CN1673231A - Monoclonal antibody of SARS coronavirus N protein and its application - Google Patents
Monoclonal antibody of SARS coronavirus N protein and its application Download PDFInfo
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- CN1673231A CN1673231A CN 200410052873 CN200410052873A CN1673231A CN 1673231 A CN1673231 A CN 1673231A CN 200410052873 CN200410052873 CN 200410052873 CN 200410052873 A CN200410052873 A CN 200410052873A CN 1673231 A CN1673231 A CN 1673231A
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Abstract
The present invention relates to one kind monoclonal antibody with specificity on the serial proteins shown in SEQ ID No. 2, hybrid tumor cell train generating the monoclonal antibody and its preparation process. The monoclonal antibody of the present invention may be used in diagnosing and treating diseases caused by SARS virus infection, especially SARS. As the application, the present invention also proposes at least one kind of diagnosis kit with the monoclonal antibody of the present invention.
Description
Technical field
The present invention relates to biological technical field, proteic monoclonal antibody of particularly a kind of sars coronavirus N and application thereof.
Background technology
Year February in November, 2002 to 2003, agnogenic atypical pneumonia (atypicalpneumonia) is broken out in Guangdong in China, occur similar case all over the world successively in China Hong Kong, Vietnam, Canada, Singapore, the U.S. and China etc. subsequently, cause showing great attention to of global common people's fear and national governments and scientific research personnel.On March 5th, 2003, The World Health Organization (WHO) has issued the health alarm with regard to atypical pneumonia to the whole world, and with this sick called after SARS (Severe Acute Respiratory Syndrome) (severe acute respiratory syndrome, SARS).Scientist studies show that SARS (Severe Acute Respiratory Syndrome) (SARS) is to be caused by a kind of new coronavirus (sars).This new coronavirus is the positive chain RNA virus of a coating, and genome total length 29,727 Nucleotide have 11 open reading frame, respectively coding: 4 kinds of structural protein (S, M, N, E albumen), the RNA polymerase that depends on RNA, 5 kinds of agnoproteins.It is reported that by on July 11st, 2003,32 countries and regions, the whole world have 8437 people to infect sars virus, and 813 people's death are wherein arranged.Whole world lethality rate is approximately 10.5%.In order to suppress the propagation diffusion of virus, a kind of test kit of effective diagnosis SARS virus is essential.Existing several detection method is broadly divided into four classes:
1 molecular detecting method (PCR detection)
PCR detects has higher specificity but sensitivity is relatively poor, and the experimental result that this means a feminine gender can not be got rid of the patient and SARS virus not occur and express, and experimentation was about 4-5 hour, pollutes (false positive and false negative result promptly occurring) easily.And owing to reasons such as virus mutation takes place frequently, design of primers, the recall rate of molecular detecting method is still waiting further raising.
2 immunological methods (antibody test)
Its detection principle is based on viral protein and removes to detect antibody in patient or the suspected patient blood, and studies show that serum antibody, after the SARS patient morbidity, antibody IgM the earliest occurs will be about 7 days, peak in the time of 10 days, descend about 15 days, IgG antibody produced after 10 days, peaked about 20 days.General patient is at firm adstante febre, blood interior disease poison, but antibody does not also generate.Sometimes the SARS patient is in latent period, do not occur symptom or symptom as yet and is not true to type, and makes the doctor be difficult to judge, produces antibody in the body by the time, and when symptom was clear and definite, the state of an illness also increased the weight of thereupon, incured loss through delay treatment and isolates.
3 cytology methods (cell cultures)
Cell culture processes is the laboratory detection means that develops the earliest, from sample that SARS patient gathered (as respiratory secretions, blood or ight soil) in virus, can and produce virus by infectious cell cultures, can under electron microscope, observe directly virion, thus very accurate.But the technical difficulty height, complicated operation, required time is long, to the equipment requirements height, is unsuitable for a large amount of detections.
4. protein chip method (antibody test)
The protein chip method also detects antibody, and is the same with above-mentioned immunological method detection antibody, and certain defective (seeing above-mentioned) is all arranged.
Present detection method all exists certain problem, still needs a kind of more early stage, sensitiveer, faster, more effective detection method, carries out early stage differential diagnosis.Thereby we press for preparation one specific specificity height, the strong monoclonal antibody of avidity and based on the diagnosis type test kit of the ELISA detection virus antigen of double antibody sandwich method.
Summary of the invention
The purpose of this invention is to provide a kind of anti-SARS virus N proteic antibody, especially monoclonal antibody.
Another object of the present invention provides a kind of immunologic detection method of early detection SARS virus.
A further object of the present invention provides a kind of diagnostic kit of early detection SARS virus.
An aspect of of the present present invention provides a kind of antibody of anti-SARS virus, the protein bound of described antibodies specific ground and aminoacid sequence shown in the SEQ ID NO:2.Preferable, described antibody is monoclonal antibody.In one embodiment, described antibody is the cell strain secretion of CCTCC NO:-C200407 by preserving number.Preferably, described antibody subtype is IgG1.
