CN1557838A - SARS coronavirus nucleocapsid protein monoclonal antibody, hybridoma for producing the same, detection agent containing the same and use thereof - Google Patents
SARS coronavirus nucleocapsid protein monoclonal antibody, hybridoma for producing the same, detection agent containing the same and use thereof Download PDFInfo
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Abstract
The present invention discloses the specific monocloneal antibody of SARS-CoV nuclear capsid protein, hybrid tumor producing the antibody, reagent containing the monocloneal antibody and reagent kit therewith. The monocloneal antibody is secreted and produced with the cell line including hybrid tumor 1E8A11 of preservation number of CCTCC-C200401, hybrid tumor 1E8A17 of preservation number of CCTCC-C200402, hybrid tumor 10E4A4 of preservation number of CCTCC-C200403, and hybrid tumor 14A3A3A19 of preservation number of CCTCC-C200404. The reagent kit established with the monocloneal antibody may be used in early diagnosis of SARS-CoV infection and has the features of simplicity, convenience, fastness, high sensitivity, powerful specificity, etc.
Description
Technical field
The present invention relates to monoclonal antibody, particularly relate to the proteic monoclonal antibody specific of generation sars coronavirus nucleocapsid (N), produce the hybridoma and the application of this monoclonal antibody in diagnosis SARS of this antibody with hybridoma technology production.
Background technology
Severe acute respiratory syndrome claims that again (Severe AcuteRespiratory Syndrome SARS), is a kind of emerging transmissible disease to severe acute respiratory syndrome.SARS's is pathogenic former on April 10th, 2003, is defined as a kind of new coronavirus, called after SARS-CoV by the World Health Organization.From in November, 2002 at first after area, China Guangdong takes place, at more than 30 countries and regions spread and epidemics, this sick mortality ratio is 6.4～16.5%.According to available data, this disease is mainly closely relayed by modes such as the respiratory tract spittle, body fluid and dirts, and the characteristics of clustering of disease in family and hospital's aggregation are arranged.It is anxious that its main clinical is characterized as onset, and fever, cough, pulmonary shadow, peripheral blood leucocyte do not raise or reduce.Although the discovery in pathogenic agent has obtained remarkable progress, in the Pathogen Biology characteristic, pathogenesis, the research of aspects such as early stage effective diagnosis reagent, specific treatment does not obtain breakthrough progress as yet, still has a series of problem to need to solve.Especially the SARS early diagnosis is that the puzzlement clinician finds early, isolates this disease and a series of difficulties such as control, because can show different Clinical symptoms with a kind of virus infection Different Individual, and identical Clinical symptoms can appear in different virus infectiones, so, only depend on clinical symptom and epidemiologic data to be difficult to determine the real cause of disease of virus infection, final making a definite diagnosis often needs breadboard diagnosis, and sophisticated objective laboratory diagnosis standard is not arranged at present as yet.Because most of virus diseases still do not have the specific treatment medicine, laboratory diagnosis has become the important means that the control virus disease is popular and spread.The task of top priority is to study effective diagnosis reagent as early as possible, diagnosis early, isolation early, cut-out route of transmission, and the control epidemic situation spreads.
Laboratory diagnosis for sars coronavirus infects mainly comprises following several method: at present based on the molecular diagnosis of PCR and biochip technology; Serodiagnosis based on antibody detection; Pathogen Biology diagnosis based on external virus culture.
At present, the laboratory early diagnosis that sars coronavirus is infected mainly is to measure based on the viral nucleic acid of PCR.The current experiments data presentation is approximately 70% in the susceptibility of 14 days left and right sides viral nucleic acids of morbidity, because viral RNA is degraded easily, detected result is subject to the influence of sample collection and processing, and sample contamination can cause false positive, therefore, need strict quality and technician skillfully to operate through training.Need special detecting instrument in addition and cost an arm and a leg,, have a lot of hospitals still can not carry out in view of above these factors.
Based on the immunology diagnosis of antibody detection, whether the existence that detects antibody according to known antigens infers further whether pathogenic agent exists.The time that dissimilar antibody produces is different with the dynamic process that level changes, and can not check antibody in the early stage inspection of disease, and IgM will just detect at metainfective about 10 days, and I gG will just detect reliably at metainfective about 21 days.Currently used method has two kinds: enzyme-linked immunosorbent assay (enzyme linked immunosorbant assay, ELISA) and immunofluorescence technique (immunofluroescence assay, IFA).ELISA detected is IgM and IgG antibody in experimenter's serum.IFA is generally used for detecting IgM, but also can be used for detecting IgG, and this method need be fixed on SARS-CoV on the slide, after the second antibody of fluorescence molecule mark is handled, uses fluorescence microscope more then.Positive findings represents before to have SARS-CoV to infect, and serum antibody is transferred to the positive and represented more than 4 times that from the antibody titers increase of acute phase to decubation infection is taking place in the recent period by feminine gender.Fall ill and all can not detect antibody in back 21 days and represent not have SARS-CoV to infect.Because specific antibody begins to occur in morbidity 7～10, therefore, antibody serum is learned and is detected the early diagnosis that can not be used for SARS.
Based on the Pathogen Biology diagnosis of external virus culture, being separated to SARS-CoV from patient's sample is laboratory diagnosis " gold standard ", but because the restriction of test conditions etc. is general not as conventional method.And the technical difficulty height, complicated operation, required time is long, to the equipment requirements height, is unsuitable for clinical hospital and uses.
Based on the etiological diagnosis of specific antibody, be method to the early diagnosis of SARS laboratory, be with virus antigen in the known specific antibody test sample.Commercialization sars coronavirus antigen diagnose reagent kit is not arranged at present as yet.The technical difficulty of setting up the virus antigen diagnostic kit with specific antibody is to need very high monoclonal antibody of specificity or polyclonal antibody, ripe hybridoma technology has played enormous function in the immunology diagnosis of multiple disease, because monoclonal antibody has the immunological properties of high special, can make up high responsive special antigen detecting agent, this is confirmed to have high specificity, good reproducibility, easy to operate and low cost and other advantages by multiple virus antigen detection reagent in the past.
By analysis to the SARS-CoV genome structure, SARS-CoV genes encoding structural protein are by nail glycoprotein (spike glycoprotein, S), coating small protein (small envelope, E), membranin (membrane, M) and nucleocapsid protein (nucleocapsid protein N) forms.Although the function to these structural protein it be not immediately clear, but find that from the cell of animal coronavirus infection owing to the albumen on the peplos is easy to morph, and nucleocapsid protein is relatively stable, its antigenicity is stronger than other structural protein, and is the main antigen part of virus.For this reason, the invention provides one group produce can with the hybridoma cell strain of the new monoclonal antibody of SARS-CoV N protein-specific bonded.And the relevant quick diagnosis reagent kit of development, be used for early diagnosis SARS infect provide a kind of fast, the method for high, the high specificity of susceptibility, lay the foundation for SARS-CoV biological character, protein science and Study on Pathogenesis simultaneously.
