CN1590409A - Antibody pointed at SARS coronavirus N protein antigen and its use in detecting SARS coronavirus or its antigen - Google Patents
Antibody pointed at SARS coronavirus N protein antigen and its use in detecting SARS coronavirus or its antigen Download PDFInfo
- Publication number
- CN1590409A CN1590409A CN 03156361 CN03156361A CN1590409A CN 1590409 A CN1590409 A CN 1590409A CN 03156361 CN03156361 CN 03156361 CN 03156361 A CN03156361 A CN 03156361A CN 1590409 A CN1590409 A CN 1590409A
- Authority
- CN
- China
- Prior art keywords
- antibody
- sars
- sample
- sars coronavirus
- protein
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Landscapes
- Peptides Or Proteins (AREA)
Abstract
An antibody against the nucleocapsidprotein of SARS coronavirus, the method for using said antibody to detect the SARS coronavirus or its antigen, and the reagent kit and relative products for said method are disclosed.
Description
Technical field
The present invention relates to antibody, utilize sars coronavirus or its antigenic method that has situation in this antibody test sample at sars coronavirus nucleocapsid protein-N albumen (nucleocapsid protein).The invention still further relates to the test kit and the corresponding product that are used for these methods.
Background technology
(Severe Acute Respiratory Syndrome SARS) is a kind of acute fatal viral infection disease to the severe acute respiratory syndrome.Infect the report in Chinese Guangdong province from the first, only the time of several months is popular 32 countries in the world that infect.Till 11 days June in 2003,8400 persons of being contaminted are arranged in the world, 789 examples dead (WHO statistics).Because its quick propagation, high mortality and no effective methods of treatment make it become the disease of the viral infection that threatens the whole world.The infectivity of this disease is strong, has a strong impact on patient's body health, and higher lethality rate is arranged.
Have definite evidence to show, SARS by do not know in the past, temporary transient called after sars coronavirus (SARS coronavirus, SARS-CoV) viral caused.From several (about 35) SARS-CoV analysis of gene sequence, and with the correlated result of other known coronavirus, SARS-CoV is the positive chain RNA genome of a 29.7kb, has system's generation and mutability.Its genetic expression have very complicated transcribe, translation and posttranslational modification mechanism, its molecule mechanism remains further to be illustrated.
The clinical SARS-CoV detection reagent that has had at present has the enzyme-linked immunoassay method of pair Serum Antibody Detection and the RT-PCR method that viral genetic is detected.Produce the window phase that needs 1-3 week owing to infect back antibody, and the latent period from infecting to falling ill very short when SARS infects (within the general week), so antibody test is nonsensical to early diagnosis.Present in addition antibody test has higher false positive rate, and specificity can't be satisfied the demand.The method and technology of RT-PCR requires high, and the operating time is long, and sample needs special processing and corresponding apparatus, and its weak point is also arranged.Thereby above-mentioned two kinds of methods all are not accepted as the index that SARS diagnoses by WHO and U.S. CDC at present.Need especially in the prior art a kind of can infect early stage fast and the method that detects delicately.Specific diagnostic antigen method, especially quick, responsive, special SARS-CoV diagnostic antigen method has irreplaceable meaning in the SARS diagnosis.Still there is not report at present in the world about SARS diagnostic antigen reagent.Do not see with N antigen as detecting antigenic report yet.
The invention provides and to diagnose the method for SARS at the SARS antigen in infecting the early stage patient of mensuration sample, thereby satisfied this demand of the prior art.
Summary of the invention
One aspect of the present invention provides the antibody at sars coronavirus N proteantigen.
The present invention provides on the other hand and has utilized in the described antibody test sample that sars coronavirus or its are antigenic to be existed situation or carry out quantitative methods.
In yet another aspect, the invention provides the test kit that is applicable to aforesaid method.
Description of drawings
Fig. 1 has shown the detected result of test strip testing method of the present invention to each dilution sars coronavirus lysate and recombinant N protein.Wherein the anti-SARS-N protein polyclone antibody of goat (4mg/ml) is as trapping antibody, and the monoclonal antibody (#1 and #10) of anti-respectively C end and N end is puted together antibody as Radioactive colloidal gold and is used in combination, and sheep anti-mouse igg is as positive control.
Embodiment
The nucleocapsid protein nucleotide sequence total length of SARS-CoV (SARS coronavirus) has 1269 bases (base), is shown among the SEQ ID NO:1.The protein sequence total length has 422 amino acid, is shown in SEQ ID NO:26.Its antigen-specific is analyzed, with the proteic RNA sequence of SARS virus N (genbank locus:AY307165,1269bp, RNA linear VRL 09-Jun-2003; Definition:SARS coronavirusnucleocapsid protein (NP) gene, complete cds.; Accession:AY307165; Version:AY307165.1, GI:31540948; Protein_ID:AAP49024.1) with GenBank+EMBL+DDBJ+PDB in all known totally 1,878,949 nucleotide sequences, 8 of registering, 890,866,776 bases (base) are analyzed, and find that this nucleic acid sequences to proteins does not repeat with any known nucleotide sequence.When being analyzed with all known protein sequences, find that SARS N albumen only has 35.7% sequence identical with the N albumen of mouse hepatitis coronavirus between the 40-175 of aminoacid sequence site, between the 252-361 of sequence site, have 27.3% sequence identical.Further, known 35 kinds of SARS virus nucleotide sequences are being compared the back discovery, N albumen is almost completely identical in all virus isolated strains.
And, have data to show, N albumen has two kinds of molecular weight (molecular size) in the expression process, can with the proteic monoclonal antibody reactive of anti-N.Wherein short N albumen is relevant with the composition of virus, and long molecule is present in the cytoplasm, does not participate in the assembling of virus, is called residue N albumen.The two at the intracellular expression amount of virus infection much at one.In employing virus cracking liquid being carried out Westerm Blot analysis, the 25-50 that long and short type N expressing quantity is spinous process albumen (Spike protein, S albumen) and membranin (M albumen) expression amount doubly.Above phenomenon makes the virus antigen immunodetection object that N albumen might be favourable as a comparison.Not only, be easy to detect owing to its shared protein content height in virus is formed; Simultaneously owing to contain the higher proteic expression of residue N that does not participate in the virus assembling in the virus infected cell, might be in the process that virus strain discharges, or in infected process of cell death, this kind of N albumen can be discharged into the extracellular thereupon, enter body fluid and blood, as the amynologic index that detects virus infection and duplicate.
The present invention finds unexpectedly, has situation or content at what the proteic specific antibody of sars coronavirus N can be used for working sample sars coronavirus or its corresponding antigens, and can reach very high sensitivity and stability.
In the present invention, have concrete openly sequence of N albumen and produce the antibody of the present invention, can also adopt its variant or fusion polypeptide or immunogenic fragments to produce as antigen except using.
In the context of the present invention, can cause after sars coronavirus N proteic " immunogenicity part " is meant in being inoculated into individual body immunne response and equally can with the part of the antibodies that produces at sars coronavirus N albumen.Therefore, protein immunogenicity part available antibodies described herein is identified in conjunction with test.These test any the carrying out in the known several different methods of common available those of ordinary skills, for example, at Harlow and Lane, the method described in " antibody: laboratory manual " (cold spring harbor laboratory, cold spring port, New York, 1988).For example, can utilize described albumen to produce mono-clonal and polyclonal antibody, to be used for detecting SARS patient or suspected patient blood or this proteic situation that exists of other sample.
Polypeptide used herein " variant " is only to be that with the difference of described polypeptide conservative property substitutes and/or modifies and still kept the immunogenic polypeptide of polypeptide.Polypeptide variants and the polypeptide of having identified preferably have at least about 80%, more preferably at least about 90%, most preferably at least about 95% identity.For immunocompetent N albumen is arranged, its variant can be identified by the aminoacid sequence of for example modifying one of aforementioned polypeptides and the immunocompetence of assessing this modified polypeptide.
Used herein " conservative property substitutes " is meant that certain monoamino-acid is substituted by the amino acid of another tool similar characteristics, and the chemistry of peptides those of skill in the art can predict not material alterations of the secondary structure of this polypeptide and water-wet behavior.Usually in fact, following amino acid group has been represented the conservative property change: (1) L-Ala, proline(Pro), glycine, L-glutamic acid, aspartic acid, glutamine, l-asparagine, Serine, Threonine; (2) halfcystine, Serine, tyrosine, Threonine; (3) Xie Ansuan, Isoleucine, leucine, methionine(Met), L-Ala, phenylalanine; (4) Methionin, arginine, Histidine; (5) phenylalanine, tyrosine, tryptophane, Histidine.
Variant can be simultaneously or is only comprised other modification, comprises deletion or adds this polypeptide antigen, secondary structure and the small amino acid of water-wet behavior influence.For example, can be with signal (or leading) sequence at proteinic N-terminal and conjugation of polypeptides, this sequence can be translated with protein, and instructs proteinic transfer after translation.Polypeptide also can be puted together with joint or other sequence, and polypeptide is synthetic to be easy to, purifying or evaluation (as the poly Histidine) or strengthen combining of polypeptide and solid support.For example, the FC district of polypeptide and immunoglobulin (Ig) can be puted together.
" polypeptide " used herein also comprises combination or fusion polypeptide." combination polypeptide " is to contain at least one above-mentioned immunogenicity part and one or more extra immunogenic SARS antigenic polypeptides, and they connect into an amino acid strand by peptide bond.These sequences can directly link together (promptly not having the amino acid that inserts), maybe can link together by can obviously not weakening the immunogenic catenation sequence of polypeptide fraction (as Gly-Cys-Gly).
In the available several different methods well known in the art any obtains SARS albumen of the present invention and this type of protein DNA molecule of coding.Be equivalent to one of code book invention SARS virus N albumen gene (or its part) dna sequence dna can by the SARS virus RNA through reverse transcription is checked order and compare with known SARS virus nucleotide sequence after obtain.The partial dna sequence that obtains like this can be used for the design oligonucleotides primer, with amplification full length DNA sequence in RT-PCR, used technology be well known in the art (visible as people such as Mullis, Cold Spring Harber Symp.Quant.Biol., 51:263,1987; Erlich compiles, round pcr, Stockton Press, New York, 1989).In case obtained the dna sequence dna of coded polypeptide, promptly the induced-mutation technique of available standard as the oligonucleotide mediated site-directed mutagenesis of being instructed in (DNA, 2:183,1983) such as Adelman, is introduced above-mentioned any modification easily.Sequence of the present invention also can directly be synthesized.