Another aspect of the present invention provides a kind of hybridoma cell strain of secreting above-mentioned antibody.Preferably, the preserving number of described hybridoma cell strain is CCTCC NO:-C200407.
Another aspect of the present invention provides a kind of immunologic detection method of early detection SARS virus, and described detection method is used said monoclonal antibody.Preferable, described antibody is the hybridoma cell strain secretion of CCTCC NO:-C200407 by preserving number.
Another aspect of the present invention provides a kind of detection kit that detects SARS virus, and described detection kit contains and marker bonded said monoclonal antibody; Marker comprises fluorescent marker, radioactively labelled substance and enzyme labelling thing.Preferable, described antibody is the hybridoma cell strain secretion of CCTCC NO:-C200407 by preserving number.
Another aspect of the present invention provides the purposes of above-mentioned antibody in diagnosis SARS virus infection associated diseases.Preferably, described disease is a SARS (Severe Acute Respiratory Syndrome).
Another aspect of the present invention provides the purposes of said monoclonal antibody in the preparation detection kit, and detection kit contains and marker bonded monoclonal antibody; Marker comprises fluorescent marker, radioactively labelled substance and enzyme labelling thing.Preferable, described antibody is the hybridoma cell strain secretion of CCTCC NO:-C200407 by preserving number.
Another aspect of the present invention provides the purposes of said monoclonal antibody in the pharmaceutical composition of preparation treatment SARS virus infection associated diseases, and described pharmaceutical composition comprises monoclonal antibody of the present invention, also comprises pharmaceutically acceptable carrier or vehicle.Preferably, described antibody is the hybridoma cell strain secretion of CCTCC NO:-C200407 by preserving number.
SARS virus N albumen of the present invention, N albumen, sars N albumen all refer to the albumen of sequence shown in the SEQ NO:2.The antibody of anti-SARS virus refers to and the protein-specific bonded antibody of sequence shown in the SEQ NO:2, comprises monoclonal antibody and polyclonal antibody, preferred monoclonal antibody.
Monoclonal antibody of the present invention can produce by hybridoma of the present invention, is to obtain from the nutrient solution of cultivating hybridoma of the present invention therefore.Yet, produce monoclonal antibody method of the present invention and be not particularly limited, if but genetically engineered antibody capable combine with SARS virus N protein-specific, it is also within the scope of the invention.
Hybridoma of the present invention produces identification SARS virus N albumen as antigenic monoclonal antibody, and can be by merging acquisition through the splenocyte of the animal of N protein immunization or lymph-node cell and myeloma cell.Hybridoma cell strain of the present invention is deposited in Chinese typical culture collection center (Wuhan) on April 21st, 2004, and preserving number is CCTCC NO:-C200407.
Hybridoma of the present invention can produce with cell-fusion techniques known in the art.Therefore, as the animal of immunogen immune except the people, splenocyte or lymph-node cell and myeloma cell's fusion with this animal produce hybridoma with N albumen, select the hybridoma that produces the proteic monoclonal antibody of identification N from it, thereby obtained hybridoma of the present invention.
Above-mentioned immunogen is not particularly limited, but can be any proteic immunogen of SARS virus N that contains.For example can perhaps contain the proteic fusion rotein of SARS virus N by the proteic engineering strain of culture expression SARS virus N on suitable medium.
The animal that produces hybridoma of the present invention through immunity is not particularly limited, and includes but not limited to: goat, sheep, cavy, mouse, rat and rabbit.Mouse preferably therein.
Whether monoclonal antibody of the present invention can detect existence that SARS virus infects.
The Application Areas of monoclonal antibody of the present invention is not particularly limited, but can be used for immunoassay, is used to judge the infection of SARS virus.
Based on said monoclonal antibody, the present invention proposes the immunoassay of early detection SARS virus, and immunoassay of the present invention is carried out with at least a monoclonal antibody of the present invention, and method can only be carried out with a kind of antibody.
Usually as the monoclonal antibody that is used for method of immunity, consider that the superb monoclonal antibody of sensitivity makes preferred antibody, because the polyclonal antibody that ground unrest is high with specificity is low compares, its specificity height and ground unrest is low.
As immunoassay of the present invention, comprise technology such as enzyme immunoassay (EIA), enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), fluorescence immunoassay (FIA), western blotting, immunochromatography.Above-mentioned various immunoassay can be used for competition law or sandwich assay, with antigen or the TPPA target antigen or the antibody of marker mark.
In above-mentioned various immunoassays, ELISA and immunochromatography technique are preferred.
Above-mentioned competitive method is based on the SARS virus N albumen of the mark of SARS virus and known quantity and monoclonal antibody quantitative competitive association reaction of the present invention in the test specimen.In the above-mentioned competitive method of mentioning especially, what add predetermined amount in containing the sample solution of SARS virus is fixed on the antibody on the carrier and the SARS virus N albumen with the marking agent mark of predetermined amount.Then, it is active or be not retained in marking agent activity on the carrier to measure the marking agent be retained on the carrier.To this, preferably almost add antibody and labelled antigen simultaneously.