Summary of the invention
But first purpose of the present invention provides one group of specificity in conjunction with the proteic monoclonal antibody of SARS-CoV N;
Second purpose of the present invention provides the hybridoma that can produce this group monoclonal antibody;
The 3rd purpose of the present invention provides this group monoclonal antibody and forms the test kit that detects the antigenic reagent of SARS-CoV and set up with this reagent.
The 4th purpose of the present invention provides the application that this group monoclonal antibody is used for detecting SARS-CoV antigen reagent.
To achieve the above object, the present invention realizes by following technical scheme.
The invention provides one group of monoclonal antibody, but its specificity and relates to following technical characterictic in conjunction with SARS-CoV N albumen:
1, western blotting method is measured and is shown that this group monoclonal antibody specific recognition SARS-CoV N proteantigen molecular weight is 46 kilodaltons.
2, described monoclonal antibody, be number to comprise hybridoma 1E8A11:CCTCC-C200401 by having to preserve, hybridoma 8E1A17:CCTCC-C200402, hybridoma 10E4A4:CCTCC-C200403, the clone secretion of hybridoma 14A3A3A19:CCTCC-C200404 produces.
3, described monoclonal antibody belongs to immunoglobulin class IgG1 or IgG2b.
4, immunoblotting shows that but the nucleocapsid protein molecular weight is that 46 kilodaltons react in specificity and the sars coronavirus split product.
5, immunoblotting shows that but the nucleocapsid protein molecular weight of specificity and gene recombination is that 46 kilodaltons react.
6, be coated on the specific carrier as antigen with the Vero cell (African green monkey kidney cell) that infects SARS-CoV, show that this group monoclonal antibody specificity combines with SARS-CoV antigen-specific on the cell that is infected by SARS-CoV but detect with indirect immunofluorescence method.
7, in fluoroimmunoassay, be used for recognizing cells or tissue sars coronavirus nucleocapsid protein cAg.
8, in the immunohistochemical analysis method, be used for recognizing cells or tissue sars coronavirus nucleocapsid protein cAg.
9, described monoclonal antibody does not have reactivity to people's coronavirus 229E and the nucleocapsid protein of OC43; And there is not cross reaction with other ani mal coronavirus and Respirovirus.Therefore, this group monoclonal anti physical efficiency that the present invention relates to is used to diagnose the disease that is infected by SARS-CoV, therefrom can distinguish the disease that is caused by other people relevant with SARS-CoV and coronavirus.
The present invention also provides the hybridoma that produces above-mentioned monoclonal antibody, is to have the hybridoma cell strain that preserving number is CCTCC-C200401, CCTCC-C200402, CCTCC-C200403 and CCTCC-C200404.
The invention provides 3 hybridoma cell strainses of this group hybridoma cell strain is immunoglobulin G 1 positive (1E8A11,8E1A17,10E4A4), and 1 hybridoma cell strains is the immunoglobulin G 2b positive (14A3A3A19).
The preparation method of above-mentioned hybridoma cell strain, comprising the albumen of giving animal cross immunity inoculation gene recombination and natural viral protein, thereby obtain to produce efficiently the immune spleen cell of specificity at SARS-CoV N protein antibodies, use conventional cell-fusion techniques, splenocyte that this is immune and the myeloma cell of mouse are merged, and successfully obtain to produce this group monoclonal antibody specificity at the proteic hybridoma of SARS-CoV N.In the mouse body, obtain high ascites of tiring by hybridoma cell strain, through obtaining highly active specificity at the proteic monoclonal antibody of SARS-CoV N with sad-ammonium sulfate precipitation method purifying.
The present invention specifically utilizes the mode of cell-fusion techniques, prepares one group and has specific hybridoma cell strain at SARS-CoV N albumen and comprise 10E4A4,8E1A17,1E8A11 and 14A3A3A19.Its method is for utilizing the cytogamy agent of generally acknowledging, polyoxyethylene glycol (PEG) for example, myeloma cell strain and the bone-marrow-derived lymphocyte that produces anti-SARS-CoV N protein antibodies are merged, with HAT screening hybridoma cell strain, utilize ELISA, indirect immunofluorescence and immunoblotting to analyze the specificity of antibody in the hybridoma culture supernatant again, select SARS-CoV N albumen is had specific individual plant hybridoma cell strain, the abdominal cavity that this group individual plant hybridoma cell strain is injected mouse is to produce ascites.Marker enzyme or fluorescein behind this group monoclonal antibody purifying be can be used for detecting SARS-CoV antigen in body fluid or the tissue in immunological technique.Immunogen used in the present invention is with the fusion N proteantigen of gene engineering method preparation and the natural virus antigen of deactivation.
The present invention also provides a kind of antibody in the immunoenzyme test right, it is characterized in that making up respectively the pairing of the antibody of antibody that said monoclonal antibody catches and mark, make up as the monoclonal antibody of catching with 1E8A11,8E1A17 and 10E4A4, the monoclonal antibody that serves as a mark with 14A3A3A19 antibody
The present invention passes through behind each strain labeling of monoclonal antibodies, determine the antigen recognition site of each monoclonal antibody by the combined crosswise test, and by the blocking-up inhibition analysis of the anti-N protein antibodies male serum in SARS the infected's blood to monoclonal antibody, and therefrom screening obtains to catch and detect the antibody optimum antibody to being used for the structure of test kit.
The present invention relates in addition can be labeled by above-mentioned one group of monoclonal antibody that SARS-CoV N albumen is had a specific reaction, promptly on monoclonal antibody in conjunction with a kind of labelled reagent, comprise peroxidase, alkaline phosphatase etc. as enzyme, Radioactive colloidal gold and fluorescein etc., can separately or mutually combine with these labelled reagents, or be combined in the reagent that immunological technique (various enzyme linked immunosorbent assay, immunofluorescence, immunochemiluminescence or the like) catches as diagnosis SARS-CoV with polyclonal antibody.
Therefore the present invention also provides a kind of sars coronavirus nucleocapsid protein antigen detecting agent, it is characterized in that comprising the above-mentioned monoclonal antibody that is labeled.
The present invention also relates to a kind of sars coronavirus nucleocapsid protein antigen detecting agent box, it is characterized in that comprising above-mentioned reagent.
The present invention relates to can provide a kind of qualitative or detection by quantitative pharynx examination by foregoing description, collutory, ight soil, sputum and serum, blood plasma or other body fluid, the proteic enzyme immunization method of SARS-CoV N in the biological fluid, this measuring method be with one group of monoclonal antibody solid phase at the different antigen sites of SARS-CoV N albumen on microwell plate, the monoclonal antibody or the polyclonal antibody that cooperate a kind of enzyme labelling, by antibody sandwich method principle, SARS-CoV antigen in the working sample, adopting this measuring method to measure SARS-CoV N protein content susceptibility is 10-50pg/ml, and the titer determination susceptibility of virus is less than 1.3 * 10
2PFU/ml.By susceptibility is measured, the sensitive performance of this measuring method detects the virus antigen of trace in the sample.