Also available synthetic or recombination form produces sars coronavirus N albumen disclosed herein or its immunogenicity part.Be less than about 100 amino acid and be less than about 50 amino acid whose synthetic polypeptide usually and can prepare with the technology that those of ordinary skills know.For example, this class polypeptide can be with any synthetic for the solid phase technique that utilizes, as the Merrifield solid phase synthesis process, wherein amino acid be added to successively on the amino acid chain that is prolonging (be found in as, Merrifield, U.S. chemical institute magazine (J.Am.Chem.Soc.85:2149-2146,1963).The polypeptide automatic DNA synthesizer DNA can be available from as branch of Perkin Elmer/ applying biological system (Foster city, producer such as CA), and can operating by the indication of manufacturers.
The generation of all can recombinating of any aforementioned polypeptides, the dna sequence dna that is about to coded polypeptide inserts in the expression vector, and in suitable host marking protein.In all expression vectors known to those of ordinary skills any all can be used to express recombinant polypeptide of the present invention.Transform or transfection contain in any suitable host cell of expression vector of recombinant polypeptide coding DNA molecule and all can express.Proper host cell comprises prokaryotic cell prokaryocyte, yeast and higher eucaryotic cells.Preferred used host cell is intestinal bacteria, yeast or cells of mamma animals system, as Chinese hamster ovary celI.The naturally occurring polypeptide of dna sequence dna codified of Biao Daing, the natural part that has polypeptide in this way, or their other variant.
Can utilize sars coronavirus N albumen of the present invention or its variant or immunogenicity partly to prepare its specific antibody by several different methods known in the art, comprise monoclonal antibody and polyclonal antibody.Antibody of the present invention also can be strand, chimeric, transplant CDR's or humanized.Such antibody can be by numerous technology preparations well known by persons skilled in the art.Consult, as Harlow and Lane, " antibody: laboratory manual ", cold spring harbor laboratory, 1988.In a kind of this type of technology, the immunogen that at first will contain antigenic polypeptide is injected in any of multiple Mammals (as rabbit, sheep, horse, donkey and goat etc.) or in the chicken.In this step, polypeptide of the present invention need not modified and promptly can be used as immunogen.Perhaps, particularly for short relatively polypeptide, polypeptide and carrier proteins (as bovine serum albumin or keyhole worm relative hemocyanin) can be linked together, to excite stronger immunne response.Immunogen is injected into the animal host,, injects the one or many booster immunization again, and regularly animal is got blood preferably according to the preset time table.The polyclonal antibody special to polypeptide can obtain by the affinity chromatography purifying that has for example utilized the polypeptide that is coupled on the suitable solid phase upholder from antiserum(antisera).
Be different from the ordinary method of preparation polyclonal antibody in laboratory animal and domestic animal, can utilize known Hybridoma Cell Culture technology to prepare the proteic monoclonal antibody of anti-N.In general, this method comprises that preparation produces the fused cell system of antibody, as with former generation splenocyte and compatible myelomatosis continuous cell line merge, by a large amount of culture techniques of cell or change the myeloma cell over to and originate and cultivate in animal or the compatible animal strain, make the fused cell growth.Compare with the antibody that immune animal produces, this kind antibody has more advantage, and for example they have excellent specificity, susceptibility, compare on immunochemistry merely.The immunocompetence fragment of these antibody also belongs within the scope of the present invention, as the humanized monoclonal antibody F of part (ab) fragment.
Chimeric and modified antibodies
Chimeric antibody comprises from the fusion between the constant region in the variable region in an immunoglobulin (Ig) source and another immunoglobulin (Ig) source.The variable region derives from the immunoglobulin (Ig) of another kind usually, perhaps is the people.This technology is understood thoroughly in this area.European patent application EP-A-0125 for example, 023 (Cabilly/Genetech) and EP-A-0120,694 and United States Patent (USP) NO4,816,567.These patents have disclosed with recombinant DNA method and have prepared the immunoglobulins molecular variant.
Another kind of preparation approach chimeric or modified antibodies is that the immunity district with monoclonal antibody is connected with another NIg molecule, and generation chimeric molecule derivative (referring to WO86/01533, (Veuberger and Rabbits/Celltech).Another kind method is gomphosis immunoglobulin of preparation, make it have different specificity (referring to EP68763) in different variable regions, also having a kind of method is to introduce a sudden change in the DNA of coding monoclonal antibody, do not changing some character that its specific substantially situation changes it, this aspect can be finished by rite-directed mutagenesis and other technology of being understood thoroughly in this area.
Utilize recombinant DNA technology, Winter patent application EP-A-0239400 has disclosed the another kind of method for preparing antibody derivatives, substitutes a kind of complement determining area (CDRs) of immune globulin variable region with the complement determining area of another kind of specific immunoglobulin.Like this, combine with the change in frame zone, the CDR implantation technique can make the antibody humanization.
Preparation people antibody also can be rebuild human immunity system in the mouse body that lacks its natural immune system, immune mouse is with the former special human antibodies that creates antagonism then.
Operate or change the gene of any known antibodies or encoding antibody, produce the technology of the antibody of deriving, by being understood thoroughly this area.
Be specific to the monoclonal antibody of purpose antigenic polypeptide, for example, can use Kohler and Milstein, European Journal of Immunology, 6:511-519,1976 the technology and the preparation of improving one's methods.Briefly, these methods relate to the permanent cell line that preparation can produce and had required specificity the antibody of the reactivity of desired polypeptides (just with).This clone can be obtained by the spleen cell of for example above-mentioned immune animal.Then with the spleen cell immortalization, for example by with myeloma cell's fusion partners, preferably merge with the homologous myeloma cell's fusion partners of immune animal.Can adopt multiple integration technology.For example, with spleen cell and myeloma cell and nonionic surface active agent mixed for several minutes, be tiled in low density then and can support hybrid cell growth and can not support on the selective medium that the myeloma cell grows.Utilized HAT (xanthoglobulin, aminopterin-induced syndrome, thymidine) screening in the preferred selection technology.Through the enough time, after about 1 to 2 week, just can be observed the heterozygote colony usually.The single colony of picking detects that it is active with combining of polypeptide.Preferably have high reaction activity and specific hybridoma.
Monoclonal antibody can be separated from the hybridoma colony supernatant liquor of growing and obtained.In addition, can adopt multiple technologies to improve output, for example hybridoma cell line is expelled in the abdominal cavity of suitable vertebrate host such as mouse, can from ascites or blood, gather in the crops monoclonal antibody then.Pollutent in the antibody can be removed with routine techniques, as chromatography, gel-filtration, precipitation and extracting.Polypeptide of the present invention can be used for for example affinity chromatography step in purge process.
The mensuration mode of the polypeptide marker in the known multiple use antibody test sample of this area routine techniques personnel.For example see Harlow and Lane, " antibody: laboratory manual ", cold spring harbor laboratory, 1988.In a preferred embodiment, mensuration comprises that the antibody that use is fixed on the solid support separates in conjunction with the N proteantigen and with its rest part with sample.The available subsequently second antibody that contains reporter group of bonded N albumen-antibody complex detects.Perhaps, can utilize competitive analysis, wherein polypeptide is by reporter group institute mark, and after sample and the common insulation of antibody it is attached on the immobilized antibody.The reactive behavior that the degree of component inhibition labeling polypeptide and antibodies has indicated sample and immobilized antibody in the sample.
Solid support can be the material that those of ordinary skills are known, antibody can be attached to it.For example, solid support can be test hole or nitrocellulose filter or other suitable membrane on the microtiter plate.Perhaps, upholder can be pearl or dish, such as glass, fiberglass, latex, Radioactive colloidal gold or plastic material, as polystyrene or polyvinyl chloride.Upholder also can be magnetic-particle or fiber optic sensor, for example is disclosed in U.S. Patent number 5,359, those materials in 681.Antibody can be fixed on the solid support with multiple technologies well known by persons skilled in the art, and these technology have sufficient description in patent and scientific literature.In the context of the present invention, " immobilization " speech refers to non-covalent combination (as absorption), and covalent attachment (can be direct connection the between functional group on antibody and the upholder, or the connection by cross-linking reagent).By on the hole that is adsorbed onto microtiter plate or on the film and immobilized method is preferred.In this class situation, can be by in the damping fluid that is fit to, make antibody contact one suitable period with solid support and finish absorption.Change with temperature duration of contact, but normally between about 1 hour to 1 day.In general, with plastic microtiter (as polystyrene or polyvinyl chloride) aperture with about 10ng-10 μ g, preferably the antibody of about 100ng-1 μ g contacts, and is enough to the fixedly wedding agent of appropriate amount.
Generally can be by bifunctional reagent and upholder be at first reacted the covalent attachment of finishing antibody and solid support, described difunctional dose can with the two reaction of functional group's (as hydroxyl or amino) on upholder and the wedding agent.For example, utilize benzoquinones, or by between the aldehyde radical on the solid support and amido on the antibody and active hydrogen, condensation taking place, can antibody is covalently bound with the upholder with suitable polymer coating.[consult Pierce ImmunotechndogyCatalog and Handbook, 1991, in A12-A13].
In certain embodiments, can measure the exist situation or the content of the sars coronavirus in the sample or its corresponding antigens by the double-antibody sandwich analysis.The antibody that at first will be fixed on the solid support (often being the hole of microtiter plate) contacts with sample, so that the polypeptide in the sample is attached on the immobilized antibody, thereby finishes this analysis.Subsequently unconjugated sample is removed from immobilization polypeptide-antibody complex body, and adding can be in conjunction with the second antibody (containing a reporter group) in another site on the polypeptide.Measure the amount that still is incorporated into the second antibody on the solid support with the method that is suitable for the particular report group then.
More particularly, in case antibody is fixed on the upholder as mentioned above, seal protein binding site remaining on the upholder usually.Many suitable encapsulants are known to those of ordinary skills, as bSA or polysorbas20
TM(Sigma chemical company, the St. Louis, MO).Then immobilized antibody and sample are incubated jointly, make polypeptide and antibodies.Available suitable diluted sample before the incubation is as phosphate buffered saline buffer (PBS).In general, suitable duration of contact (being soaking time) is to be enough to determine that there is the time of situation in sars coronavirus N albumen in the sample.Preferred duration of contact be enough to make in conjunction with level reach combination and during not in conjunction with the polypeptide balance in conjunction with time of at least 95% of level.Those of ordinary skills will appreciate that, by in for some time in conjunction with level, can measure easily and reach the balance time necessary.During room temperature, about 30 minutes soaking time is just enough usually.
