present has complement fixation test (CFT), EUSA, IFA and capillary tube agglutination test; (Gonzalez E F such as card agglutination test and plate agglutination test; Long R F; Todorovic R.A.Comparisons of the complement-fixation; Indirectfluorescent antibody, and card agglutination tests for the diagnosis ofbovine anaplasmosis.Am J Vet Res 1978,39 (9): 1538-41; Duzgun A, Schuntner C A, Wright I G, et al.A sensitive ELISA technique for thediagnosis of Anaplasma marginale infections.Vet Parasitol, 1988,29 (1): 1-7; Schindler K; Ristic M and Wokatsch R.Vergleichende Untersuchungenmit Anaplasma marginale and A.centrale.Z Tropenmed Parasitol; 1966; (17): 337-360), these methods exist various drawbacks and shortcoming in medical diagnosis on disease, and cross reaction is especially outstanding.
This shows, do not cultivate in external being difficult to, bring very big difficulty therefore for its Study on etiology because the edge has slurry; The method that present domestic detection edge does not have slurry mainly is blood smear method, serological method and molecular biology method, but the blood smear method obscure mutually with Eperythrozoon easily, be difficult to differentiate; Serological method mainly is that the test kit of using external import detects, and costs an arm and a leg, and is not suitable for clinical extensive use; The molecular biology method specificity is high, the result is certain; But to having relatively high expectations of experiment condition and operator, therefore be used for laboratory study more, be difficult to get into the clinical application stage.Enzyme immunoassay technique combines the high-level efficiency and the immunoreactive height specificity of enzymatic reaction; Have highly sensitive, high specificity,, advantages such as cost low, simple and efficient to handle, "dead" pollution, level of automation high, reagent shelf time long less demanding to plant and instrument; Be applicable to terrain testing in enormous quantities, possibly become the diagnostic method that has promotional value.At present; Because the edge has the research at home of slurry disease and does not just carry out, and does not also have the report of its monoclonal antibody aspect, therefore to a great extent limit monoclonal antibody do not have the application of slurry disease in clinical diagnosis on the edge of; Therefore development detects the edge does not have the sick ELISA diagnostic kit of slurry to this sick epidemic monitoring, epidemiology survey and improve all significant (Zhou Jie such as immunization strategy; Ginger suddenly, Zhang Hongying etc. CDV reorganization nucleocapsid protein MONOCLONAL ANTIBODIES SPECIFIC FOR and evaluation [J], Chinese Preventive Veterinary Medicine newspaper; 2007,29 (7): 528-532).In this process, monoclonal antibody is indispensable instrument.
Summary of the invention
One of the object of the invention provides a strain can secrete the hybridoma cell strain of resisting anaplasma marginale MSP 5 protein monoclonal antibody respectively;
Two of the object of the invention is that the resisting anaplasma marginale MSP 5 protein monoclonal antibody that above-mentioned hybridoma cell strain is secreted is applied to diagnosis or the prevention edge does not have the slurry disease;
Above-mentioned purpose of the present invention realizes through following technical scheme:
The present invention adopts behind the anaplasma marginale MSP 5 protein purifying of escherichia coli expression as immunogen; The immunity BALB/c mouse; Get its SPL and SP2/0 myeloma cell and merge, obtain the hybridoma cell strain of the anti-MSP5 protein monoclonal antibody of a strain stably excreting through screening.
One strain can be secreted the hybridoma cell strain of resisting anaplasma marginale MSP 5 protein monoclonal antibody respectively, its called after of classifying: the hybridoma cell strain of secretion resisting anaplasma marginale MSP 5 protein monoclonal antibody; Its microbial preservation number is: CGMCC NO.3205; The classification name is: the hybridoma of secrete monoclonal antibody; The preservation address is: Datun Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica; Depositary institution is: China Committee for Culture Collection of Microorganisms common micro-organisms center; The preservation time is: on July 23rd, 2009.By the secreted resisting anaplasma marginale MSP 5 protein monoclonal antibody of this hybridoma cell strain, called after 1D8.
The present invention adopts indirect ELISA method to measure the titration of monoclonal antibody 1D8, measures the result and shows that the ascites that monoclonal antibody 1D8 inducing mouse produces is tired and is respectively 10
6, tiring of cell conditioned medium reaches 10
4
The hypotype qualification result shows that the heavy chain of monoclonal antibody 1D8 of the present invention is IgG2b, and light chain is the κ chain.