Monoclonal antibody of the present invention also can separately or mutually combine, or is combined in the immunological technique (various enzyme linked immunosorbent assay, chemiluminescence immune assay or the like) with polyclonal antibody and is used to detect SARS-CoV antigen.
Monoclonal antibody of the present invention can be used for from the material of natural or recombinant sources affinity purification and isolates SARS-CoV N proteantigen in immunological technique, purified N albumen can be used for the research of serodiagnosis and protein function.
The monoclonal antibody of hybridoma cell strain provided by the present invention, anti-SARS-CoV N proteantigen, except can be applicable to above-mentioned detection kit, also can be applicable in other relevant field of immunology, for example, fast immune chromatographic method (one-step strip), immunoblotting (Western blot), immunoprecipitation, immunofluorescence dyeing, immune group histochemical stain, original position demarcation (in situ labeling) etc. all are within category of the present invention.
Described beneficial effect of the present invention is the monoclonal antibody of prepared specificity at SARS-CoV N proteantigen, comprise the reagent of this antibody and the detection kit of setting up with this reagent, can detect SARS-CoV antigen simple and easy, easily and fast and accurately, be used at present early diagnosis viral nucleic acid detection method compare have high specificity, good reproducibility, easy to operate, cost is low, be fit to advantages such as grass-roots unit's use.
Study of Monoclonal Antibodies successfully has very important scientific value to early diagnosis, the examination of epidemic disease source animal and the research of SARS-CoV protein science that research SARS-CoV infects, and can also be used for SARS pathogenesis, SARS-CoV biologic activity.
Be deposited in Chinese typical culture collection center (CCTCC on January 19th, 2004 by the hybridoma that the present invention relates to, China, the Wuhan City), preserving number is respectively: hybridoma 1E8A11:CCTCC-C200401, hybridoma 8E1A17:CCTCC-C200402, hybridoma 10E4A4:CCTCC-C200403, hybridoma 14A3A3A19:CCTCC-C200404.
Description of drawings
Fig. 1: be the immunoblotting result, show that Hybridoma Cell Culture supernatant specificity of the present invention is in conjunction with gene recombination SARS-CoV N albumen.
Fig. 2: be the immunoblotting result, show that Hybridoma Cell Culture supernatant specificity of the present invention is in conjunction with the N albumen in the full SARS-CoV virus.
Fig. 3: the indirect IF staining result shows that Hybridoma Cell Culture supernatant specificity of the present invention is in conjunction with the virus antigen that is infected by SARS-CoV on the Vero E6 cell.
Fig. 4: detect the proteic typical curve of N for showing the double-monoclonal antibody sandwich ELISA method detection kit that monoclonal antibody of the present invention is set up.Adopting this measuring method to measure SARS-CoV N protein content susceptibility is 10-50pg/ml.
Fig. 5: the N antigen that detects the SARS-CoV in the cell cultures for the double-monoclonal antibody sandwich ELISA method detection kit that shows monoclonal antibody foundation of the present invention.The titer determination susceptibility of virus is less than 1.3 * 10
2PFU/ml.
Fig. 6: detect SARS-CoV antigen in patient's SARS lung tissue for showing monoclonal antibody of the present invention: lung mononuclear macrophage viral inclusion body SARS-CoV stained positive.
Fig. 7: detect SARS-CoV antigen in patient's SARS lung tissue for showing monoclonal antibody of the present invention: Bronchio-serous gland epithelial cell SARS-CoV stained positive.
Fig. 8: the N antigen that detects the SARS-CoV among the SARS patients serum for the double-monoclonal antibody sandwich ELISA method detection kit that shows monoclonal antibody foundation of the present invention.Be mensuration male sample greater than critical line.
Embodiment
The present invention is described in further detail below in conjunction with drawings and Examples, but these embodiment illustrate as an example, is not in order to limit scope of the present invention.
Embodiment 1:SARS-CoV nucleocapsid protein (hereinafter to be referred as N albumen) MONOCLONAL ANTIBODIES SPECIFIC FOR method.
1, the preparation of immunizing antigen
The immunogen that the present invention is used to prepare monoclonal antibody is gene recombination SARS-CoV N albumen and the natural virus antigen of deactivation, and gene recombination SARS-CoV N egg is to be that a kind of coli strain is prepared with the engineering strain that carries SARS-CoV N protein gene.Announce that with PUBMED sars coronavirus HKU-39849 strain (or other strains) nucleocapsid gene order is consistent through the sars coronavirus nucleocapsid gene order that order-checking confirmation bacterial classification carries.Reorganization SARS-CoV N protein Preparation is carried out according to a conventional method, obtains N fusion rotein antigen through carry out purifying with the method for nickel-triglycollamic acid metal affinity chromatography, and at length the preparation method can consult and use handbook.Behind N fusion rotein purifying, with Xylene Brilliant Cyanine G (Coomassie) analysis of protein reagent (PIERC, Cat, No.ED62976) quantitative.The natural virus antigen of deactivation is to obtain from the virus host cell (Vero, a kind of monkey embryonic kidney cells) that SARS-CoV infects.
2, immune mouse
Get 6 the week age BALB/c mouse, adopt RIBI adjuvant (MPL+TDM Adjuvant System, Sigma:M6536) with the emulsification of equal-volume antigen mixing, first inferior to the mouse peritoneal injecting immune, subcutaneous and two metapedes pad portions immunity in nape portion after 21 days, reach two metapedes pad portions injection booster immunization after 12 days once more in the abdominal cavity, each antigen injection volume is the antigen of every mouse 50 micrograms, altogether immune 10 mouse.
3, immune serum titration
Setting up indirect elisa method mensuration immune serum tires.Prepare the 50mM pH9.6 carbonate buffer solution of 1 mcg/ml recombinant N protein, bag is by little 96 orifice plates of polystyrene, and 0.1 milliliter/hole, 4 ℃ are spent the night.Next day, contain 1% bovine serum albumin (BSA, spend the night for 4 ℃ in 0.3 milliliter/hole of confining liquid Sigma), contain 10% saccharose treatment coated slab with the 10mM phosphate buffered saline buffer next day again, vacuum-drying 2～12 hours with the vacuum-packed 4 ℃ of preservations of aluminum foil bag, is used for the titration of mouse immune serum.In eye socket blood sampling in immune back 10 days for the third time, the mouse immune serum was with containing 1%BSA10mM PBS with 10
3～10
6Doubly dilution, add 96 orifice plates, 37 ℃ in 0.1 milliliter/hole 30 minutes is after 10mM PBS contains 0.1% Tween-20 washings and washes plate five times, add 1: 2000 times of dilution horseradish peroxidase-labeled goat anti-mouse igg (U.S. ZYMEDLABORATORIES, INC.), 37 ℃ in 0.1 milliliter/hole 30 minutes, the same wash plate after, add and contain 0.05% (W/V) TMB and 0.06% (W/V) dioxygen pH5.0 citrate buffer solution, 0.1 milliliter/hole, room temperature lucifuge 10 minutes adds 0.1 milliliter/hole 2M H
2SO
2Termination reaction is surveyed the 450nm absorption value, as negative control, judges the tiring of immune serum so that measured value and control value are must be than 〉=2.1 positive with mice serum before the immunity.