Then, available suitable damping fluid (as contains 0.1% polysorbas20
TMPBS) clean solid support and remove not in conjunction with sample.The second antibody that contains reporter group can be added on the solid support subsequently.Preferred reporter group comprises enzyme (as horseradish peroxidase), substrate, cofactor, inhibitor, dyestuff, radionuclide, luminophore, fluorophor and vitamin H.The standard method of puting together known to available those of ordinary skills of antibody and reporter group is finished.
Then with second antibody and immobilized antibody-polypeptide complex body incubation sufficiently long time, to detect related polypeptide.Usually according to determining the suitable incubation time in conjunction with level after test for some time.Then remove unconjugated second antibody, and detect the bonded second antibody by reporter group.The method of examining report group depends on the character of reporter group.For the radioactivity group, scintillation counting technique or radioautograph method are normally feasible.Spectrography can be used to detect dyestuff, luminophore and fluorophor.Vitamin H can be with coupling the avidin of other reporter group (normally radioactivity or fluorophor or enzyme) detect.Usually by adding substrate (generally lasting specified time), with spectrography or other methods analyst reaction product the enzyme reporter group being detected again.
Have situation or content for what determine sars coronavirus in the sample or its corresponding antigens, the signal and the typical curve of the reporter group that still is incorporated into solid support that will record usually compare.In a preferred examples, typical curve is by with sars coronavirus lysate or the corresponding antigens incubation of immobilized antibody with serial dilution, and detected signal and employing virus cracking liquid or antigen titre/concentration mapped obtains.
In a related example, can carry out this analysis by circulating form.Wherein antibody is fixed on film, on nitrocellulose.In process of the test, when sample flow during through film, the polypeptide in the sample promptly combines with immobilized antibody.Then, when the solution stream that contains second antibody during through film, the second antibody of mark promptly combines with the antibody-polypeptide complex body.The detection of bonded second antibody can be carried out according to aforesaid method.
In another related example, can carry out this analysis by the test strip test form.Wherein, the end immersion that combines the film of antibody is contained in the solution of sample.Sample moves along film, through containing the zone of first antibody, to the zone of trapping antibody (second antibody).First antibody is in the concentrated explanation virus in the zone of trapping antibody or the existence of corresponding antigens.Usually, first antibody is at the concentrated visible pattern that produced in this site, as a line.Not having this pattern explanation is negative findings.In one embodiment, the film that is used for the test strip test is a nitrocellulose filter, nylon membrane or analogue etc.First antibody can be a polyclonal antibody, also can be one or more monoclonal antibody, perhaps its combination.Second antibody also can be a polyclonal antibody, perhaps one or more monoclonal antibody, perhaps its combination.The pairing service condition of first antibody and second antibody can determine that this will more specifically describe hereinafter through cooperating experiment.First antibody can be attached on colloid gold particle, magnetic-particle, the microballon etc.Colloid gold particle preferably, its size can be 10-100nm, preferred 20-60nm, especially for example 40nm.Preferably, test sample is further handled, its antigenicity is improved.For example, sample is carried out cracking handle, to expose its N proteantigen.Cracking can be adopted for example 0.001-0.2%SDS, preferably 0.005-0.15%SDS, more preferably 0.01-0.1%SDS perhaps boiled 5-10 minute in room temperature treatment.When detecting SARS virus, can also before detection, carry out the Vero cell amplification to virus, detect then, like this sars coronavirus in the sample is detected lower limit and can reach lower.Usually, the amount of selecting for use that is fixed on the antibody on the film is, when containing in the biological sample when being enough to produce the polypeptide level of positive signal in the double-antibody sandwich test, but also can produce the pattern that naked eyes are discovered in above-mentioned detection mode.Preferably, the antibody amount that is fixed on the film is about 25ng-1 μ g, more preferably about 50ng-500ng.This class detects and can biological sample very in a small amount be carried out.This immunity test strip of SARS fast bar method has the advantage of sensitivity, quick, special, easy (operation does not need special technique and equipment) and early diagnosis, but also can reduce the generation of pattern detection waste (liquid), the advantage that has other detection method not possess in the isolation of disease, aspect anti-pollution.
Be applicable to that antibody of the present invention can be varied, as long as it has enough reactive behavioies and the reactive of recombinant N protein and natural sars coronavirus lysate quite (preferably equated).The selection of pairing antibody can be carried out according to ordinary method, for example common double antibody sandwich experiment.In brief, first antibody is fixed in the hole of 96 hole microtiter plates, adding standard N proteantigen is the enough time of incubation with it, so that fully combination.For example add second antibody then, incubation through horseradish peroxidase-labeled.Washing is removed after the unconjugated second antibody, with the substrate colour developing of respective markers.Selecting second antibody can be in conjunction with combination (being antagonistic action can not take place between first antibody and the second antibody) thereon.In one embodiment, described first antibody and second antibody all are polyclonal antibodies.In another embodiment, described first antibody is the mixture of a kind of monoclonal antibody or two or more monoclonal antibodies, and described second antibody is a polyclonal antibody.In another embodiment, described first antibody and second antibody are the monoclonal antibodies that can match.
In the context of the present invention, " antibody that can match " be meant described antibody at be epi-position different in the N proteantigen, with the combination of N proteantigen in the antibody combination of mutual interference mutually or antagonism does not take place.
Antibody among the present invention cooperates test for example to carry out according to following method: as trapping antibody (being sprayed on the film), put together antibody (gold conjugate) with other the individual plant monoclonal antibody or the combination of many strains monoclonal antibody as Radioactive colloidal gold with the combination of individual plant monoclonal antibody or many strains monoclonal antibody.Also can be with polyclonal antibody (goat-anti SARS-N antibody) as trapping antibody (being sprayed on the film), individual plant or many strains monoclonal antibody are as puting together antibody.Spray film line in contrast with the anti-IgG of 1mg/ml.The consumption of antibody of serving as a mark is decided by each single characteristic of planting antibody itself, with can be behind 10% sodium-chlor that adds 10%v/v colloid gold particle cohesion does not take place is standard, this can be definite by the serial dilution degree.
The consumption of the antibody that mark is good can determine the susceptibility of reagent, and is general so that the OD of the Radioactive colloidal gold of traget antibody (40nm colloid gold particle commonly used is in the absorbancy at 520-530nm wavelength place) is relevant with the amount on being used in golden film (gold pad).In the production Radioactive colloidal gold being puted together antibody is applied to and two kinds of methods is arranged, promptly traditional infusion method and improved spray embrane method on the golden film.The former generally uses OD
530Suspension between 1-10 is by the consumption of the limited aignment mark antibody of OD concentration; The latter generally uses OD
530=50 suspension spray film, but be concentrated to OD Radioactive colloidal gold can't being puted together antibody complex
530The next OD that adopts of the situation of=50 suspension
530The maximum concentration that can be concentrated to below＜50.Its consumption is to decide with the volume that is sprayed onto on the golden film.But the volume of general working range is between 1-2.5ul/cm.In SARS N proteantigen detection reagent development of the present invention, all system testings between 0.5-3.0ul/cm of all traget antibodies can reach suitable susceptibility at 1-2.5ul/cm usually, reach best susceptibility between 2.0-3.0.The characteristic that also is decided by different monoclonal antibodies itself as the consumption of trapping antibody, whether as having between the avidity of antibody, antibody purity, the antibody add up mutually or antagonistic action or the like, the consumption of polyclonal antibody is decided by the titre of antibody, method of purification factors such as (whether lean on through affinity chromatography and purify or be the clear IgG fraction of simple immunology).General antibody consumption between 0.5mg/ml-5mg/ml, preferred 1mg/ml-3mg/m.High sensitive will be decided by the combination and the mated condition of antibody (trapping antibody and Radioactive colloidal gold are puted together antibody).
The inventor finds, utilizes the proteic antibody of SARS coronary virus resistant N, mode that can test strip easily in the working sample SARS virus have a situation, and can reach and the RT-PCR detection sensitivity of having reported so far (1000pfu/ml).Preferably, described first antibody is labeled Radioactive colloidal gold.The size of colloid gold particle can be for example 20-60nm, especially 40nm.In the present invention, can use size and inhomogenous colloid gold particle.Can put together with first antibody and Radioactive colloidal gold by ordinary method.Such method has numerous scientific and technical literatures can be for reference in the prior art, also is found in for example webpage of Arista Biologicals Inc. of many colloid gold particles manufacturer.
The mark environment PH has direct influence to labeling effciency.Polyclonal antibody generally carries out at pH7.2-7.5 the mark of Radioactive colloidal gold.For monoclonal antibody, mark environment pH value generally is than the about 0.1-0.5 of its pI (iso-electric point) meta-alkalescence.This can determine by the pI value of measuring concrete monoclonal antibody.Perhaps, can measure monoclonal antibody labeling effciency, selection the best to Radioactive colloidal gold under different pH values and determine by pH value gradient is set.The usefulness of the enough marks of the consumption of traget antibody gets final product, and is too much unsuitable, otherwise can increase cost, and even more serious is can the interference measurement reaction.Directly detection method be with can be behind the 10%NaCl solution that adds 1/10 volume room temperature leave standstill 10 minutes colloid gold particles cohesion does not take place be standard.Select for use the minimum antibody concentration that can reach this degree to carry out the mark of colloid gold particle.The colloid gold particle suspension that mark is good (common OD
530nmReading between the 1.0-10) can be applied to by traditional direct infusion method on the golden film (gold pad).Perhaps, can concentrate the good colloid gold particle suspension of mark, for example 2400g-4400g, 4 ℃ were condensed into OD in centrifugal 30-60 minute
530nmReading reach 50 or its peaked suspension, be sprayed onto on the golden film by the consumption of improved spray embrane method then with 0.5-3.0 μ l/cm, preferred 1.0-3.0 μ l/cm, for example 1-2.5,2.0-3.0 μ l/cm.