Above-mentioned sandwich assay comprises: SARS virus is clipped between fixed monoclonal antibody of the present invention and the monoclonal antibody of the present invention or polyclonal antibody with the marking agent mark, add substrate at marker (for example enzyme) etc. then, Show Color etc., thereby the SARS virus in the detection sample.
The above-mentioned marking agent of mentioning, can be radio isotope (for example
125I), enzyme, enzyme substrates, phosphorus, fluorescent substance, vitamin H and coloring material.In with these marking agents and antigen or antibodies, can use maleimide method (J.Biochem. (1976), 79,233), activation vitamin H method (J.Am.Chem.SOC. (1978), 100,3585) or hydrophobic bond method.
The above-mentioned enzyme of mentioning comprises peroxidase, alkaline phosphatase, beta-galactosidase enzymes and glucose oxidase.For the substrate that is used for this occasion, can select to be fit to the substrate of the used enzyme of this occasion, for example comprise: ABTS, luminol,3-aminophthalic acid cyclic hydrazide-H
2O
2, o-phenylenediamine one H
2O
2(at peroxidase), p-nitrophenyl phosphate, phosphoric acid methyl are paid shape ester, 3-(2 '-volution diamantane)-4-methoxyl group-4-(3 '-phosphorus acyloxy) phenyl-1,, 2-dioxetane (at alkaline phosphatase), p-nitrophenyl-BETA-D-semi-lactosi (at beta-galactosidase enzymes).
Can be 4-40 ℃ of reaction 1 minute-18 hours, color or fluorescence, phosphorescence or the colour developing amount of measurement result demonstration are carried out above-mentioned test then.In addition, can use so-called speed trial, it is incubated in 4-40 ℃ of scope carries out.
With commercially available Bolton-Hunter reagent can be easily to the radio-labeling that carries out of above-mentioned antigen or antibody.For example, can be by Bolton-Hunter reagent being added in the solution (this solvent prepares by antigen or antibody are dissolved in the 0.1M sodium bicarbonate aqueous solution), passed through then 1-2 hour, and removed unreacted Bolton-Hunter reagent part with G-25 desalting column etc.
In addition, can use chloramine T method, the former method of iodine etc., carry out easily
125The I radio-labeling.
The above-mentioned phosphorus of mentioning has for example different luminol,3-aminophthalic acid cyclic hydrazide and bifurcation pyridine ester etc., and the above-mentioned fluorescent substance of mentioning has for example fluorescein and rhodamine etc.To this, can use active ester method or isocyanic ester method (" enzyme immunoassay technique ", IgakuShoin1987 publishes) to carry out mark easily.The above-mentioned coloring material of mentioning has for example painted latex particle and Radioactive colloidal gold.
In order to carry out immunoassay of the present invention with above-mentioned competitive method, add the SARS virus sample that the people is contained unknown quantity in monoclonal antibody of the present invention in by currently known methods physics or chemically combined solid phase, reaction is carried out.Simultaneously, add SARS virus N albumen, reaction is carried out with the predetermined amount of marking agent mark.
Implementing in the immunoassay of the present invention with above-mentioned sandwich assay, the monoclonal antibody among the present invention is being added the people by currently known methods physics or Chemical bond contain in the sample of SARS virus of unknown quantity on solid phase, reaction is carried out.Then, add monoclonal antibody of the present invention or polyclonal antibody, reaction is carried out with the marking agent mark.
In both cases, need the thorough washing solid phase, measure and labelled reagent bonded activity.When marking agent is radio isotope, measure with hole counter or liquid phase scintillometer.When marking agent is enzyme, add substrate, after dyeing, use colorimetric and fluorometric assay enzymic activity.When marking agent is fluorescent substance, phosphorus or coloring material, can measure by methods known in the art respectively.
Immunoassay of the present invention is used monoclonal antibody of the present invention, therefore has outstanding characteristic, can discern the N albumen epi-position that is present in the SARS virus, and not because cross reaction and therefore other materials of error-detecting can carry out the very high mensuration of specificity.
In order to implement immunoassay of the present invention, the present invention proposes a kind of diagnostic kit.This diagnostic kit contains at least a monoclonal antibody of the present invention, and can only contain a kind of monoclonal antibody.The monoclonal antibody that is used for this diagnostic kit is not particularly limited, and can be the proteic monoclonal antibody of any identification SARS virus N, also can be the degradation production of monoclonal antibody of the present invention, as F (ab ')
2, Fab ', Fab etc.
Diagnostic kit of the present invention can detect the sample that contains SARS virus with immunological method, and as blood, therefore oral secretion water or SARS virus can judge the infection of SARS virus.
In diagnostic kit of the present invention, but monoclonal antibody predetermined fixed of the present invention on solid phase, monoclonal antibody of the present invention can also be with above-mentioned marking agent mark.
The solid phase that is used for diagnostic kit of the present invention is not particularly limited, and includes but not limited to polymkeric substance (as polystyrene), granulated glass sphere, magnetic-particle, microtiter plate, immunochromatography filter paper, glass filter or other insoluble carriers.