4, hybridoma preparation
Select serum antibody titer to reach 1 * 10
6Mouse, in merging preceding 3 days tail vein injection 10 microgram antigens.The aseptic mouse spleen of getting, the murine myeloma cell strain NS-1 that makes splenocyte suspension and logarithmic phase is by 10: 1 mixed, with merging under 50% polyoxyethylene glycol (PEG, MW4000, Sigma catalog number (Cat.No.), the lot number) effect.By following step polyglycol solution is added cell.Under 37 ℃, added 1.5 milliliter of 50% polyglycol solution in the half at the 1st minute; In the 2nd minute, add 1.5 milliliters of serum-free RPMI-1640 substratum (GIBCO), at the 3rd minute, add 3 milliliters of serum-free RPMI-1640 substratum, in the 4th minute, add 8 milliliters of above-mentioned solution again.The 5th with the 6th minute in add solution identical on 10 milliliters again.Centrifugal 5 minutes of the speed that this mixed solution changes with per minute 800 with whizzer, the supernatant in the sucking-off centrifuge tube has hanged cell gently with 50 to 60 milliliters of substratum that contain 15% foetal calf serum again.This cell suspension is added on 6 96 well culture plates, and temperature is that 37 ℃, carbon dioxide content are in 5% the CO2gas incubator in CO2gas incubator.After one day, add 100 microlitres in every hole and contain xanthoglobulin, aminopterin-induced syndrome-Thymine deoxyriboside (HAT, Sigma) screening culture medium.。Changed liquid once with this screening culture medium to culture in per 3 days later on, and formed up to clone cell.
Embodiment 2: the hybridoma of screening secretion SARS-CoV nucleocapsid protein monoclonal antibody
For detecting the existence that produces antibody cloning, the method of tiring with embodiment 1 survey immune serum is that indirect elisa method detects cells and supernatant, be summarized as follows: the hybridoma culture supernatant is added bag by in the good hole, 0.1 37 ℃ in milliliter/hole 30 minutes, after washings is washed plate five times, add 1: 2000 times of dilution horseradish peroxidase-labeled goat anti-mouse igg (U.S. ZYMED LABORATORIES, INC.), 0.1 37 ℃ in milliliter/hole 30 minutes, the same wash plate after, add substrate TMB, 0.1 milliliter/hole, room temperature lucifuge 10 minutes adds 0.1 milliliter/hole 2M H
2SO
2Termination reaction is surveyed the 450nm absorption value.Select strong positive clone hybridization oncocyte to carry out formal cloning,, obtain the hybridoma cell strain of 4 strain stably excreting antibody altogether with the continuous cloning of limiting dilution assay 2～3 times.Difference called after 8E1A17,1E8A11,10E4A4 and 14A3A3A19.Positive rate after the cloning is reached 100% cell amplification cultivate the back liquid nitrogen cryopreservation.
Embodiment 3:SARS-CoV nucleocapsid protein monoclonal antibody subclass detects
4 clones that detect acquisition in the present embodiment with the indirect elisa method of describing among the embodiment 1 are to determine the antibody subclass of its generation.The hybridoma culture supernatant is added bag by in the good hole, 0.1 37 ℃ in milliliter/hole 30 minutes, after washings is washed plate five times, these antibody of the different subclass specific immunoglobulin of the anti-mouse of rabbit that add 1: 1000 times of dilution horseradish peroxidase-labeled comprise the anti-mouse IgG1 of rabbit (U.S. ZYMED LABORATORIES, INC, catalog number (Cat.No.) 61-0120), the anti-mouse IgG2a of rabbit (the same, catalog number (Cat.No.) 61-0220), anti-mouse IgG2b is (the same for rabbit, catalog number (Cat.No.) 61-0320), the anti-mouse IgG3 of rabbit (the same, catalog number (Cat.No.) 61-0420), anti-mouse IgM is (the same for rabbit, catalog number (Cat.No.) 61-6820), 37 ℃ in 0.1 milliliter/hole 30 minutes, the same wash plate after, add substrate TMB, 0.1 milliliter/hole, room temperature lucifuge 10 minutes adds 0.1 milliliter/hole 2M H
2SO
2Termination reaction is surveyed the 450nm absorption value.Detected result 1E8A11,8E1A17, a 10E4A4 hybridoma cell strain are immunoglobulin G 1 positive, and 1 hybridoma of 14A3A3A19 is the IgG2b positive.
Embodiment 4: preparation and the purifying and the determination of activity of anti-SARS-CoV nucleocapsid protein monoclonal antibody ascites
Induce legal system in the employing body and be equipped with monoclonal antibody among the present invention, promptly in the mouse body, inoculate hybridoma, preparation ascites, in every mouse peritoneal, inject 0.5 milliliter of pristane (2,6,10 with 25 or No. 27 standard needle, the 10-tetramethyl-pentadecane is available from the Aldrich chemical company).The processing of pristane can be grown oncocyte with the ascitic tumor form at intraperitoneal.After about 1～2 week, 10
6The hybridoma that is suspended in serum-free RPMI1460 substratum about individual injects the mouse peritoneal of handling with pristane.Each clone is injected into mouse peritoneal by said procedure.The injection hybridoma is put ascites with No. 16 syringe needles after about 1～2 week, can collect repeatedly for several times.Ascites is frozen behind centrifugal clarification.The method that the ascites titration adopts embodiment 1 indirect elisa method of describing mensuration immune serum to tire is carried out, and ascites is made doubling dilution.The ascites of result's 4 strain monoclonal antibodies of the present invention is tired and is seen Table 1.
Table 1: the immunological characteristic of anti-SARS-CoV N protein monoclonal antibody
| The hybridoma cell strains title | Subclass | Ascites is tired | Antibody purification activity (mcg/ml) | Affinity costant | The enzymic-labelled antibody working concentration |
| ??8E1A17 | ??IgG1 | ??1∶200000 | ????0.78 | ?2.7×10 -9M | ??1∶400000 |
| ??10E4A4 | ??IgG1 | ??1∶1000000 | ????1.56 | ?7.9×10 -8M | ??1∶400000 |
| ??1E8A11 | ??IgG1 | ??1∶400000 | ????0.78 | ?1.9×10 -9M | ??1∶400000 |
| ??14A3A3A19 | ??IgG2b | ??1∶10000000 | ????1.56 | ?7×10 -9M | ??1∶50000 |
The purifying of ascites antibody adopts sad-ammonium sulfate precipitation method, ascites 60mmol/ milliliter, 2 times of pH5.0 acetate buffer solution dilutions, with 0.1N hydrochloric acid adjust pH to 4.8, liquid is muddy by limpid change, it is sad to add in dropwise slow while stirring 30 minutes under the room temperature, to add 33 microlitres sad for ascites before every milliliter of dilution, precipitation in a large number occurs, and 4 ℃ left standstill 2 hours, 10000g, 4 ℃ centrifugal 30 minutes, get supernatant, add the pH7.4 10mmol/ ml phosphate buffer of 1/10 volume, and with 0.1N sodium hydroxide adjust pH to 7.4, ice bath stirs and slowly adds 0.277 grams per milliliter ammonium sulfate down, is 45% saturation ratio, perhaps directly adds pH value 7.4 saturated ammonium sulphates and reaches final concentration 45%, 4 ℃ of standing over night, 10000g, 4 ℃ centrifugal 30 minutes, abandon supernatant, precipitation is dissolved in an amount of 10mmol/ ml phosphate buffer, with same liquid, 4 ℃ of dialysed overnight are changed liquid three times.With Xylene Brilliant Cyanine G (Coomassie) analysis of protein reagent (PIERC, Cat, No.ED62976) quantitative.-80 ℃ of preservations behind the mensuration protein concentration.The method that the monoclonal antibody determination of activity adopts embodiment 1 indirect elisa method of describing mensuration immune serum to tire is carried out, and antibody purified is made doubling dilution to 0.1 pico-gram/ml, and the activity of result's 4 strain monoclonal antibodies of the present invention sees Table 1.