The present invention adopts Western blot to analyze the immunocompetence of a strain monoclonal antibody, and the Westernblot detected result shows that monoclonal antibody 1D8 of the present invention can combine with the rMSP5 protein-specific of prokaryotic expression.
Monoclonal antibody 1D8 of the present invention can react with anaplasma marginale MSP 5, and the while can not have slurry with Niu Bianyuan and the sheep edge does not have slurry generation specific reaction, and does not react with Mycoplasma, neospora and Eperythrozoon etc.Monoclonal antibody of the present invention can be used for differentiating that the edge does not have the slurry cause of disease relevant with other, for set up a kind of quick, simple and easy, accurately diagnostic method and detect in immunologic mechanism research, immunologic function, the research of the aspects such as foundation of detection method provides material base
Description of drawings
Fig. 1 monoclonal antibody subgroup identification of the present invention result.
Fig. 2 Western blot detects the immunocompetence result of monoclonal antibody of the present invention.
Embodiment
Further describe the present invention below in conjunction with specific embodiment, advantage of the present invention and characteristics will be more clear along with description.But these embodiment only are exemplary, scope of the present invention are not constituted any restriction.It will be understood by those skilled in the art that and down can make amendment with form or replace without departing from the spirit and scope of the present invention, but these modifications and replacing all fall in protection scope of the present invention the details of technical scheme of the present invention.
Cell, laboratory animal and biochemical reagents
(1) recombinant plasmid pQE30-MSP5, SP2/0 cell are preserved by this laboratory;
(2) 8 experiments in age in week are provided by Harbin Veterinary Medicine Inst., China Academy of Agriculture's Experimental Animal Center with female BALB/c mouse;
(3) Ni-NTA His-Bind Resins is available from Amersco company; Standard foetal calf serum, substratum DMEM purchase the company in Gibco; Freund's complete adjuvant, Freund's incomplete adjuvant, selective medium HAT and HT, cytogamy are purchased the company in Sigma with PEG (MW4000), monoclonal antibody hypotype identification kit, horseradish peroxidase-labeled sheep anti-mouse igg (HRP-IgG); DAB colouring reagents box is available from Wuhan doctor's moral biotech firm.
Embodiment 1 MONOCLONAL ANTIBODIES SPECIFIC FOR
1, the purifying of recombinant protein and detection
Albumen rMSP5 after inducing is gathered in the crops bacterium liquid, 6000rpmmin behind 5h
-1Centrifugal 10min adds the binding buffer liquid of 10mL.Resuspended bacterium liquid places ice bath ultrasonic treatment (30% power) cracking 3s, interval 15s, ultrasonic to liquid-transparent with sample.11000rpmmin then
-1Centrifugal 20min removes cell debris, and supernatant is moved in the new pipe.Get supernatant and deposition respectively and carry out SDS-PAGE to confirm whether expressed proteins is soluble proteins.The His-tag selectivity label that utilizes the pQE30 carrier to have is used Novagen company specification sheets purifying protein, and concrete purification step is following:
1) 3mL aseptic deionized water and resin are mixed, treat that resin sinks fully after, with the supernatant sucking-off.Wash twice.The combination liquid (pH8.0) of 3ml and resin are mixed, treat that resin sinks fully after, with the supernatant sucking-off, wash twice.
2) 3mL lysate supernatant and 1.5mL resin are mixed, 4 ℃ on shaking table vibration hatch 2h or spend the night (will guarantee that resin suspend).
3) with the vertical static placement of nickel post, treat that resin sinks fully after, with the supernatant sucking-off.The resuspended resin of BufferB scavenging solution (pH8.0) that adds 3mL.After treating that resin sinks fully, liquid is flowed out from the below, repeat twice.
4) use the resuspended resin of scavenging solution (pH6.3) of 3mlBufferC then, treat that resin sinks fully after, liquid is flowed out triplicate from the below.
5) add the resuspended resin of 600ul scavenging solution (pH5.9), treat that resin sinks fully after, liquid is flowed out from the below, repeat 4 times.
6) add the resuspended resin of 500ul elutriant (PH4.5), treat that resin sinks fully after, with 2mLmin
-1Flow velocity collect liquid.And the pipe liquid that will collect numbering, carry out SDS-PAGE and analyze.