Diagnostic kit of the present invention also can contain other assemblies.
Above-mentioned other assemblies are not particularly limited, and include but not limited to enzyme, its substrate, radio isotope, phosphorus, fluorescent substance, coloring material, damping fluid and culture dish that mark is used, and the above-mentioned composition of mentioning.
Though the form of diagnostic kit of the present invention is not particularly limited, the integration shape diagnostic kit that contains diagnostic kit all components of the present invention is preferred, so that fast, simply and easily diagnose.
Above-mentioned integrated diagnostic kit is not particularly limited, and for example can be box type, core pattern etc.
Embodiment as above-mentioned box type, use immunochromatography technique, for example can mention: a kind of embodiment, it comprises: a kind of film, an end be fixed on (downstream side) on the described film monoclonal antibody of the present invention, be fixed on the chromophoric solution on the film of side (upstream side), contain at above-mentioned marking agent and be arranged near the chromophoric solution and at the liner of the substrate in its downstream side be placed in the monoclonal antibody of containing on the film intermediary liner with above-mentioned marking agent mark.
Literary composition is given an example in the described diagnostic kit with box type embodiment in the use, sample is coated on the liner that contains with the monoclonal antibody of the present invention of above-mentioned marker mark, make SARS virus contained in the sample and monoclonal antibody with the marking agent mark of the present invention carry out combination then, by shift form in monoclonal antibody fixed of the present invention position in conjunction with product, make chromophoric solution colour developing mentioned above, in this stationkeeping monoclonal antibody of the present invention is arranged, form SARS virus N albumen, polyclonal antibody and immobilized monoclonal antibody with the marking agent mark form mixture.Then, above-mentioned labelled reagent and above-mentioned substrate reactions are with Show Color etc.Measure the color of demonstration etc., thereby carry out the judgement that SARS virus infects.
For with capacity chromophoric solution dilution sample, and the solution that obtains dripped to embodiment on the film, do not need in advance colour-developing solvent to be placed on the film.When during as above-mentioned marker, not needing substrate, can judge whether monoclonal antibody fixed of the present invention site forms above-mentioned mixture according to the colour developing of coloring material with coloring materials such as painted latex particles.
For example, for the above-mentioned core pattern embodiment that reacts with above-mentioned competitive method, can use core with a plurality of holes etc., they are integrally formed, comprise that (a) contains the hole of monoclonal antibody of the present invention, (b) contain the hole (as buffered soln) of liquid reagent, the SARS virus N albumen that contains above-mentioned labelled reagent mark in this liquid reagent, (c) contain the hole of liquid reagent (as buffered soln), this liquid reagent contains the substrate at above-mentioned labelled reagent, in order to implement reaction with sandwich assay, mention a kind of core etc., have a plurality of integrally formed holes, comprise that (a) contains the hole of insoluble carrier, is fixed with monoclonal antibody of the present invention on carrier, (b) contain the hole of liquid reagent (as buffered soln), this reagent contains with the monoclonal antibody of above-mentioned labelled reagent mark and (c) contains the hole of liquid reagent (as damping fluid), contains the substrate at above-mentioned marking agent.
In using the core pattern diagnostic kit of the invention described above for example, reaction and test are carried out with the same way as that be at war with method or sandwich assay use usually.
Diagnostic kit of the present invention can be the test kit that is used to implement above-mentioned competitive method or above-mentioned sandwich assay.Yet preferably use the test kit of sandwich assay.Above-mentioned sandwich assay has advantage, and is promptly highly sensitive, and the reaction times that needs is short, the accuracy rate height.Use above-mentioned immunochromatography technique, can prepare a kind of test kit, it makes testing method convenient and simple, and can be simply by the visual inspection judged result with it.When using above-mentioned sandwich assay with elisa technique, therefore the formation of enzyme reaction product can set up a kind of system by the decision of test objective amount of substance, can pass through colour developing etc. easily, and it is positive to determine that with visual inspection SARS virus infects.
The used sample of diagnostic kit of the present invention is not particularly limited, and includes but not limited to: blood, collutory and digestive tube movement, preferred blood.
Because diagnostic kit of the present invention uses monoclonal antibody, have high-caliber detection sensitivity, do not produce false negative or false positive problem, batch with criticize between without any difference.
According to the present invention, as antigen, can eliminate the influence that is present in other material in the sample with monoclonal antibody identification SARS virus N albumen, thus the existence of sensitivity that can be very high and specific detection SARS virus.Since set up the hybridoma that produces at the proteic monoclonal antibody of SARS virus N, now can the same monoclonal antibody of almost semi-permanent generation.Use the diagnostic kit of monoclonal antibody of the present invention therefore can infect by simple and convenient effective detection SARS virus N albumen, and patient not caused any misery with collutory as sample.In addition, under the situation of only using a kind of monoclonal antibody, use the diagnostic kit of monoclonal antibody of the present invention can stably obtain very high accuracy rate, and batch with batch between without any difference, therefore can be specific and the detection SARS virus infection of pin-point accuracy.Above-mentioned diagnostic kit is easy to measure, and is simple to operate.