Embodiment 5: the monoclonal antibody affinity costant is measured
The antibody affinity costant is measured and is selected for use indirect ELISA method to measure, and at JImmunol Methods, 100:173-179 has comprehensive description in (1983) to this method with reference to people such as Beatey.According to this method, with 50mM pH9.6 carbonate buffer solution antigen is wrapped by little 96 orifice plates of polystyrene with 1 mcg/ml and 10 little shells/milliliter respectively, 0.1 milliliter/hole, 4 ℃ are spent the night.Next day, contain 1% bovine serum albumin (BSA, Sigma) spending the night for 4 ℃ in 0.3 milliliter/hole of confining liquid, adds the monoclonal antibody of different weaker concns, 37 ℃ in 0.1 milliliter/hole 30 minutes, after 10mM PBS contains 0.1% Tween-20 washings and washes plate five times, add 1: 2000 times of dilution horseradish peroxidase-labeled goat anti-mouse igg (U.S. ZYMEDLABORATORIES, INC.), 37 ℃ in 0.1 milliliter/hole 30 minutes, the same wash plate after, the OD value is surveyed in TMB colour developing.Logarithm with antibody concentration is an abscissa, with the OD value is ordinate, every kind of monoclonal antibody draws 2 response curves, with the OD value of every curve upper planar section as 100%, corresponding antibody concentration when on curve, finding the 50%OD value, press Beatty derivation formula: Kaff=(n-1)/2 (n[Ab '] t-[Ab] t) calculate affinity costant.Result's 4 strain monoclonal antibody affinity costants of the present invention see Table 1.
Embodiment 6: horseradish peroxidase-monoclonal antibody binding substances preparation
The horseradish peroxidase-labeled Antibody Preparation adopts improvement sodium periodate method, with 5mg horseradish peroxidase stirring and dissolving in 1 ml distilled water, add 0.2 milliliter and newly joined the 0.1M sodium periodate 30 minutes, put in the 1mM pH4.4 sodium-acetate buffer, 4 ℃ of dialysed overnight, add the 10mg antibody of pH 9.5 dialysis equilibriums in the 0.01M carbonate buffer solution in advance after adding 20 μ l 0.2M pH9.5 carbonate buffer solutions next day, stirred gently 2～3 hours in the room temperature lucifuge, add 0.1 milliliter and newly join 4mg/ milliliter sodium borohydride, 4 ℃ of lucifuges are spent the night, lucifuge stirs and dropwise to add equal-volume saturated ammonium sulphate (ammonium sulfate with preceding elder generation with ammoniacal liquor accent pH to 7.0-7.2) down on the ice bath, put 4 ℃ 6 hours.10000g, 4 ℃ centrifugal 30 minutes, abandon supernatant, the precipitation be dissolved in an amount of 10mM phosphate buffered saline buffer, with same liquid, 4 ℃ of dialysed overnight are changed liquid three times.Collect binding substances and add 2%BSA PBS 50% glycerine protective material, divide device-70 ℃ to preserve and carry out the enzyme labelled antibody working concentration and measure.The method that the enzyme labelled antibody titration adopts embodiment 1 indirect elisa method of describing mensuration immune serum to tire is carried out, and enzyme labelled antibody is made doubling dilution.The working concentration of the enzyme labelled antibody of result's 4 strain monoclonal antibodies of the present invention sees Table 1.
Embodiment 7: the specificity of identifying SARS-CoV N protein monoclonal antibody
1, indirect elisa method carries out the monoclonal antibody specificity analyses
With the antigen coated microwell plate of purifying SARS-CoV totivirus lysate, setting up the step of measuring the indirect elisa method that immune serum tires by embodiment 1 carries out, the Hybridoma Cell Culture supernatant liquor that in the microwell plate of bag quilt, adds this patent invention, hatched again 1 hour for 37 ℃, add 1: 2000 dilution horseradish peroxidase-labeled goat anti-mouse igg (U.S. ZYMED LABORATORIES, INC.), 37 ℃ in 0.1 milliliter/hole 30 minutes, add TMB colour developing liquid chamber temperature lucifuge 10 minutes, add 0.2M H
2SO
2Termination reaction is surveyed the 450nm absorption value.Table 2 result shows that the SARS-CoV monoclonal antibody of this patent invention and SARS-CoV antigen produce very strong specific immune response.
Table 2:SARS-CoV monoclonal antibody and sars coronavirus totivirus lysate antigen are anti-
Answer the indirect ELISA result
| The SARS-CoV monoclonal antibody | Irrelevant antibody | ||||
| ??8E1A17 | ??10E4A4 | ??1E8A11 | ??14A3A3A19 | ||
| ??A450 | ??1.731 | ??2.364 | ??1.592 | ??3.446 | ????0.108 |
2, carry out the monoclonal antibody specificity analyses with indirect immunofluorescence
Infect the Vero-E6 cell with SARS-CoV, when pathology appears in 2/3 cell, collecting cell, 1 * PBS with precooling washes cell two times, then cell is dripped on aseptic exsiccant slide, after the drying, be prepared into smear, thorough drying, fix 30 minutes with cold acetone, respectively wash 3 times with PBS and deionized water, dry up, the Hybridoma Cell Culture supernatant is dropped in the different holes with 10 μ l respectively by different extent of dilution, establish feminine gender and positive control serum simultaneously, put in 37 ℃ of water baths, act on after 45 minutes, taking-up is put in the antigen sheet in the staining jar and respectively washes 3 times with 0.01mM pH7.2 PBS and distilled water, dries up, and adds the fluorescent mark goat anti-mouse igg antibody, put in 37 ℃ of water baths, effect after 45 minutes, taking-up is put in the antigen sheet in the staining jar and respectively washes 3 times with 0.01mmol/LpH7.2PBS and distilled water, dries up, fluorescent microscope is observed fluoroscopic image down, carry out the result with intensity of fluorescence and dyeing form and judge, detect antibody intensity and count the positive with (+～++ ++), antibody intensity (±) and (-) count feminine gender.As Fig. 3 and table 3 result in the accompanying drawing develop the color the SARS-CoV monoclonal antibody be fixed on slide on the SARS-CoV antigen-specific combine.