As the second antibody of " trapping antibody ", its consumption depends on the character and the purity of antibody itself, and with the mated condition of first antibody.For example with the avidity of monoclonal antibody, antibody purity, antibody between whether have add up mutually or factor such as antagonistic effect relevant.As polyclonal antibody, its consumption is decided by titre, method of purification of antibody for example etc.Generally between 1mg/ml-5mg/ml.Can determine best Di Yikangti ﹠amp by ordinary method; Second antibody combination and consumption.For example, after described method has been determined the combination of suitable first antibody and second antibody according to preamble, on 96 hole microtiter plates with the first antibody bag of serial dilution by respective aperture, again with sars coronavirus N proteantigen enough time of incubation of proper concn.Fully after the combination, remove incubation liquid, add the mark second antibody incubation of serial dilution.Colour developing afterwards.Selection falls into the Di Yikangti ﹠amp of the numerical range that the ELISA reader is fit to read; Second antibody cooperates consumption.Can adopt the scope of determining like this in laboratory afterwards or the clinical detection.By standard N proteantigen or SARS virus lysate reading to series concentration, make typical curve, double-antibody sandwich detection method of the present invention can also be used for the N proteantigen of sample or SARS virus content are carried out quantitative assay.
In the method for the invention, first antibody and second antibody need not refining and can use by simple ammonium sulfate precipitation with after dialysing.Certainly, undoubtedly, also can be used among the present invention through the antibody of for example affinity purification.
In test strip measuring method of the present invention, except colloid gold particle, can also use magnetic-particle, latex particle through the first antibody mark to wait and carry out.The consumption of wherein corresponding particulate mark, consumption, second antibody etc. all can be determined routinely.
Can also adopt for example method such as kapillary, sars coronavirus in double antibody sandwich method test sample disclosed by the invention or corresponding N proteantigen.Such mensuration mode all is as known in the art, and has numerous documents can be for reference.
What detect in the measuring method of the present invention is N proteantigen, i.e. nucleocapsid protein.In complete virion, be coated on granule interior.Therefore, if without cracking, in complete S ARS coronavirus be detect less than.Yet, result of study shows, contain the higher proteic expression of residue N that does not participate in the virus assembling in the virus infected cell, might be in the process that virus strain discharges, or in infected process of cell death, be discharged into the extracellular thereupon, enter body fluid and blood vessel, as the amynologic index that detects virus infection and duplicate, thus direct, easy by method of the present invention, detect apace.
If detect the N proteantigen of intact virus, then can use 0.001-0.2%, preferred 0.005-0.15% simply, more preferably 0.01-0.1%, for example 0.05%SDS handle sample, so that the lytic virus particle, release N proteantigen detects.
In the test strip experiment, can simply test strip be immersed in sample or the lysate and detect.This has simplified the processing of sample greatly, has saved the time, and important meaning is arranged in clinical diagnosis.
Certainly, exist and to be suitable for the test method used with antibody of the present invention in a large number.Foregoing description just illustrates.
In another example, sars coronavirus N albumen can be used as the mark use of monitoring SARS conditions of patients.In this example, above-mentionedly diagnose described mensuration to carry out in time, and assess the change situation of reactive polypeptide level with regard to SARS.For example, can be during 1 month to 1 year once measure in every 24-48 hour, carry out on demand later on.Usually, through antibody test, the polypeptide level continues to increase, and illustrates that SARS is developing always in patient's body.On the contrary, when the level of reaction polypeptide reduced in time, SARS no longer developed.
Antibody of the present invention and/or method can be used for suitable patient specimen is detected, as the index of observation of curative effect and whether have infectivity and whether need isolated index.One of them embodiment is to provide monitoring scheme for therapeutic intervention.Preferably, periodicity is monitored sars coronavirus N protein level in therapeutic process, before the treatment beginning or after the treatment end periodicity of sars coronavirus N protein level is measured and may be highly profitable.
" patient " described herein is meant any warm-blooded animal, people preferably, and the patient can be ill, or does not suffer from perceptible disease.
In the context of the present invention, " sample " can be anyly may contain SARS virus or its antigenic sample, comprises for example serum, whole blood, sputum, oral cavity/nasopharyngeal secretions or washing lotion, urine, ight soil, pleural effusions and ascites, cerebrospinal fluid and tissue sample.
The invention provides the diagnostic kit that a physiological sample that has SARS virus to pollute to suspection detects.This test kit comprises that (a) is of the present invention at the proteic antibody of sars coronavirus N, for example one or more monoclonal antibodies and/or polyclonal antibody; (b) Ren Xuan sample preparation solution, for example 0.01-0.2%SDS; (c) working instructions.Also can contain suitable detectable (for example horseradish peroxidase) and corresponding mark and detection reagent in the diagnostic kit of the present invention.In one embodiment, comprise the first antibody and the second antibody that can be used in the described test kit.Preferably, described first antibody is puted together mutually with Radioactive colloidal gold, magnetic-particle, latex etc.
The present invention further provides the method for SARS virus in a kind of detection or the working sample.This method comprises and will randomly mix with SARS coronary virus resistant N protein antibodies of the present invention through the sample of handling (for example utilizing 0.01-0.2%SDS to cross in room temperature treatment), form a kind of antibody and antigenic divalence mixture of comprising, the mixture that forms is carried out qualitative detection and quantitative assay.What the existence of this complex body or quantity had been indicated SARS virus exists situation or content.Antibody of the present invention comprises human or animal's monoclonal antibody, polyclonal antibody preparation, and antibody fragment or synthetic antibody.Synthetic antibody comprises recombinant antibodies and chimeric antibody, and it comprises humanized antibody again, antiidiotypic antibody and derivative thereof.
The present invention is further illustrated by following examples.These embodiment are exemplary, and do not limit the present invention in any way.
N protein nucleic acid sequence total length is 1269bp, and the protein sequence total length has 422 amino acid.Because SARS virus is the pathogenic agent of communicable disease of the serious threat life of air-borne transmission, with the synthetic safety and simple from security consideration.(Locus:AY307165 Protein_id=" AAP49024.1 " synthesizes whole coding region sequence at sars coronavirus nucleocapsid protein (NP) gene complete coding region nucleotide sequence that GenBank delivers to be dependent on June 19th, 2003, this sequence is shown in SEQ ID NO:1, and the aminoacid sequence of its deduction is shown in SEQ ID No:26.
N protein coding gene synthesis strategy is as follows:
1??????3????5????7????9????11????13????15????17????19
5’????-????-????-????-????-?????-?????-?????-?????-????3’
3’????-????-????-????-????-?????-?????-?????-?????-????5’
2??????4????6????8????10???12????14????16????18????20
For finishing the synthetic of complete nucleotide sequence, at first synthetic following oligonucleotide:
1) 5 ' atgtctgataatggaccccaatcaaaccaacgtagtgccccccgcattacatttgg tggacccacagatt 3 ' (SEQ ID NO:2 is corresponding to sequence shown in the Nucleotide of 1-70 position among the SEQ ID NO:1);
2) 5 ' ctgttttggccttgccccattgcgtcctccattctggttattgtcagttgaatctg tgggtccaccaaat 3 ' (SEQ ID NO:3 is corresponding to the complementary sequence of sequence shown in the Nucleotide of 51-140 position among the SEQ ID NO:1);
3) 5 ' ccgaccccaaggtttacccaataatattgcgtcttggttcacagctctcactcagc atggcaaggaggaacttagatt 3 ' (SEQ ID NO:4 is corresponding to sequence shown in the Nucleotide of 123-200 position among the SEQ ID NO:1);
4) 5 ' tagccaatttggtcatctggaccactattggtgttgattggaacgccctggcctcg agggaatctaagttcctccttgcc 3 ' (SEQ ID NO:5 is corresponding to the complementary sequence of sequence shown in the Nucleotide of 181-260 position among the SEQ ID NO:1);
5) 5 ' ccagatgaccaaattggctactaccgaagagctacccgacgagttcgtggtggtga cggcaaaatgaaagagctcagccccag 3 ' (SEQ ID NO:6 is corresponding to sequence shown in the Nucleotide of 241-323 position among the SEQ IDNO:1);
6) 5 ' ttgttagcgccgtagggaagtgaagcttctgggccagttcctaggtaatagaagta ccatctggggctgagctctttca 3 ' (SEQ ID NO:7 is corresponding to the complementary sequence of sequence shown in the Nucleotide of 305-383 position among the SEQ ID NO:1);
7) 5 ' ttccctacggcgctaacaaagaaggcatcgtatgggttgcaactgagggagccttg aatacacccaaagaccacattggcac 3 ' (SEQ ID NO:8 is corresponding to sequence shown in the Nucleotide of 365-446 position among the SEQ ID NO:1);
8) 5 ' tttggcaatgttgttccttgaggaagttgtagcacggtggcagcattgttattagg attgcgggtgccaatgtggtctttgg 3 ' (SEQ ID NO:9 is corresponding to the complementary sequence of sequence shown in the Nucleotide of 428-509 position among the SEQ ID NO:1);
9) 5 ' tcaaggaacaacattgccaaaaggcttctacgcagagggaagcagaggcggcagtc aa gcctcttctcgctc ctcatcacgtagtcgcg 3 ' (SEQ ID NO:10 is corresponding to sequence shown in the Nucleotide of 488-577 position among the SEQID NO:1);
10) 5 ' tcgagcaggagaatttcccctactgctgccaggagttgaatttcttgaattaccgc gactacgtgatgagg 3 ' (SEQ ID NO:11 is corresponding to the complementary sequence of sequence shown in the Nucleotide of 560-630 position among the SEQ ID NO:1);
11) 5 ' ggaaattctcctgctcgaatggctagcggaggtggtgaaactgccctcgcgctatt gctgctagacagatt gaaccagcttg 3 ' (SEQ ID NO:12 is corresponding to sequence shown in the Nucleotide of 613-694 position among the SEQ IDNO:1);
12) 5 ' gatgcctcagcagcagatttcttagtgacagtttggccttgttgttgttggccttt accagaaactttgctctcaagctggttcaatctgtc 3 ' (SEQ ID NO:13 is corresponding to the complementary sequence of sequence shown in the Nucleotide of 676-767 position among the SEQ ID NO:1);
13) 5 ' aatctgctgctgaggcatctaaaaagcctcgccaaaaacgtactgccacaaaacag tacaacgtcactcaag catttgggagacgtg 3 ' (SEQ ID NO:14 is corresponding to sequence shown in the Nucleotide of 749-835 position among the SEQID NO:1);
14) 5 ' atcagttccttgtctgattaggtcttggtccccgaaatttccttgggtttgttctg gaccacgtctcccaaatgcttg 3 ' (SEQ ID NO:15 is corresponding to the complementary sequence of sequence shown in the Nucleotide of 817-894 position among the SEQ ID NO:1);
15) 5 ' ctaatcagacaaggaactgattacaaacattggccgcaaattgcacaatttgctcc aagtgcctctgcattc tttggaatgtca 3 ' (SEQ ID NO:16 is corresponding to sequence shown in the Nucleotide of 874-957 position among the SEQ IDNO:1);
16) 5 ' taatggctccatgataagtcagccatgttcccgaaggtgtgacttccatgccaatg cgtgacattccaaagaatgcaga 3 ' (SEQ ID NO:17 is corresponding to the complementary sequence of sequence shown in the Nucleotide of 937-1015 position among the SEQ ID NO:1);
17) 5 ' gacttatcatggagccattaaattggatgacaaagatccacaattcaaagacaacg tcatactgctgaacaa gcacatt 3 ' (SEQ ID NO:18 is corresponding to sequence shown in the Nucleotide of 996-1074 position among the SEQ ID NO:1);
18) 5 ' gctctgttggtgggaatgttttgtatgcgtcaatgtgcttgttcagcagtat 3 ' (SEQID NO:19 is corresponding to the complementary sequence of sequence shown in the Nucleotide of 1054-1105 position among the SEQ ID NO:1);
19) 5 ' acattcccaccaacagagcctaaaaaggacaaaaagaaaaagactgatgaagctca gcctttgccgcagagacaaaagaagcagcccactgtgactcttcttcctgcggctg 3 ' (SEQ IDNO:20 is corresponding to sequence shown in the Nucleotide of 1087-1198 position among the SEQ ID NO:1);
20) 5 ' ttatgcctgagttgaatcagcagaagctccactcatggaattttgaagttgtctgg agaaatcatccatgtcagccgcaggaagaagag 3 ' (SEQ ID NO:21 is corresponding to the complementary sequence of sequence shown in the Nucleotide of 1181-1269 position among the SEQ IDNO:1).