The SARS antigen detecting agent box that uses in the embodiment of the invention is by the N albumen among the double-antibody sandwich elisa principle detection patients serum, can realize early stage diagnosis.SARS antigen detecting agent box detects principle, with the proteic monoclonal antibody of anti-sars N and the proteic polyclonal antibody of anti-N as immobilization antibody and enzyme labelled antibody, if there is the N antigen of sars virus in the testing sample, then form antibody-antigen-hrp-antibody complex, add tmb substrate and produce color reaction, otherwise then do not have color reaction.SARS antigen detecting agent box can in early days just can detect N antigen in patient infection virus, thereby diagnosis early goes out virus infection person in time finds in time to treat isolation, and what ward off disease spreads.
SARS virus N albumen combines with virus genome RNA and forms nucleocapsid, and many functions are arranged: provide the into signal of nuclear, the growth of interference cell, virus replication and RNA packing.Bibliographical information, behind the infection coronavirus, N albumen is the relevant albumen of the maximum virus of quantity, and the proteic antigenicity of N is stronger, these features make N albumen become a well diagnosis candidate target spot.And the double-antibody sandwich elisa method is a kind of very sensitive virus detection techniques, and is higher than other serological method specificity when detecting most of virus.And ELISA susceptibility height, easy and simple to handle, the development of necessary instrument equipment make schedule of operation standardization and automatization, thereby have further improved stability.
In addition, the invention allows for the purposes of said monoclonal antibody in the pharmaceutical composition of preparation treatment SARS virus infection associated diseases.Described pharmaceutical composition comprises monoclonal antibody of the present invention, also comprises pharmaceutically acceptable carrier or vehicle.
Description of drawings
The detection of Fig. 1, three immunity back rabbit antiserum titres.
Fig. 2, double-antibody sandwich elisa detect the N albumen and the JC albumen of different concns
The detected result (see figure 2) shows that the N protein concentration of detection and OD value are linear dependence, and its sensitivity is: 62.5ng/ml, promptly when the proteic concentration of N was 62.5ng/ml, detected result was still positive.And nothing to do with albumen JC becomes negative reaction.By further optimization experiment condition, detection sensitivity may further improve.
Fig. 3, double-antibody sandwich elisa detect three dilution 10 portions of normal human serums of difference.
Fig. 4, double-antibody sandwich elisa detect three dilution 10 parts of tumour patient serum of difference.
Fig. 5, double-antibody sandwich elisa detect three dilution 10 parts of SLE diseases of difference.
Fig. 6, double-antibody sandwich elisa detect three dilution 10 parts of patients with syphilis serum of difference.
Fig. 7, double-antibody sandwich elisa detect three dilution 10 parts of HBV patients serums of difference.
Fig. 8, double-antibody sandwich elisa detect three dilution 10 parts of HCV patients serums of difference.
Embodiment
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to people such as normal condition such as Sambrook, Russell, molecular cloning: laboratory manual III (New York:Cold Spring HarborLaboratory Press, 2001) condition described in, or the condition of advising according to manufacturer.
The proteic expression of embodiment 1:SARS N
After the proteic full-length gene of SARS N (SEQ ID NO:1) is inserted into expression vector pETs, transfection Escherichia coli BL21 (DE3).Adding IPTG is that 0.5mmol/L induces bacterial expression N albumen to final concentration, then according to operational manual behind the Ni-NTA affinitive layer purification, SDS-PAGE detects.
Embodiment 2: Polyclonal Antibody Preparation
Animal immune: with three immunizing rabbits of N albumen of embodiment 1 expression and purification.Immune for the first time with emulsification behind Fu Shi Freund's complete adjuvant and the N albumen equivalent mixing, subcutaneous multi-point injection, 1mg/, second and third time is immune with emulsification behind freund 's incomplete adjuvant and the N albumen equivalent mixing, and immunization method and consumption are immune for the first time together.Each immunity about one month at interval, blood was got in one week in the immunity back for the third time.
Indirect enzyme-linked immunosorbent adsorption experiment (indirect ELISA) detects sero-fast tiring:
With N albumen bag by the ELISA check-out console, contain the PBS confining liquid sealing of 10% bovine serum, 0.1%Tween, add different dilution antiserum(antisera)s then, hatched 2 hours for 37 ℃, rabbit anteserum adds the antibody of the goat-anti rabbit Ig of horseradish peroxidase (HRP) mark at last as negative control before establishing identical dilution immunity simultaneously.Hatched 1 hour for 37 ℃, add substrate tetramethyl benzidine (TMB) colour developing behind the thorough washing and measure 450nm place absorbance value.
Detected result such as Fig. 1, it is 1,024,000 that indirect ELISA detects sero-fast tiring, promptly still positive when antiserum(antisera) dilutes 1,024,000 times.
The separation of antibody and HRP enzyme labelling:
Antibody with in the ammonium sulfate salting-out process separation rabbit anti-serum with HRP and its coupling, is prepared into enzyme labelled antibody by the glutaraldehyde two-step approach.Demarcate the optimal dilution (method is the same) of its use with ELISA.ELISA result shows that the optimal dilution of enzyme labelled antibody is 1: 500.