Test-results: albumen has higher purity and not significantly loss of protein content behind the purifying.
2, mouse immune
As immunogen, 7 ages in week, female BALB/c mouse are carried out 3 immunity with purified recombinant MSP5 albumen.Immunization protocol is following: head exempts from, and every mouse is mixed and made into emulsifying agent, subcutaneous multiple spot of nape portion and abdominal injection with reorganization MSP5 albumen and the equivalent FCA of 50 μ g; Two avoid carrying out in 2 weeks after head exempts from, and change FCA into FICA, and dosage and method are the same; Three avoid two exempts from back carrying out in 2 weeks, and dosage and method are exempted from two.Cytogamy was carried out booster immunization in preceding 3 days, and method is abdominal injection 100 μ g purified recombinant MSP5 albumen.
3, cytogamy
2d prepares feeder cell before merging, and gets the BALB/c mouse peritoneal macrophage according to ordinary method and is laid in the 96 porocyte culture plates for use.Disconnected neck is put to death the mouse of waiting to get spleen; Aseptic its splenocyte of getting; Merge with PEG in splenocyte and 5: 1 ratio of SP2/0 myeloma cell, the cell after the fusion is laid on (Kohler G, Milstein C.Continuous cultures of fused cellsecreting antibody of predefined specificity.Nature on the ready feeder cell; 1975,256 (5517): 495-497.).
4, the screening of positive hybridoma cell strain and subclone
Get the rMSP5 of purifying, with carbonate buffer solution (0.225M Na
2CO
38mL, 0.2M NaHCO
317mL adds deionized water to 100mL, and pH 9.6) be diluted to the best and encapsulate concentration; Add in the elisa plate with 50 μ L/ holes, 37 ℃, 3h; PBST (0.01M, pH 7.3, and PBS+0.05%Tween-20) washing is 3 times, 5min/ time; Add PBST/FCS (0.01M, pH 7.3, PBST+10%FCS), 200 μ L/ holes, 37 ℃ of sealings 3h or 4 ℃ spend the night; PBST washes 3 times, and 5min/ time, clap and do, deposit subsequent use for-20 ℃ or 4 ℃.Confirm antigenic optimum diluting multiple through the square formation test, be about to the rMSP5 fusion rotein and encapsulate 96 orifice plates, carry out the square formation test, obtain the extension rate of antigenic working concentration and prokaryotic expression protein rMSP5 immunized mice positive serum.Negative mice serum is set simultaneously as negative control.Select antigenic the best to encapsulate concentration according to reaction result.Detect the Hybridoma Cell Culture supernatant by conventional indirect elisa method.Enzyme plate is placed the value of reading on the ELISA ELIASA, with P/N >=2.1 as positive criterion.Pick out the hybridoma cell clone hole of anti-rMSP5.
By above-mentioned screening method, the positive hybridoma cell that screening is obtained carries out subclone, and subclone adopts limiting dilution assay, divides 96 porocyte plates again after the archioporus cell is diluted with the HT substratum, and an archioporus cell divides a plank.After accomplishing, subclone notes observing the number of cells and the state in each hole.It is stable and be that the subclone second time is carried out in single clone's hole as far as possible to get secretory antibody behind the subclone.Positive rate through the detected result of former monoclonal before this subclone plank behind three subclones should reach 100%.Obtained 1 strain can the stably excreting specificity to the hybridoma cell strain of the proteic monoclonal antibody of MSP5 (called after 1D8).
5, a large amount of preparations of monoclonal antibody
Give the healthy BALB/c mouse abdominal injection whiteruss about 10 ages in week, 0.5mL/, 1w pneumoretroperitoneum injection 10
5Individual hybridoma extracts ascites when the mouse web portion extreme expansion behind the 7-10d, take out once at a distance from 2d, with the centrifugal 10min of ascites 10000r/min that extracts, removes upper strata grease and deposition, and supernatant is sub-packed in-20 ℃ or-70 ℃ of preservations.
The evaluation of Test Example 1 monoclonal antibody
1, the titration of antibody
Use indirect ELISA method, be about to cell conditioned medium or ascites and carry out behind the doubling dilution as being coated with in the antigenic enzyme plate anti-an adding, and the positive is set, negative mice serum is done contrast.With the positive criterion in P/N >=2.1, the positive cell strain culture supernatant and the ascites that obtain are carried out the antibody titer detection.