The immunofluorescence detected result of table 3, monoclonal antibody
| The hybridoma clone | Hybridoma culture supernatant extent of | ||||
| 1∶10 | ????1∶20 | ????1∶40 | ????1∶80 | ????1∶160 | |
| ??8E1A17 | +++ | ????++ | ????++ | ????+ | ????- |
| ??1E8A11 | +++ | ????++ | ????+ | ????+ | ????- |
| ??10E4A4 | +++ | ????++ | ????+ | ????- | ????- |
| ??14A3A3A19 | +++ | ????++ | ????++ | ????+ | ????+ |
3, the immunoblotting assay method is carried out the monoclonal antibody specificity analyses
N albumen with deactivation SARS-CoV nutrient solution or reorganization, with one times of 2 * SDS sample loading buffer dilution, sample is added in the 10%SDS-polyacrylamide gel, electrophoretic separation protein, make at the protein transduction of separating on the gel by electroelution and to print on the nitrocellulose membrane, transfer film contains 7% skimmed milk and 3%BSA with 1 * PBS, 4 ℃ were sealed 6 hours, transfer film is cut into several little, add respectively in the Hybridoma Cell Culture supernatant, 4 ℃ of reactions are spent the night, and behind the 1 * PBS washing film that contains 0.5%Tween 20, add the HRP mark sheep anti-mouse igg of 1: 500 times of dilution, put room temperature reaction 1 hour, behind same washings washing film, after the AEC colour developing, use the deionized water color development stopping.
Fig. 1 of immunoblotting result such as accompanying drawing and Fig. 2 show that 4 strain monoclonal antibodies of the present invention combine with the N protein-specific, and conjugated protein relative molecular mass is 46 kilodaltons.SARS-CoV liquid is the visible single protein immunity combined belts of 46 kilodaltons at molecular weight, and the specific protein combined belt is that 46 kilodaltons are consistent with predicted molecular weight.Illustrate that the monoclonal antibody that obtains can specific recognition SARS-CoV antigen.
Embodiment 8: the analysis in monoclonal antibody identification native antigen site
Adopt the indirect competition of solid phase antigen to suppress method, key step is as follows:
The 50mM pH9.6 carbonate buffer solution that (1) 1 mcg/ml recombinant N protein is 0.1 milliliter, bag is by little 96 orifice plates of polystyrene, and 4 ℃ are spent the night.Next day, contain 1% bovine serum albumin (BSA, Sigma) after spending the night in 4 ℃ in 0.3 milliliter/hole of confining liquid, (2) serum of the culture supernatant of A:50 microlitre hybridoma and 50 microlitre SARS-CoV N district positive patients, hatched jointly 1 hour for 37 ℃, this step is called the inhibition of vying each other, after 37 ℃ of the serum of B:100 microlitre SARS-CoV N district positive patients are hatched 1 hour, with the serum sucking-off, the culture supernatant that adds 100 microlitre hybridomas then, hatched 1 hour for 37 ℃, this step is called competition inhibition successively again.(3) add 1: 2000 dilution horseradish peroxidase-labeled goat anti-mouse igg (U.S. ZYMED LABORATORIES, INC.), 0.1 37 ℃ in milliliter/hole 30 minutes, add TMB colour developing liquid chamber temperature lucifuge 10 minutes, add 0.2M H2S02 termination reaction, survey the 450nm absorption value, as suppressing negative control, be judged as with serum OD measured value/normal human serum OD measured value≤05 of SARS-CoV N district positive patients and suppress the positive with normal human serum.Table 4 result shows: when 12 parts of SARS-CoV antibody positive patients' serum is vied each other inhibition test the present invention's 4 strain monoclonal antibodies are not produced restraining effect, and when successively competing inhibition test, this 4 strain cell is produced the obvious suppression effect, show that thus the antibody that produces after the natural infection can suppress the reaction of monoclonal antibody and recombinant antigen, illustrate that the site that this group monoclonal antibody is discerned is present on the native antigen, also point out simultaneously 4 strain monoclonal antibodies of the present invention because the avidity height, can with patient's the serum solid phase antigen of vying each other.
Table 4:SARS positive serum is respectively to the restraining effect of 4 strain monoclonal antibodies
| Clone number | The inhibition of vying each other | Competition successively suppresses | ||
| Suppress number | Inhibiting rate (%) | Suppress number | Inhibiting rate (%) | |
| ??14A3A3A19 | ????1 | ????8.3 | ????8 | ????66.6 |
| ??10A4A1 | ????10 | ????83.3 | ????12 | ????100 |
| ??8E1A17 | ????0 | ????0 | ????10 | ????83.3 |
| ??1E8A11 | ????10 | ????83.3 | ????12 | ????100 |
Embodiment 9: the monoclonal antibody recognition site is analyzed
The 50mM pH9.6 carbonate buffer solution that 5 mcg/ml recombinant N proteins are 0.1 milliliter, bag is by little 96 orifice plates of polystyrene, and 4 ℃ are spent the night.Next day, contain 1% bovine serum albumin (BSA, Sigma) after spending the night in 4 ℃ in 0.3 milliliter/hole of confining liquid, add earlier monoclonal antibody 10 mg/ml, 50 microlitres and after add 1: 100,1: 200,1: 400,1: 800,1: 1600,1: 2,000 50 microlitre of serial dilution HRP mark monoclonal antibody, 37 ℃, sealed 1 hour; PBST adds substrate 100 microlitres/hole after washing five times, and 10～20 minutes, OD450 measured absorption value.With monoclonal antibody the monoclonal antibody of same HRP mark being suppressed is 100%, to the negative contrast of the inhibition of mark monoclonal antibody, calculates the inhibiting rate between each monoclonal antibody with known irrelevant monoclonal antibody.Be that inhibiting rate is (1-measured value OD/ negative control value OD) * 100.Inhibiting rate＞75% is for relevant, and＞50% is not exclusively relevant, and＜50% is uncorrelated, and＜25% for uncorrelated fully.Table 5 result shows 4 incomplete same antigen sites of 4 strain monoclonal antibodies identification.