Embodiment 3: the foundation of the proteic hybridoma cell strain of anti-N and the preparation of monoclonal antibody thereof
1, animal immune: with three immune balb/c mices of N albumen of gene recombination.The same rabbit of immunization method, immune for the third time back one month, recalling stimulated mouse.Detecting sero-fast tiring with indirect elisa method is 64000.
2, the preparation of hybridoma and screening: fetch and recall the mouse spleen cell and NS-1, the SP2/0 myeloma cell that stimulate after 3 days and merge with 50%PEG according to a conventional method, behind indirect ELISA method screening and clone, obtain hybridoma cell strain, wherein the S-39-2 clone is used for this work (preserving number is CCTCC NO:-C200407).This cell strain excretory antibody subtype is IgG1.
3, the preparation of ascites and purifying antibody: every mouse peritoneal injection 0.5ml pristane, pneumoretroperitoneum injection 10 in 7-10 days
6Individual hybridoma was got ascites after 7-10 days.Behind the ascites elder generation ammonium sulfate salting-out process preliminary purification, be further purified with protein G immune-affinity chromatography again.
4, the affinity constant of monoclonal antibody is measured: with reference to method (the Beatty JD of Beatty, Beatty BG, VlahosWG.Measurement of monoclonal antibody affinity by non-competitive enzymeimmunoassay.J Immunol Methods, 100 (1987) 173-9), with N albumen respectively with 0.625,1.25,2.5, the concentration bag quilt of 5ug/ml, the purified monoclonal antibody (seeing present embodiment the 3rd point) that adds doubling dilution, the antibody that adds the goat-anti mouse Ig of HRP enzyme labelling again, OD is surveyed in the TMB colour developing
450Value.Logarithm with antibody concentration is an X-coordinate, OD
450Value is ordinate zou, gets a growth saturation curve, and 4 kinds of antigen concentrations obtain 4 different curves, the par OD of each curve
450Value is obtained 50%OD as 100%
450The antibody concentration that value is corresponding, it is K that the formula by Beatty calculates affinity costant
Aff=3.1 * 10
9L/mol.
Embodiment 4: the sensitivity and the specificity that detect this test kit
1, the double-antibody sandwich elisa method detects N albumen and a kind of irrelevant vegetable-protein JC albumen of gene recombination
Wrap by the ELISA check-out console with the anti-N albumen monoclonal antibody (about 5ug/ml) behind the PBS dilution purifying that contains 0.01%Thimerosal, the 100ul/ hole, 4 ℃ are spent the night.Wash on the plate machine at Thermol that (1M Tris-HCl contains 0.1%Tween, 0.01%Thimerosal) washes 5 times with washings.With diluent (PBS that contains 10% calf serum, 0.01%Thimerosal) sealing, 200ul/ hole, 37 ℃, 2 hours.Wash again, add the N albumen and the JC albumen (with the PBS dilution that contains 0.01%Thimerosal) of different dilution gene recombination, 100ul/ hole, 37 ℃, 2 hours.After the washing, the enzyme that adds dilution in 1: 500 is marked the proteic polyclonal antibody of anti-N (using diluted), 100ul/ hole, 37 ℃, 1 hour.Washing then adds tmb substrate, and adding 2M H after 10 minutes is reacted in the 100ul/ hole
2SO
4, 100ul/ hole, termination reaction.On the Thermol microplate reader, read OD
450Value.
The detected result (see figure 2) shows that the N protein concentration of detection and OD value are linear dependence, and its sensitivity is: 62.5ng/ml, promptly when the proteic concentration of N was 62.5ng/ml, detected result was still positive.And nothing to do with albumen JC becomes negative reaction.By further optimization experiment condition, detection sensitivity may further improve.
2, the double-antibody sandwich elisa method detects human serum
Monoclonal antibody bag quilt, washing, sealing and enzyme mark resists reaction to be equal to embodiment 4.1, is to have added different dilution human serums when adding antigen just, and serum dilutes with the PBS that contains 0.01%Thimerosal.Negative control with figure below is a diluent, and positive control is the gene recombination N albumen of 1ug/ml.
No matter result (Fig. 3-8) shows also right and wrong SARS patients serum (patient such as HBV, HCV and the syphilis that comprise tumour, allergic patients, infective virus) and this detection system reaction that all is negative of normal human serum, has shown that this detection kit has good clinical application prospect.
The preparation of embodiment 5:ELISA detection kit
The ELISA check-out console (48 person-portion) of the proteic polyclonal antibody bag of anti-N quilt, the monoclonal antibody 5ml of HRP mark, tmb substrate 10ml, 10% calf serum diluent 20ml, 1M Tris-HCl washings 100ml.
The preparation of embodiment 6:ELISA detection kit
The ELISA check-out console (48 person-portion) of the proteic monoclonal antibody bag of anti-N quilt, the how anti-5ml of HRP mark, tmb substrate 10ml, 10% calf serum diluent 20ml, 1M Tris-HCl washings 100ml.