Utilize automatic dna synthesizer to synthesize above-mentioned oligonucleotide, and carry out gel-purified, be dissolved in dH with 20pmol/ μ l
2Among the O.
2) annealing is with gentle mixing of various each the 2 μ l (20pmol/ μ l) of oligonucleotide 1-20.Drying heater internal heating to 90 ℃ 10 minutes, be cooled to room temperature about 1 hour.
3) extend: PCR adds PCR damping fluid, MgCl in oligonucleotide mixture routinely
2, dNTP and Taq polysaccharase, extended 5 minutes in 56 ℃, be cooled to 4 ℃ then.This step will be filled and led up all breach.
4) connect: the T4 ligase enzyme (New England Biolabs), ligase enzyme damping fluid and the dH that in annealed oligonucleotide mixture, add capacity
2O doubles volume.Spend the night in 16 ℃ of connections.
5).PCR：
Using following primer, is template with the connection product that obtains in the step 4), the sars coronavirus N albumen coded sequence of pcr amplification total length and part brachymemma:
Primer 1:5 ' caccatgtctgataatggacccca 3 ' (SEQ ID NO:22), wherein italic is 5 ' the terminal Nucleotide that adds, all the other Nucleotide correspondences are 1-20 position residue in SEQ ID NO:1;
Primer 2: 5 ' tgcctgagttgaatcagcag 3 ' (SEQ ID NO:23) is corresponding to the complementary sequence of 1247-1266 position residue among the SEQID NO:1;
Primer 3:5 ' gccgtcaccaccacgaact 3 ' (SEQ ID NO:24) is corresponding to the complementary sequence of 282-300 position residue among the SEQID NO:1;
Primer 4:5 ' caccatggaagtcacaccttcgggaa 3 ' (SEQ ID NO:25), wherein italic is 5 ' the terminal Nucleotide that adds, all the other Nucleotide correspondences are in the 970-988 position residue of SEQ ID NO:1.
With primer 1 and primer 2, the product that primer 1 obtains when being used in combination with primer 2 with primer 3 and primer 4 is cloned into respectively in the pET101/D-TOPO carrier (1nvitrogen company), and clone operations is according to Invitrogen Champion
TMThe directed TOPO of pET expresses test kit and carries out.Obtain three kinds of recombinant expression plasmid pET-1, pET-2 and pET-3.Wherein pET-1 expresses 6 * His label that total length N protein sequence (422 amino acid) adds the C-end; The brachymemma N albumen (about 100 amino acid) that pET-2 expresses N-terminal adds C-end 6 * His label; The brachymemma N albumen (about 99 amino acid) that pET-3 expresses the C-end adds C-end 6 * His label.
6) order-checking: use the universal sequencing primer thing that provides in the test kit to three kinds of cloning and sequencings, select the right-on clone of sequence.
7) express: carry out according to the test kit specification sheets.
8) purifying: the standard scheme of following the recombinant protein that has been used to merge 6 * His label carries out.
Embodiment 2. utilizes goat SARS coronary virus resistant N protein polyclone antibody to carry out the double-antibody sandwich analysis
(1) Polyclonal Antibody Preparation
According to ordinary method, with the total length SARS virus N protein immunization inoculation goat of preparation among the embodiment 1, and booster immunization.Separate polyclonal antibody by immune goat serum, and, PBS is dialysed through the ammonium sulfate purifying.Precipitation.
(2) polyclonal antibody and colloid gold particle are puted together
Utilize the colloid gold label test kit of Arista Biologicals Inc., it is standby that the proteic polyclonal antibody of goat anti-SARS virus N is puted together the 40nm colloid gold particle.
(3) prepare test strip with ordinary method, wherein the goat SARS coronary virus resistant N protein polyclone antibody of puting together with Radioactive colloidal gold is a first antibody, simple goat anti-SARS virus N protein polyclone antibody is as " trapping antibody "-second antibody, the positive contrast of the anti-goat IgG of rabbit.The test strip immersion is contained SARS virus lysate (10
2, 10
3, 10
4, 10
5Pfu/ml) in the sample.Left standstill after the taking-up 5 minutes, 10
3Can obviously observe male colour developing band in the sample of the above order of magnitude of pfu/ml.
The preparation of embodiment 3 mouse-anti SARS-N protein monoclonal antibodies:
I. antigenic preparation: the total length SARS-N recombinant protein antigen of preparation among the embodiment 1 is dissolved among 1 * PBS, is prepared into the working fluid of 0.5mg/ml.
II. mouse: Balb/c female mice, 5-6 age in week.8-9 begins immunity during age in week.
III. the preparation of hybridoma:
1). the total length SARS-N albumen that with 1.5ml concentration is 1.0mg/ml mixes with equal-volume thoroughness freund's adjuvant.
2). the initial immunity mouse: every injected in mice 0.5ml, immunity is 6 altogether.
3). booster immunization: the 14th day beginning booster immunization behind the initial immunity, per then 14 days booster immunizations are once.Booster immunization adopts the mixture of antigen and incomplete Freund's adjuvant to carry out, and booster immunization is 3 times altogether.Tail vein is gathered in behind the 3rd booster immunization the 10th day.With 1 * PBS respectively with immune serum and not immune serum be diluted to different concentration, the ELISA method detects antibody titers.
Trace routine: with the N proteantigen coated elisa plate of 2 μ g/ml concentration.Every hole adds 5 μ l serum and 45 μ l sample diluents (1 * PBST).Each extent of dilution is all done diplopore.37 ℃ are incubated 1 hour.Wash plate five times.Sheep anti-mouse igg-the HRP that adds 1: 8000,37 ℃ are incubated 1 hour.Wash plate five times.37 ℃ of TMB colour developings 10 minutes.0.2N sulfuric acid termination reaction.Reading under the 450nm wavelength.The results are shown in Table 1.
Table 1 ELISA method detects the antibody titers result.
| ?1∶20 | ?1∶40 | ?1∶80 | ?1∶160 | ?1∶320 | ?1∶640 | |
| Immune mouse | ?0.803 | ?0.617 | ?0.405 | ?0.388 | ?0.261 | ?0.098 |
| Immune mouse not | ?0.059 | ?0.056 | ?0.075 | ?0.072 | ?0.082 | ?0.047 |
3). final immunity: behind initial immunity the 56th day, respectively the filtering 100 μ l antigens of 0.2 μ are not added adjuvant and carry out injecting in intravenous injection and the homonymy vola.。
4). immune mouse lymphocyte preparation: behind initial immunity the 60th day, put to death mouse.Separate inguinal lymph nodes, place 10ml to be preheated to 37 ℃ serum free medium, isolated lymphocytes.Centrifugal 5 minutes of 400g is resuspended in the 5ml serum free medium.
5). the preparation of myeloma cell Ag8.8 (can obtain): cell strain was recovered from liquid nitrogen in preceding 6 days in merging from ATCC.Merging the day before yesterday, is 5 * 10 with containing 10% fresh serum substratum with cellular segregation
5Cell/ml.In the morning on the same day of merging, in 10ml incubated overnight bottle, add isopyknic substratum that contains 20% foetal calf serum.Centrifugal 5 minutes of 400g is resuspended in the 5ml serum free medium.
6). cytogamy: the 5ml lymphocyte and the 5ml myeloma cell that prepare are merged, and centrifugal 5 minutes of 400g carefully removes supernatant.The pre-temperature of slow adding 0.2ml is that 37 ℃ 30%PEG solution makes cytogamy.Cell transfer after merging is contained to 100ml in the substratum of 20% foetal calf serum, 1 * OPI, 1 * AH, plant 10 96 well culture plates, every hole 100 microlitres place in 37 ℃ of cultivations of CO2gas incubator.After 10 days, can be observed every plate all has the dozens of clone.