Detected result is seen table 1 and table 2, and it is 10 that the ascites that monoclonal antibody 1D8 inducing mouse produces is tired
6, tiring of cell conditioned medium reaches 10
4
The mensuration that table 1 odd contradictive hydroperitoneum is tired
The mensuration that table 2 monoclonal antibody supernatant is tired
2, monoclonal antibody subgroup identification
The subclass that the ELISA test kit detects monoclonal antibody is caught in use:
(1) with epitopic features property antibody I gA, IgG1, IgG2b, IgG2a, IgG3, IgM carry out dilution in 1: 1000 with PBS respectively, and the 100ul/ hole encapsulates, and every kind of characteristic property antibody encapsulates two holes, hatches 1h for 37 ℃.
(2) with washings PBST washing 3 times, 5min/ time.
(3) every hole adds 100ul monoclonal antibody supernatant to be detected, hatches 1h for 37 ℃.
(4) with washings washing 3 times, 5min/ time.
(5) with PBST the sheep anti-mouse igg antibody (Fab fragment) of horseradish peroxidase-labeled is carried out diluting at 1: 1000, every hole 100ul is hatched 30min for 37 ℃.
(6) with washings washing three times, 5min/ time.
(7) every hole adds the substrate solution 100ul of fresh configuration, and 37 ℃ of lucifuges are hatched 20min.
(8) every hole adds 50ul 2M H
2SO
4Termination reaction.On ELIASA, read the value of OD490,
Be judged to its monoclonal antibody subclass with the OD value apparently higher than the epitopic features property antibody in other each holes.
The hypotype qualification result is seen table 3 and Fig. 1, and the heavy chain of monoclonal antibody 1D8 of the present invention is IgG2b, and light chain is the κ chain.
Table 3 monoclonal antibody subgroup identification result
3, the McAbs immunocompetence is identified
Western blot is used for the immunocompetence of analysis list clonal antibody; Western blot program is following: with purified recombinant MSP5 albumen with dye albumen Marker in advance and carry out SDS-PAGE electrophoresis (gum concentration is 12%); The electrophoresis product is transferred to nitrocellulose filter (NC); Cut the reorganization MSP5 protein band of each swimming lane and dye albumen Marker band (preserving subsequent use) in advance; To contain reorganization MSP5 proteic NC film band then and put into deionized water and clean 10min, 5% skimming milk room temperature sealing 1h, the NC film band after the sealing respectively with 1: 100 times of dilution induce ascites that mouse produces and 37 ℃ of effects of culture supernatant liquid 1h of normal SP2/0 cell by the 1-D8 monoclonal antibody; PBST (0.01mol/L, pH7.2; Contain 0.05% Tween-20) washing 3 times, with 37 ℃ of HRP-sheep anti-mouse iggs (1: 8000) effect 1h, PBST washes 5 times then, with 3,3 '-diaminobenzidine (DAB) substrate solution develops the color.
Western blot detected result shows that monoclonal antibody all can combine (Fig. 2) with the rMSP5 protein-specific of prokaryotic expression.
Claims (3)
1. the hybridoma cell strain of strain secretion resisting anaplasma marginale MSP 5 protein monoclonal antibody, its microbial preservation number are: CGMCC NO.3205.
2. by the said hybridoma cell strain excretory of claim 1 monoclonal antibody.
3. the described monoclonal antibody of claim 2 does not have the purposes in the sick reagent of slurry in preparation diagnosis or detection edge.
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| CN101415436A (en) * | 2005-11-23 | 2009-04-22 | 巴斯德研究院 | Recombinant plasmodium falciparum merozoite surface proteins 4 and 5 and their use |
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| CN101415436A (en) * | 2005-11-23 | 2009-04-22 | 巴斯德研究院 | Recombinant plasmodium falciparum merozoite surface proteins 4 and 5 and their use |
Non-Patent Citations (2)
| Title |
|---|
| 边缘无浆体MSP5基因序列分析及其融合蛋白的纯化;马米玲等;《动物医学进展》;20080831;第29卷(第8期);13-16 * |
| 马米玲等.边缘无浆体MSP5基因序列分析及其融合蛋白的纯化.《动物医学进展》.2008,第29卷(第8期),13-16. |
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