All quote in this application as a reference at all documents that the present invention mentions, just quoted as a reference separately as each piece document.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.
SEQUENCE?LISTING
＜110〉Shanghai Inst. of Life Science, CAS
＜120〉proteic monoclonal antibody of a kind of sars coronavirus N and application thereof
<130>040521
<160>2
<170>PatentIn?version?3.1
<210>1
<211>1266
<212>DNA
＜213〉SARS virus N albumen
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ggcagcagta?ggggaaattc?tcctgctcga?atggctagcg?gaggtggtga?aactgccctc 660
gcgctattgc?tgctagacag?attgaaccag?cttgagagca?aagtttctgg?taaaggccaa 720
caacaacaag?gccaaactgt?cactaagaaa?tctgctgctg?aggcatctaa?aaagcctcgc 780
caaaaacgta?ctgccacaaa?acagtacaac?gtcactcaag?catttgggag?acgtggtcca 840
gaacaaaccc?aaggaaattt?cggggaccaa?gacctaatca?gacaaggaac?tgattacaaa 900
cattggccgc?aaattgcaca?atttgctcca?agtgcctctg?cattctttgg?aatgtcacgc 960
attggcatgg?aagtcacacc?ttcgggaaca?tggctgactt?atcatggagc?cattaaattg 1020
gatgacaaag?atccacaatt?caaagacaac?gtcatactgc?tgaacaagca?cattgacgca 1080
tacaaaacat?tcccaccaac?agagcctaaa?aaggacaaaa?agaaaaagac?tgatgaagct 1140
cagcctttgc?cgcagagaca?aaagaagcag?cccactgtga?ctcttcttcc?tgcggctgac 1200
atggatgatt?tctccagaca?acttcaaaat?tccatgagtg?gagcttctgc?tgattcaact 1260
caggca 1266
<210>2
<211>422
<212>PRT
＜213〉SARS virus N albumen
<400>2
Met?Ser?Asp?Asn?Gly?Pro?Gln?Ser?Asn?Gln?Arg?Ser?Ala?Pro?Arg?Ile
1 5 10 15
Thr?Phe?Gly?Gly?Pro?Thr?Asp?Ser?Thr?Asp?Asn?Asn?Gln?Asn?Gly?Gly
20 25 30
Arg?Asn?Gly?Ala?Arg?Pro?Lys?Gln?Arg?Arg?Pro?Gln?Gly?Leu?Pro?Asn
35 40 45
Asn?Thr?Ala?Ser?Trp?Phe?Thr?Ala?Leu?Thr?Gln?His?Gly?Lys?Glu?Glu
50 55 60
Leu?Arg?Phe?Pro?Arg?Gly?Gln?Gly?Val?Pro?Ile?Asn?Thr?Asn?Ser?Gly
65 70 75 80
Pro?Asp?Asp?Gln?Ile?Gly?Tyr?Tyr?Arg?Arg?Ala?Thr?Arg?Arg?Val?Arg
85 90 95
Gly?Gly?Asp?Gly?Lys?Met?Lys?Glu?Leu?Ser?Pro?Arg?Trp?Tyr?Phe?Tyr
100 105 110
Tyr?Leu?Gly?Thr?Gly?Pro?Glu?Ala?Ser?Leu?Pro?Tyr?Gly?Ala?Asn?Lys
115 120 125
Glu?Gly?Ile?Val?Trp?Val?Ala?Thr?Glu?Gly?Ala?Leu?Asn?Thr?Pro?Lys
130 135 140
Asp?His?Ile?Gly?Thr?Arg?Asn?Pro?Asn?Asn?Asn?Ala?Ala?Thr?Val?Leu
145 150 155 160
Gln?Leu?Pro?Gln?Gly?Thr?Thr?Leu?Pro?Lys?Gly?Phe?Tyr?Ala?Glu?Gly
165 170 175
Ser?Arg?Gly?Gly?Ser?Gln?Ala?Ser?Ser?Arg?Ser?Ser?Ser?Arg?Ser?Arg
180 185 190
Gly?Asn?Ser?Arg?Asn?Ser?Thr?Pro?Gly?Ser?Ser?Arg?Gly?Asn?Ser?Pro
195 200 205
Ala?Arg?Met?Ala?Ser?Gly?Gly?Gly?Glu?Thr?Ala?Leu?Ala?Leu?Leu?Leu
210 215 220
Leu?Asp?Arg?Leu?Asn?Gln?Leu?Glu?Ser?Lys?Val?Ser?Gly?Lys?Gly?Gln
225 230 235 240
Gln?Gln?Gln?Gly?Gln?Thr?Val?Thr?Lys?Lys?Ser?Ala?Ala?Glu?Ala?Ser
245 250 255
Lys?Lys?Pro?Arg?Gln?Lys?Arg?Thr?Ala?Thr?Lys?Gln?Tyr?Asn?Val?Thr
260 265 270
Gln?Ala?Phe?Gly?Arg?Arg?Gly?Pro?Glu?Gln?Thr?Gln?Gly?Asn?Phe?Gly
275 280 285
Asp?Gln?Asp?Leu?Ile?Arg?Gln?Gly?Thr?Asp?Tyr?Lys?His?Trp?Pro?Gln
290 295 300
Ile?Ala?Gln?Phe?Ala?Pro?Ser?Ala?Ser?Ala?Phe?Phe?Gly?Met?Ser?Arg
305 310 315 320
Ile?Gly?Met?Glu?Val?Thr?Pro?Ser?Gly?Thr?Trp?Leu?Thr?Tyr?His?Gly
325 330 335
Ala?Ile?Lys?Leu?Asp?Asp?Lys?Asp?Pro?Gln?Phe?Lys?Asp?Asn?Val?Ile
340 345 350
Leu?Leu?Asn?Lys?His?Ile?Asp?Ala?Tyr?Lys?Thr?Phe?Pro?Pro?Thr?Glu
355 360 365
Pro?Lys?Lys?Asp?Lys?Lys?Lys?Lys?Thr?Asp?Glu?Ala?Gln?Pro?Leu?Pro
370 375 380
Gln?Arg?Gln?Lys?Lys?Gln?Pro?Thr?Val?Thr?Leu?Leu?Pro?Ala?Ala?Asp
385 390 395 400
Met?Asp?Asp?Phe?Ser?Arg?Gln?Leu?Gln?Asn?Ser?Met?Ser?Gly?Ala?Ser
405 410 415
Ala?Asp?Ser?Thr?Gln?Ala
420
Claims (10)
1, a kind of antibody of anti-SARS virus is characterized in that, the protein bound of aminoacid sequence shown in described antibodies specific ground and the SEQ ID NO:2.