Table 5: SARS coronary virus resistant nucleocapsid protein monoclonal antibody recognition site is measured
| Monoclonal antibody (1mg/ml) | Monoclonal antibody linked with peroxidase extent of dilution 1: 400 (inhibiting rate %) | |||
| ????1E8A11 | ????8E1A17 | ????10E4A4 | ??14A3A3A19 | |
| ??1E8A11 | ????95 | ????0 | ????0 | ????0 |
| ??8E1A17 | ????1.4 | ????95.6 | ????0 | ????0 |
| ??10A4A1 | ????0 | ????0 | ????97.6 | ????0 |
| ??14A3A3A19 | ????8.82 | ????0 | ????1.36 | ????78.08 |
The screening and the ELISA Parameter Optimization of embodiment 10 double-antibody sandwich elisa method monoclonal antibody best pairing
From the monoclonal antibody of the ascites purifying of embodiment 4 be used for carrying out one group of matrix format experiment with select be suitable for most below embodiment 11 described sandwich ELISA methods to be used as the monoclonal antibody of catching with mark right.Briefly, with 4 strain of hybridoma (being numbered 1E8A11,8E1A17,10E4A4,14A3A3A19) of sad-ammonium sulfate precipitation method purifying derive and the ascites bag that comes by 96 orifice plates, right with the recombinant N protein that embodiment obtains as antigen selection antibody.Monoclonal antibody with 4 strain horseradish peroxidase-labeled described in the embodiment 6, adopt matrix format, just above-mentioned 4 strain monoclonal antibodies are wrapped every strain to be used as and are caught or hybrid packet is used as and catches, match with each strain enzyme labelling monoclonal antibody respectively, right to catch with the monoclonal antibody of mark in the rapid screening sandwich ELISA.Preliminary experiment shows that monoclonal antibody 1E8A11,8E1A17,10A4A1 and 10E4A4 respectively as capture antibody, when matching with enzyme labelling 14A3A3A19 monoclonal antibody, can produce stronger signal.And on this basis, make up the pairing of the antibody of antibody that above-mentioned 4 strain monoclonal antibodies catch and mark respectively, with strength of signal and specificity, make up as the monoclonal antibody of catching with 1E8A11,8E1A17 and 10E4A4, monoclonal antibody with 14A3A3A19 antibody serves as a mark produces the strongest signal.
Embodiment 11: detect the antigenic double-antibody sandwich elisa method of SARS-CoV test kit with anti-SARS-CoV N protein monoclonal antibody and set up
The operating process that detection SARS-CoV antigenic reagent box of the present invention is set up is described below:
1, monoclonal antibody 1E8A11 of the present invention, 8E1A17 and 10E4A4 are diluted to 10 mcg/ml with 50mM carbonate buffer solution (pH9.6), by polystyrene 96 microwell plates, spend the night in 4 ℃ with 0.1 milliliter/hole bag.With 0.05%Tween 20/10mMPBS flushing 1 time, pat dry then.(BSA, confining liquid Sigma) spend the night with the sealing nonspecific binding site in 4 ℃ 1% bovine serum albumin that the adding of every hole is 0.3 milliliter.Contain 10% saccharose treatment coated slab with the 10mM phosphate buffered saline buffer, vacuum-drying 2～12 hours, standby with the vacuum-packed 4 ℃ of preservations of aluminum foil bag.
2, with various samples to be measured with after the sample diluting liquid dilution, each adds 100 microlitres in polystyrene 96 hole trace test plates, every duplicate samples is established multiple hole, 37 ℃ of incubations 1 hour, after containing the 0.1%Tween-20 washings and wash plate five times with 10mM PBS, with the enzyme diluted horseradish peroxidase-labeled 14A3A3A19 monoclonal antibody is done 1: 1000 times of dilution, every hole adds 100 microlitres, 37 ℃ 1 hour, the same wash plate after, add and to contain 0.05% (W/V) TMB and 0.06% (W/V) hydrogen peroxide pH5.0 citrate buffer solution, every hole adds 100 microlitres, 37 ℃ of lucifuges 10～15 minutes, every hole adds 100 microlitre 2MH
2SO
2Termination reaction.
3, the result judges: with the blank well zeroing, measure the A value in the 450nm wavelength.Positive control mean value 〉=0.50, negative control mean value≤0.10, experiment is set up.Sample A value 〉=negative control A value mean value * 2.1 then is judged to the positive.Otherwise it is negative.
The analysis that double-antibody sandwich elisa method of the present invention detects SARS-CoV antigenic reagent box result is described below:
1, antibody sandwich ELISA method test kit special to the SARS-CoV strain of different sources and different virus
The property determination and analysis:
Get from Guangzhou, the isolating SARS-CoV strain in area of Beijing and Hong Kong, ani mal coronavirus and other viral cultures, detect respectively with above-mentioned double-antibody sandwich elisa method.Table 6 result shows, separation SARS-CoV strain to different areas all has specific reaction, to human corona virus (OC43 and 229E), other ani mal coronavirus (fowl coronavirus IBV and canine coronavirus CCV), Respirovirus (influenza virus, adenovirus, respiratory syncytial virus, rhinovirus) and Chlamydia pneumoniae, the equal no cross reaction of mycoplasma pneumoniae.Illustrate that the double-antibody sandwich elisa method of setting up with monoclonal antibody of the present invention has the specificity of height to SARS-CoV.
Table 6: double-antibody sandwich elisa method test kit detects the SARS-CoV antigen of cell cultures
| The SARS-CoV strain | Discretely | The culture extension rate | The result | |
| Positive | Negative | |||
| Hong Kong strain | Hong Kong | ????1∶10000 | ????+ | ????0 |
| Beijing strain | Beijing | ????1∶10000 | ????+ | ????0 |
| The Guangdong strain | Guangdong | ????1∶1000 | ????+ | ????0 |
| ????WHO | ????1∶1000 | ????+ | ????0 | |
| U.S.'s strain | ????1∶10000 | ????+ | ????0 | |
| Fowl coronavirus (IBV) | Cultivate stoste | ????0 | ????- | |
| Canine coronavirus (CCV) | Cultivate stoste | ????0 | ????- | |
| Influenza virus | Cultivate stoste | ????0 | ????- | |
| Adenovirus | Cultivate stoste | ????0 | ????- | |
| Respiratory syncytial virus | Cultivate stoste | ????0 | ????- | |
| Rhinovirus | Cultivate stoste | ????0 | - | |
| Cytomegalovirus | Cultivate stoste | ????0 | - | |
| Human corona virus (OC43) | Cultivate stoste | ????0 | - | |
| Human corona virus (229E) | Cultivate stoste | ????0 | - |
2, the sensitivity check and analysis of antibody sandwich ELISA method test kit detection
With the SARS-CoV N recombinant protein of purifying as standard detection product (0,50,100,200,400,800 and 1600pg/ milliliter), with irrelevant albumen as negative control, detect respectively with above-mentioned double-antibody sandwich elisa method, the drawing standard curve is shown in Fig. 4 result in the accompanying drawing.This test kit sensitivity can reach the 10-50pg/ milliliter.By the OD value of test sample, the content of calculation sample from typical curve.