7). the screening of hybridoma: merged the back the 10th day, with ELISA method screening positive clone.N albumen difference coated elisa plate with 2 μ g/ml total length N antigens, N end and C end.Every hole adds 20 μ l substratum supernatants and 30 μ l sample diluent (1X PBS, 2%BSA, 0.05%NP-40,0.01%NaN
3) 37 ℃ of incubations 60 minutes.Wash plate five times.Sheep anti-mouse igg-the HRP that adds 1: 8000.37 ℃ of incubations 60 minutes.Wash plate five times.TMB colour developing 10 minutes.0.2N sulfuric acid termination reaction.The 450nm reading.Select all that total length N albumen and N end fragment are the male hybridoma and all are the male hybridoma to total length N albumen and C end fragment.Observe according to ELISA result and microscopically, random choose goes out the cell in 40 holes, is incubated at 24 orifice plates.After 4 days, with the screening of ELISA method.Concrete grammar is the same.Select 30 strain hybridomas altogether.
8). the supernatant with cell culture medium is further made the Western trace again.With total length N albumen, N end fragment and C end fragment carry out SDS-PAGE, transfer on the nitrocellulose filter, with the sealing of 5% skim-milk, washing, add the substratum supernatant of different cell strains, and washing adds sheep anti-mouse igg-HRP, adds chromogenic reagent at last.Select ELISA and WesternBlot all to reflect the cell strain preservation that stronger, N end fragment and full-length proteins or C end fragment and full-length proteins are all reacted, and pass through the further subclone of limiting dilution, obtain 14 strain mono-clonals.They are numbered #1-#14 respectively, and wherein #1-#6 reacts N end fragment and full-length proteins, and #7-#14 reacts C end fragment and full-length proteins.
9). the cultivation of mono-clonal hybridoma and storage:
Stable hybridoma is containing 2% inactivated fetal bovine serum, 2mML-glutamine, 100U/ml penicillin-Streptomycin sulphate, 1mM Sodium.alpha.-ketopropionate, 5.5 * 10
-5Carrying out subculture in vitro separately in the HB101 substratum of M 2 mercapto ethanol cultivates.Cell concn is 2-2.5 * 10
5/ ml is stored in 37 ℃ of incubators that contain 8% carbonic acid gas.After cell changes liquid, be suspended from the minimum medium that contains 20%FCS and 10%DMSO 5 * 10
6Individual/the ml/ pipe, slowly frozen in liquid nitrogen.
10). Purification of Monoclonal Antibodies: collect monoclonal cell substratum supernatant, use 50% saturated ammonium sulphate, dialysis in PBS (pH7.2) immediately, centrifugal absorption supernatant, the sodiumazide of adding 0.02% is stored in-70 ℃.
11). the evaluation of monoclonal antibody:
Western Blot:
The antigen samples electrophoresis
The preparation antigen samples, concentration is 10 μ g/ml, and electrophoretic separation.Electrotransfer afterwards seals with 5ml confining liquid (0.05% skim-milk) to nitrocellulose filter.Change to then with confining liquid by the first antibody that diluted at 1: 1000, at room temperature continued to shake 30～60 minutes.Take out film and wash film four times, each 10-15 minute with TTBS.Add again with confining liquid by the HRP-sheep anti-mouse igg antibody conjugate that diluted at 1: 2000.Make the film colour developing according to a conventional method, band should occur in 10-30 minute.
Direct ELISA method: bag is by the N proteantigen in enzyme plate, and bag is 2ug/ml by concentration.Sealing back adds the anti-N protein monoclonal antibody of serial dilution, and 37 ℃ of incubations 30 minutes are washed the sheep anti-mouse igg that adds the HRP mark behind the plate again, 37 degree incubations 30 minutes, TMB colour developing.
The #1-#14 hybridoma that above-mentioned experiment confirm obtained is all secreted the monoclonal antibody with N proteantigen specific reaction.
The immunoreactive detection of embodiment 4 anti-N protein antibodies and virus antigen
For determining that whether the immunoreactivity between anti-N protein antibodies and recombinant N protein antigen and the natural N proteantigen of SARS virus there are differences, and need guarantee to use the recombinant antigen and the natural antigen of equal parts.For this reason, recombinant full-lenght N proteantigen 500ng and SARS virus split product series titre liquid are carried out the SDS-PAGE electrophoresis, determine the SARS virus titre suitable with the recombinant protein consumption by the brightness of N protein band.Wrap respectively by plate with the recombinant full-lenght N proteantigen of equal parts and the SARS virus lysate of handling through SDS, with #1-#10 authentic monoclonal antibody and its reaction of HRP mark, select the antibody that response intensity equates or close antibody conduct is finally used between the two.But the equal intensity of all the other 12 strain authentic monoclonal antibodies that the result obtains except that #2 and #7 is considerably reacted with recombinant N protein antigen and SARS virus split product.
Embodiment 5 anti-SARS virus N protein polyclone antibodies and monoclonal antibody are used in combination to detect recombinant N protein
The anti-N protein monoclonal antibody # 1 that selects among the random choose embodiment 4, #3, #5, #8, #9 and #12 put together according to ordinary method and Radioactive colloidal gold as first antibody, anti-SARS virus N protein polyclone antibody 4mg/ml is used in combination as " trapping antibody "-second antibody, to test to each extent of dilution of recombinant N protein (5pg/ml, 10pg/ml, 20pg/ml, 50pg/ml, 100pg/ml, 200pg/ml, reactivity 500pg/ml).Found that these antibody all can reach visual inspection and detect the antigenic susceptibility of 20pg/ml reorganization N.
Two kinds of monoclonal antibodies of embodiment 6 anti-respectively N albumen n end fragment and C end fragment are used to detect recombinant N protein antigen and sars coronavirus lysate
The pairing experiment of coated antibody and enzyme len antibody
The anti-N protein monoclonal antibody of selecting (#1, #3-#6 and #8-#14) is all used PierceHRP labelling kit (Pierce Biotechnology, Inc., Customer ServiceDepartment, P.O.Box 117, Rockford, IL.61105 U.S.A, cat.no.31490) mark.
Use the said monoclonal antibody coated elisa plate.Antibody is dissolved in BBS with 1ug/ml.With the 3%BSA closure plate that is dissolved among the PBS that contains 0.02%NaN3 (sodiumazide), add N proteantigen 100pg/ml.Each bag is at first done compatibility test with the monoclonal antibody of the anti-C end of HRP mark by the anti-N of usefulness end monoclonal antibody, and then respectively with other underlined anti-N-HRP antibody cooperate, select to have the monoclonal antibody of peak signal as pairing antibody.Otherwise the monoclonal antibody with anti-C end as coated antibody is, at first the monoclonal antibody with the anti-N end of HRP mark cooperates, and then with other underlined antibody cooperate.# 1 that the selective reaction signal is the strongest and #10, #3 and #9 are as cooperating antibody.
With reference to ordinary method # 1 and #9 are puted together with colloid gold particle respectively, concentrate the back with OD
530nm=50 spray films, consumption is 2.5ul/cm.#3 and #10 antibody are fixed on the corresponding test strip as trapping antibody with 2.0mg/ml, and with sheep anti-mouse igg as positive control.Test strip is immersed the recombinant N protein of serial dilution and in the SARS virus lysate sample of 0.05%SDS deactivation, detect.Found that two kinds of combinations all can detect the susceptibility of 10pg/ml reorganization N antigen and 1000pfu/ml SARS virus.
The inventor has also tested with the anti-SARS-N protein polyclone antibody of goat (4mg/ml) as trapping antibody, the monoclonal antibody (#1 and #10) of anti-C end and the N end cooperation of puting together antibody as Radioactive colloidal gold respectively.The Radioactive colloidal gold conjugate final concentration of every kind of monoclonal antibody is OD
530nm=50, the injection volume is 2.5ul/cm.Detected result is seen Fig. 1.Wherein upper strap portion is a mountain sheep anti-mouse igg positive control, and lower strip is the anti-SARS-N protein polyclone antibody of trapping antibody-goat.Can reach the extent of dilution that visual inspection detects 10pg/ml (and being lower than 10pg/ml) the reorganization antigenic susceptibility of N and detection reaches 1000PFU (and being lower than 1000PFU) to deactivation of SARS virus with this understanding.This susceptibility is the level of sensitivity that present RT-PCR method detects SARS virus.
Conclusion
Detection sensitivity can reach the 10pg/ml recombinant N protein at 10 minutes detection time for method of the present invention, detected deactivation SARS-CoV and can reach 1000PFU/ml or higher extent of dilution.
That method of the present invention has is simple, quick, easy to use, do not need special expertise or equipment and be easy to preserve (generally can in room temperature preservation 18-24 month), be used for the SARS diagnosis, present method is applicable to the generation by the pollutent (as the washing lotion in the ELISA method) of non-voluntary personnel operation and minimizing detection generation itself in non-medical institutions (as airport, station, family etc.) in addition.Can be used in early diagnosis have highly sensitive and specificity SARS colloid gold reagent/method has all incomparable advantage of other any method.Can find out that by The above results the susceptibility of the inventive method can reach the level of ELISA method, and can be by carrying out detection by quantitative, observe and judge thereby whether the curative effect of SARS treatment is promptly had infectivity with the typical curve comparison.
In illustrative mode the present invention is illustrated above, but the present invention never is limited to this.Those of ordinary skills clearly fully can do various changes to the present invention after having read entire description, and still without departing from the spirit and scope of the present invention.