2, antibody according to claim 1 is characterized in that, described antibody is monoclonal antibody.
3, antibody according to claim 2 is characterized in that, described monoclonal antibody is the cell strain secretion of CCTCC NO:-C200407 by preserving number.
4, antibody according to claim 3 is characterized in that, described antibody subtype is IgG1.
5, a kind of hybridoma cell strain of secreting the described antibody of claim 2, the preserving number of this hybridoma cell strain is CCTCCNO:-C200407.
6, a kind of immunologic detection method of early detection SARS virus is characterized in that, uses claim 2 or 3 described monoclonal antibodies.
7, a kind of detection kit of early detection SARS virus is characterized in that, contains a kind of as claim 2 or the described monoclonal antibody of claim 3 at least.
8, detection kit according to claim 7 is characterized in that described monoclonal antibody combines with marker, and this marker comprises fluorescent marker, radioactively labelled substance and enzyme labelling thing.
9, detection kit according to claim 8, it is characterized in that comprising: a kind of film, an end be fixed on monoclonal antibody of the present invention on the described film, be fixed on the chromophoric solution on the film of side, contain at above-mentioned marking agent and be arranged near the chromophoric solution, at the liner of the substrate in its downstream side be placed in the monoclonal antibody of containing on the film intermediary liner with above-mentioned marking agent mark.
10, the purposes of a kind of monoclonal antibody as claimed in claim 2 in the pharmaceutical composition of preparation treatment SARS virus infection associated diseases.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200410052873 CN1673231A (en) | 2004-07-15 | 2004-07-15 | Monoclonal antibody of SARS coronavirus N protein and its application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200410052873 CN1673231A (en) | 2004-07-15 | 2004-07-15 | Monoclonal antibody of SARS coronavirus N protein and its application |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN1673231A true CN1673231A (en) | 2005-09-28 |
Family
ID=35046033
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 200410052873 Pending CN1673231A (en) | 2004-07-15 | 2004-07-15 | Monoclonal antibody of SARS coronavirus N protein and its application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN1673231A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111153991A (en) * | 2020-02-26 | 2020-05-15 | 北京博奥森生物技术有限公司 | Human SARS-CoV-2 monoclonal antibody and its preparation method and use |
| CN111337668A (en) * | 2020-02-27 | 2020-06-26 | 四川省人民医院 | Novel coronavirus antigen colloidal gold rapid diagnosis kit and preparation method thereof |
| WO2021159703A1 (en) * | 2020-02-13 | 2021-08-19 | 北京华科泰生物技术股份有限公司 | Immunochromatographic kit for rapidly detecting novel coronavirus n protein, and preparation method and application thereof |
-
2004
- 2004-07-15 CN CN 200410052873 patent/CN1673231A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2021159703A1 (en) * | 2020-02-13 | 2021-08-19 | 北京华科泰生物技术股份有限公司 | Immunochromatographic kit for rapidly detecting novel coronavirus n protein, and preparation method and application thereof |
| CN111153991A (en) * | 2020-02-26 | 2020-05-15 | 北京博奥森生物技术有限公司 | Human SARS-CoV-2 monoclonal antibody and its preparation method and use |
| CN111337668A (en) * | 2020-02-27 | 2020-06-26 | 四川省人民医院 | Novel coronavirus antigen colloidal gold rapid diagnosis kit and preparation method thereof |
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