Sequence table
<110>Ma，Jie
＜120〉detection of sars coronavirus or its related antigen
<130>idc030083-ccpit
<160>26
<170>PatentIn?version?3.1
<210>1
<211>1269
<212>DNA
<213>
<400>1
atgtctgata?atggacccca?atcaaaccaa?cgtagtgccc?cccgcattac?atttggtgga????60
cccacagatt?caactgacaa?taaccagaat?ggaggacgca?atggggcaag?gccaaaacag????120
cgccgacccc?aaggtttacc?caataatatt?gcgtcttggt?tcacagctct?cactcagcat????180
ggcaaggagg?aacttagatt?ccctcgaggc?cagggcgttc?caatcaacac?caatagtggt????240
ccagatgacc?aaattggcta?ctaccgaaga?gctacccgac?gagttcgtgg?tggtgacggc????300
aaaatgaaag?agctcagccc?cagatggtac?ttctattacc?taggaactgg?cccagaagct????360
tcacttccct?acggcgctaa?caaagaaggc?atcgtatggg?ttgcaactga?gggagccttg????420
aatacaccca?aagaccacat?tggcacccgc?aatcctaata?acaatgctgc?caccgtgcta????480
caacttcctc?aaggaacaac?attgccaaaa?ggcttctacg?cagagggaag?cagaggcggc????540
agtcaagcct?cttctcgctc?ctcatcacgt?agtcgcggta?attcaagaaa?ttcaactcct????600
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
caggcataa????????????????????????????????????????????????????????????1269
<210>2
<211>70
<212>DNA
＜213〉synthetic oligonucleotide
<400>2
atgtctgata?atggacccca?atcaaaccaa?cgtagtgccc?cccgcattac?atttggtgga????60
cccacagatt???????????????????????????????????????????????????????????70
<210>3
<211>70
<212>DNA
＜213〉synthetic oligonucleotide
<400>3
ctgttttggc?cttgccccat?tgcgtcctcc?attctggtta?ttgtcagttg?aatctgtggg????60
tccaccaaat???????????????????????????????????????????????????????????70
<210>4
<211>78
<212>DNA
＜213〉synthetic oligonucleotide
<400>4
ccgaccccaa?ggtttaccca?ataatattgc?gtcttggttc?acagctctca?ctcagcatgg????60
caaggaggaa?cttagatt??????????????????????????????????????????????????78
<210>5
<211>80
<212>DNA
＜213〉synthetic oligonucleotide
<400>5
tagccaattt?ggtcatctgg?accactattg?gtgttgattg?gaacgccctg?gcctcgaggg????60
aatctaagtt?cctccttgcc????????????????????????????????????????????????80
<210>6
<211>83
<212>DNA
＜213〉synthetic oligonucleotide
<400>6
ccagatgacc?aaattggcta?ctaccgaaga?gctacccgac?gagttcgtgg?tggtgacggc????60
aaaatgaaag?agctcagccc?cag????????????????????????????????????????????83
<210>7
<211>79
<212>DNA
＜213〉synthetic oligonucleotide
<400>7
ttgttagcgc?cgtagggaag?tgaagcttct?gggccagttc?ctaggtaata?gaagtaccat????60
ctggggctga?gctctttca?????????????????????????????????????????????????79
<210>8
<211>82
<212>DNA
＜213〉synthetic oligonucleotide
<400>8
ttccctacgg?cgctaacaaa?gaaggcatcg?tatgggttgc?aactgaggga?gccttgaata????60
cacccaaaga?ccacattggc?ac?????????????????????????????????????????????82
<210>9
<211>82
<212>DNA
＜213〉synthetic oligonucleotide
<400>9
tttggcaatg?ttgttccttg?aggaagttgt?agcacggtgg?cagcattgtt?attaggattg????60
cgggtgccaa?tgtggtcttt?gg?????????????????????????????????????????????82
<210>10
<211>89
<212>DNA
＜213〉synthetic oligonucleotide
<400>10
tcaaggaaca?acattgccaa?aaggcttcta?cgcagaggga?agcagaggcg?gcagtcaagc????60
ctcttctcgc?tcctcatcac?gtagtcgcg??????????????????????????????????????89
<210>11
<211>71
<212>DNA
＜213〉synthetic oligonucleotide
<400>11
tcgagcagga?gaatttcccc?tactgctgcc?aggagttgaa?tttcttgaat?taccgcgact????60
acgtgatgag?g?????????????????????????????????????????????????????????71
<210>12
<211>82
<212>DNA
＜213〉synthetic oligonucleotide
<400>12
ggaaattctc?ctgctcgaat?ggctagcgga?ggtggtgaaa?ctgccctcgc?gctattgctg????60
ctagacagat?tgaaccagct?tg?????????????????????????????????????????????82
<210>13
<211>92
<212>DNA
＜213〉synthetic oligonucleotide
<400>13
gatgcctcag?cagcagattt?cttagtgaca?gtttggcctt?gttgttgttg?gcctttacca????60
gaaactttgc?tctcaagctg?gttcaatctg?tc??????????????????????????????????92
<210>14
<211>87
<212>DNA
＜213〉synthetic oligonucleotide
<400>14
aatctgctgc?tgaggcatct?aaaaagcctc?gccaaaaacg?tactgccaca?aaacagtaca????60
acgtcactca?agcatttggg?agacgtg????????????????????????????????????????87
<210>15
<211>78
<212>DNA
＜213〉synthetic oligonucleotide
<400>15
atcagttcct?tgtctgatta?ggtcttggtc?cccgaaattt?ccttgggttt?gttctggacc????60
acgtctccca?aatgcttg??????????????????????????????????????????????????78
<210>16
<211>84
<212>DNA
＜213〉synthetic oligonucleotide
<400>16
ctaatcagac?aaggaactga?ttacaaacat?tggccgcaaa?ttgcacaatt?tgctccaagt????60
gcctctgcat?tctttggaat?gtca???????????????????????????????????????????84
<210>17
<211>79
<212>DNA
＜213〉synthetic oligonucleotide
<400>17
agacgtaaga?aaccttacag?tgcgtaaccg?taccttcagt?gtggaagccc?ttgtaccgac????60
tgaatagtac?ctcggtaat?????????????????????????????????????????????????79
<210>18
<211>79
<212>DNA
＜213〉synthetic oligonucleotide
<400>18
taatggctcc?atgataagtc?agccatgttc?ccgaaggtgt?gacttccatg?ccaatgcgtg????60
acattccaaa?gaatgcaga?????????????????????????????????????????????????79
<210>19
<211>52
<212>DNA
＜213〉synthetic oligonucleotide
<400>19
gctctgttgg?tgggaatgtt?ttgtatgcgt?caatgtgctt?gttcagcagt?at????????????52
<210>20
<211>112
<212>DNA
＜213〉synthetic oligonucleotide
<400>20
acattcccac?caacagagcc?taaaaaggac?aaaaagaaaa?agactgatga?agctcagcct????60
ttgccgcaga?gacaaaagaa?gcagcccact?gtgactcttc?ttcctgcggc?tg????????????112
<210>21
<211>89
<212>DNA
＜213〉synthetic oligonucleotide
<400>21
ttatgcctga?gttgaatcag?cagaagctcc?actcatggaa?ttttgaagtt?gtctggagaa????60
atcatccatg?tcagccgcag?gaagaagag??????????????????????????????????????89
<210>22
<211>24
<212>DNA
＜213〉primer
<400>22
caccatgtct?gataatggac?ccca??????????????????????????????????????????24
<210>23
<211>20
<212>DNA
＜213〉primer
<400>23
tgcctgagtt?gaatcagcag????????????????????????????????????????????????20
<210>24
<211>19
<212>DNA
＜213〉primer
<400>24
gccgtcacca?ccacgaact?????????????????????????????????????????????????19
<210>25
<211>26
<212>DNA
＜213〉primer
<400>25
caccatggaa?gtcacacctt?cgggaa?????????????????????????????????????????26
<210>26
<211>422
<212>PRT
<213>
<400>26
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?Ile?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 (16)
1. antibody at sars coronavirus N proteantigen.
2. the antibody of claim 1, it is monoclonal.
3. the antibody of claim 1, it is polyclonal.
4. the antibody of claim 1, it is a chimeric antibody.
5. the method that has situation of sars coronavirus or its corresponding antigens in the test sample comprises the steps:
(1) with each antibody incubation under appropriate condition among sample and the claim 1-4;
(2) detect in the above-mentioned steps existence in conjunction with complex body.
6. the method for claim 5, wherein said sample is patient's serum, whole blood, sputum, oral cavity/nasopharyngeal secretions or washing lotion, urine, ight soil, pleural effusions and ascites, cerebrospinal fluid and a tissue sample etc.
7. the method for claim 5 is wherein handled described sample with 0.01-0.2%SDS before incubation.
8. the method for claim 5, wherein said antibody is fixed on upholder, and for example film such as nitrocellulose, latex particle, magnetic-particle, Radioactive colloidal gold, pearl or dish are such as on glass, fiberglass or plastic material such as polystyrene or polyvinyl chloride or the fiber optic sensor etc.
9. the diagnostic method of SARS disease among the patient, comprise with among patient's sample and the claim 1-4 each the antibody incubation and detect the specificity bonded situation of described antibody.
10. a detection kit wherein comprises at proteic first antibody of sars coronavirus N and second antibody, and described first antibody has randomly carried out suitable mark, for example mark such as horseradish peroxidase or Radioactive colloidal gold.
11. the test kit of claim 10, wherein said first antibody and second antibody all are polyclonal antibodies.
12. the test kit of claim 10, wherein said first antibody is polyclonal antibody or one or more monoclonal antibody or their mixture, and described second antibody is polyclonal antibody or one or more monoclonal antibody or their mixture that can be used.
13. test strip product, wherein comprise at proteic first antibody of sars coronavirus N and second antibody, described first antibody has randomly carried out suitable mark, mark such as horseradish peroxidase or Radioactive colloidal gold for example, and described second antibody is as trapping antibody.
14. the test strip product of claim 13, wherein said first antibody and second antibody all are polyclonal antibodies.
15. the test strip product of claim 13, wherein said first antibody is polyclonal antibody or one or more monoclonal antibody or their mixture, and described second antibody is polyclonal antibody or one or more monoclonal antibody or their mixture that can be used.
16. the test strip product of claim 13 wherein also contains the positive control antibody of anti-first antibody.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 03156361 CN1590409A (en) | 2003-09-05 | 2003-09-05 | Antibody pointed at SARS coronavirus N protein antigen and its use in detecting SARS coronavirus or its antigen |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 03156361 CN1590409A (en) | 2003-09-05 | 2003-09-05 | Antibody pointed at SARS coronavirus N protein antigen and its use in detecting SARS coronavirus or its antigen |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN1590409A true CN1590409A (en) | 2005-03-09 |
Family
ID=34598393
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 03156361 Pending CN1590409A (en) | 2003-09-05 | 2003-09-05 | Antibody pointed at SARS coronavirus N protein antigen and its use in detecting SARS coronavirus or its antigen |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN1590409A (en) |
Cited By (19)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106814189A (en) * | 2016-12-20 | 2017-06-09 | 成都金思唯生物技术有限公司 | A kind of kit for detecting clostridium difficile and application thereof |
| CN108845149A (en) * | 2018-07-05 | 2018-11-20 | 郑州大学 | The colloidal gold duplex test strips and preparation method thereof of porcine reproductive and respiratory syndrome virus and swine fever virus are detected simultaneously |
| CN111024954A (en) * | 2020-03-09 | 2020-04-17 | 深圳市易瑞生物技术股份有限公司 | Colloidal gold immunochromatography device for combined detection of COVID-19 antigen and antibody and use method thereof |
| CN111060691A (en) * | 2020-03-17 | 2020-04-24 | 深圳市易瑞生物技术股份有限公司 | Fluorescence immunochromatography device for detecting COVID-19 and using method thereof |
| CN111239400A (en) * | 2020-03-17 | 2020-06-05 | 深圳市易瑞生物技术股份有限公司 | Colloidal gold immunochromatographic device for detecting COVID-19 and use method thereof |
| CN111233985A (en) * | 2020-02-09 | 2020-06-05 | 北京丹大生物技术有限公司 | Preparation method of novel coronavirus IgA antibody rapid detection test strip |
| CN111378018A (en) * | 2020-03-28 | 2020-07-07 | 江苏省疾病预防控制中心(江苏省公共卫生研究院) | Test strip for detecting novel coronavirus antibody and preparation method and application thereof |
| CN111398589A (en) * | 2020-02-13 | 2020-07-10 | 北京华科泰生物技术股份有限公司 | Immunochromatography kit for rapidly detecting novel coronavirus N protein and preparation method and application thereof |
| CN111398583A (en) * | 2020-02-13 | 2020-07-10 | 北京华科泰生物技术股份有限公司 | Kit for detecting novel coronavirus N protein and application thereof |
| CN111426844A (en) * | 2020-03-13 | 2020-07-17 | 南京农业大学 | Novel fluorescence immunochromatographic test strip for combined detection of coronavirus SARS-CoV-2 IgG-IgM antibody |
| CN111443199A (en) * | 2020-04-02 | 2020-07-24 | 杨宇 | Reagent for rapidly detecting new coronavirus antibody by magnetic nanoparticle immunochromatography and preparation method thereof |
| CN111505286A (en) * | 2020-04-28 | 2020-08-07 | 郑州伊美诺生物技术有限公司 | Novel coronavirus specific antibody double-antigen sandwich E L ISA detection kit and preparation method thereof |
| CN111505316A (en) * | 2020-05-22 | 2020-08-07 | 无锡市孚维尔生物医疗科技有限公司 | Production method of novel coronavirus 2019-nCoV antibody spectrum detection kit |
| CN111624348A (en) * | 2020-05-22 | 2020-09-04 | 无锡市孚维尔生物医疗科技有限公司 | Method for detecting novel coronavirus 2019-nCoV through multi-protein joint inspection combination |
| CN111624349A (en) * | 2020-05-22 | 2020-09-04 | 无锡市孚维尔生物医疗科技有限公司 | Novel coronavirus 2019-nCoV antibody spectrum detection test strip |
| CN111665365A (en) * | 2020-05-22 | 2020-09-15 | 无锡市孚维尔生物医疗科技有限公司 | Novel coronavirus 2019-nCoV antibody spectrum detection kit |
| 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 |
| CN113862257A (en) * | 2021-08-30 | 2021-12-31 | 南京贝思奥生物科技有限公司 | Method and kit for rapidly detecting size of viral genome |
| WO2022003192A1 (en) * | 2020-07-02 | 2022-01-06 | Leibniz-Institut Für Photonische Technologien E.V. | Agents and method for enriching or depleting and for detecting corona viruses |
-
2003
- 2003-09-05 CN CN 03156361 patent/CN1590409A/en active Pending
Cited By (21)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106814189A (en) * | 2016-12-20 | 2017-06-09 | 成都金思唯生物技术有限公司 | A kind of kit for detecting clostridium difficile and application thereof |
| CN108845149A (en) * | 2018-07-05 | 2018-11-20 | 郑州大学 | The colloidal gold duplex test strips and preparation method thereof of porcine reproductive and respiratory syndrome virus and swine fever virus are detected simultaneously |
| CN111233985A (en) * | 2020-02-09 | 2020-06-05 | 北京丹大生物技术有限公司 | Preparation method of novel coronavirus IgA antibody rapid detection test strip |
| 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 |
| CN111398589A (en) * | 2020-02-13 | 2020-07-10 | 北京华科泰生物技术股份有限公司 | Immunochromatography kit for rapidly detecting novel coronavirus N protein and preparation method and application thereof |
| CN111398583A (en) * | 2020-02-13 | 2020-07-10 | 北京华科泰生物技术股份有限公司 | Kit for detecting novel coronavirus N protein and application thereof |
| CN111024954A (en) * | 2020-03-09 | 2020-04-17 | 深圳市易瑞生物技术股份有限公司 | Colloidal gold immunochromatography device for combined detection of COVID-19 antigen and antibody and use method thereof |
| CN111426844A (en) * | 2020-03-13 | 2020-07-17 | 南京农业大学 | Novel fluorescence immunochromatographic test strip for combined detection of coronavirus SARS-CoV-2 IgG-IgM antibody |
| CN111060691A (en) * | 2020-03-17 | 2020-04-24 | 深圳市易瑞生物技术股份有限公司 | Fluorescence immunochromatography device for detecting COVID-19 and using method thereof |
| CN111239400A (en) * | 2020-03-17 | 2020-06-05 | 深圳市易瑞生物技术股份有限公司 | Colloidal gold immunochromatographic device for detecting COVID-19 and use method thereof |
| CN111378018A (en) * | 2020-03-28 | 2020-07-07 | 江苏省疾病预防控制中心(江苏省公共卫生研究院) | Test strip for detecting novel coronavirus antibody and preparation method and application thereof |
| CN111443199A (en) * | 2020-04-02 | 2020-07-24 | 杨宇 | Reagent for rapidly detecting new coronavirus antibody by magnetic nanoparticle immunochromatography and preparation method thereof |
| CN111443199B (en) * | 2020-04-02 | 2023-11-28 | 杨宇 | Reagent for rapidly detecting novel coronavirus antibody by magnetic nanoparticle immunochromatography and preparation method thereof |
| CN111505286A (en) * | 2020-04-28 | 2020-08-07 | 郑州伊美诺生物技术有限公司 | Novel coronavirus specific antibody double-antigen sandwich E L ISA detection kit and preparation method thereof |
| CN111505316A (en) * | 2020-05-22 | 2020-08-07 | 无锡市孚维尔生物医疗科技有限公司 | Production method of novel coronavirus 2019-nCoV antibody spectrum detection kit |
| CN111624348A (en) * | 2020-05-22 | 2020-09-04 | 无锡市孚维尔生物医疗科技有限公司 | Method for detecting novel coronavirus 2019-nCoV through multi-protein joint inspection combination |
| CN111624349A (en) * | 2020-05-22 | 2020-09-04 | 无锡市孚维尔生物医疗科技有限公司 | Novel coronavirus 2019-nCoV antibody spectrum detection test strip |
| CN111665365A (en) * | 2020-05-22 | 2020-09-15 | 无锡市孚维尔生物医疗科技有限公司 | Novel coronavirus 2019-nCoV antibody spectrum detection kit |
| WO2022003192A1 (en) * | 2020-07-02 | 2022-01-06 | Leibniz-Institut Für Photonische Technologien E.V. | Agents and method for enriching or depleting and for detecting corona viruses |
| CN113862257A (en) * | 2021-08-30 | 2021-12-31 | 南京贝思奥生物科技有限公司 | Method and kit for rapidly detecting size of viral genome |
| CN113862257B (en) * | 2021-08-30 | 2024-01-16 | 南京贝思奥生物科技有限公司 | Method and kit for rapidly detecting size of viral genome |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN1590409A (en) | Antibody pointed at SARS coronavirus N protein antigen and its use in detecting SARS coronavirus or its antigen | |
| CN1267452C (en) | Monoclonal antibodies, antigens and diagnosis and therapy of malignant diseases | |
| CN1279051C (en) | DNA sequence and encoded mammary-specific breast cancer protein | |
| CN1784425A (en) | Method of detecting probnp with a monoclonal antibody binding to the amino acids 38-44 | |
| CN1174998C (en) | Peptide immunogens for vaccination against and treatment of allergy | |
| CN1130910A (en) | Hapten-marked peptides | |
| CN1646910A (en) | Method of judging leukemia, pre-leukemia or aleukemic malignant blood disease and diagnostic therefor | |
| CN1884303A (en) | SARS-CoV virus structural protein infusion protein and its high yield expression and purification and uses | |
| CN1622958A (en) | Epitope regions of a thyrotrophin (TSH) receptor, uses thereof and antibodies thereto | |
| CN1678911A (en) | Method of diagnosing cancer by detecting GPC300 | |
| CN1513000A (en) | Anti-osteopontin antibody and use thereof | |
| CN1930474A (en) | Method of detecting allergen | |
| CN1639193A (en) | Antibodies against fibroblast growth factor 23 | |
| CN1968962A (en) | Novel soluble cd14 antigen | |
| CN1659440A (en) | Method for simultaneously detecting an antigen and an antibody of an infectious microorganism | |
| CN1877330A (en) | Enzyme-linked immunologic diagnosis kit for core antigen of C type hepatitis virus and method for preparing same | |
| CN1896105A (en) | Compounds and methods of early diagnosis of cervical cancer and genital condyloma with hpv, chsp60 tumor suppressor h-ras, k-ras and pten derived peptides modified | |
| CN1230549A (en) | Escape mutant of surface antigen of hepatitis B virus | |
| CN1526072A (en) | Method for the identification of extracellular domains of the epstein barr virus (ebv), tumor-associated latent membrane proteins and for the selection of antibody reagents reactive therewith | |
| CN1249781A (en) | Epitopes of protein pre-M/M of dengue virus, synthetic peptides | |
| CN1701117A (en) | Antibody against enzyme specifically cleaving VON WILLEBRAND factor and assay system using the same | |
| CN1966526A (en) | Evolved immunoglobulin binding molecule, and its preparation method and uses | |
| CN1735684A (en) | P153 and P156 antigens for the immunodiagnosis of canine and human ehrlichioses and uses thereof | |
| CN101039955A (en) | Nucleic acids, polypeptides, methods of expression, and immunogenic compositions associated with sars corona virus spike protein | |
| CN1221428A (en) | Cellular internalization of PIGR large biological molecule characteristic high structure and associated ligands |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C12 | Rejection of a patent application after its publication | ||
| RJ01 | Rejection of invention patent application after publication |