The clinical application research of embodiment 12:SARS-CoV monoclonal antibody
Detect SARS-CoV antigen in patient's SARS lung tissue:
Use the SARS-CoV monoclonal antibody, detect the antigenic expression of SARS-CoV in patient's SARS lung tissue by Immunohistochemical Staining.According to a conventional method tissue slice behind the lung tissue paraffin embedding is carried out Immunohistochemical Staining, be summarized as follows: section placed 58 ℃ of baking boxs roasting 1 hour, immerse in the dimethylbenzene to dewax, and after the conventional aquation, 3%H
2O
2 Room temperature effect 15 minutes is with the sealing endogenous peroxidase activity.Use cold 1 * PBS to develop a film then, 3 times, each 5 minutes.Add SARS-CoV monoclonal antibody ascites 1: 1000 in wet box 4 ℃ spend the night.Develop a film 3 times with cold PBS.The mark sheep anti-mouse igg of horseradish peroxidase-labeled was hatched 1 hour for 37 ℃, after fully developing a film, and the DAB colour developing, Hematorylin is redyed, after the hydrochloride alcohol differentiation, the gradient dehydration, transparent, the neutral gum sealing.The microscopically microscopy.Shown in Fig. 6,7, the result shows that monoclonal antibody of the present invention detects the SARS-CoV antigen in the alveolar epithelial cells in patient's SARS lung tissue in the accompanying drawing.Illustrate that the SARS-CoV monoclonal antibody has very high specificity and susceptibility.
Double-antibody sandwich elisa method test kit detects the SARS-CoV antigen of cell cultures
Set up the double-antibody sandwich euzymelinked immunosorbent assay (ELISA) by the anti-SARS-CoV monoclonal antibody that the invention provides high specific and detect SARS-CoV antigen, antibody 1E8A11 with embodiment 11 descriptions, 8E1A17 and 10E4A4 are as trapping antibody, on little 96 orifice plates of polystyrene, the monoclonal antibody of purifying made solid and with bovine serum albumin (BSA, Sigma) the unnecessary site of sealing, the culture supernatant of the Vero cell that infects with the SARS-CoV of deactivation is originated as SARS-CoV antigen, 37 ℃ hatch washing after, add horseradish peroxidase-labeled 14A3A3A19 monoclonal antibody, once more 37 ℃ hatch washing after, add the colour developing of TMB enzyme substrates liquid lucifuge, add H
2SO
2Termination reaction is surveyed the 450nm absorption value.Use this method, the cells and supernatant that 3 strain SARS-CoV from different areas are infected all is positive.And with the cell culture no cross reaction that does not infect SARS-CoV.Fig. 5 in the accompanying drawing provides analytical results.
Double-antibody sandwich elisa method test kit detects the SARS-CoV antigen among the patients serum.
The method that embodiment 11 sets up detects the SARS-CoV antigen among the SARS patients serum, provide 165 parts of patient's SARS acute phases and convalescent phase serum sample to detect to the CDC of Guangdong Province, detecting sars coronavirus antigen positive rate is 40%, and it is 93.3% (14/15) that sars coronavirus antigen in the blood in 10 days acute phases of 15 routine laboratory diagnosiss (decubation IgG 4 multiplications are high) is detected positive rate.Providing in the blood of 20 parts of patients SARS in 5 days acute phases sars coronavirus antigen to detect positive rate to Beijing CDC is 90% (18/20).The specificity that 2000 parts of control samples are detected is 99%.The test kit that the present invention of detected result proof sets up is in early stage first day serum of morbidity, measure SARS-CoV antigen, and find that by the detection method that the present invention sets up the antigen in 6-10 days serum of SARS morbidity peaks, begin to descend shown in Figure 8 as in the accompanying drawing after 10 days of morbidity.This discovery proof one group of monoclonal antibody of the present invention and the pathogenesis research that not only can be used for the early diagnosis of SARS but also be used for SARS with the detection method of this group monoclonal antibody foundation have very important scientific value.
Recited above with 1E8A11,8E1A17 and 10E4A4 hybrid packet by solid phase surface, the double-antibody sandwich euzymelinked immunosorbent assay (ELISA) of being formed as the detection antibody of liquid phase with horseradish peroxidase-labeled 14A3A3A19 antibody detects SARS-CoV antigen, can be used for detecting human or animal's serum or the SARS-CoV antigen in plasma sample sputum, collutory, nose swab, throat swab, urine and the ight soil, be used for the early diagnosis that SARS-CoV infects, reach early discovery, the early stage isolation.Avoid the purpose relayed.
With adopting the direct immunofluorescence method to detect in the lung cast-off cells that infected by SARS-CoV and the virus antigen in the living tissue behind the labeling of monoclonal antibodies fluorescence of the present invention, or with embodiment 7 described indirect immunofluorescence promptly use in monoclonal antibody of the present invention and the cell or tissue SARS-CoV antigen combine after, combine with monoclonal antibody in being combined in cell or tissue with the fluorescent mark goat anti-mouse igg antibody again, equally also can be used for the research of SARS-CoV diagnosis of infection.
Claims (15)
1, a kind of have atopic monoclonal antibody to the sars coronavirus nucleocapsid protein.
2, claim 1 described monoclonal antibody is characterized in that, described sars coronavirus nucleocapsid protein antigen molecular is 46 kilodaltons.
3, the described monoclonal antibody of claim 1, it is characterized in that number comprising hybridoma 1E8A11:CCTCC-C200401 by having to preserve, hybridoma 8E1A17:CCTCC-C200402, hybridoma 10E4A4:CCTCC-C200403, the clone secretion of hybridoma 14A3A3A19:CCTCC-C200404 produces.
4, claim 1 described monoclonal antibody is characterized in that described monoclonal antibody belongs to immunoglobulin class IgG1 or IgG2b.
5, the described monoclonal antibody of claim 1, but it is characterized in that immunoblotting shows that the nucleocapsid protein molecular weight is that 46 kilodaltons react in specificity and the sars coronavirus split product.
6, the described monoclonal antibody of claim 1, but it is characterized in that immunoblotting shows that the nucleocapsid protein molecular weight of specificity and gene recombination is that 46 kilodaltons react.
7, the described monoclonal antibody of claim 1, it is characterized in that can be in fluoroimmunoassay nucleocapsid protein in the African green monkey kidney cell of the infected sars coronavirus of specific recognition.
8, the described monoclonal antibody of claim 1, it is characterized in that can be in fluoroimmunoassay sars coronavirus nucleocapsid protein antigen in specific recognition patient's SARS the cell or tissue.
9, the described monoclonal antibody of claim 1 is characterized in that can being used for discerning patient's SARS cell or tissue sars coronavirus nucleocapsid protein antigen in the immunohistochemical analysis method.
10, claim 1 described monoclonal antibody is characterized in that people's coronavirus 229E and the nucleocapsid protein of OC43 are not had reactivity; And there is not cross reaction with other ani mal coronavirus and Respirovirus.
11, the described monoclonal antibody of claim 1, it is characterized in that the pairing of the antibody of the antibody that can catch and mark, make up as the monoclonal antibody of catching with 1E8A11,8E1A17 and 10E4A4, with the monoclonal antibody that 14A3A3A19 antibody serves as a mark, it is right to form a kind of antibody in the immunoenzyme test.
12, has the hybridoma cell strain that preserving number is CCTCC-C200401, CCTCC-C200402, CCTCC-C200403 and CCTCC-C200404.
13, a kind of sars coronavirus nucleocapsid protein antigen detecting agent is characterized in that comprising any one described monoclonal antibody of claim 1～11.
14, a kind of sars coronavirus nucleocapsid protein antigen detecting agent box is characterized in that comprising the described reagent of claim 13.
15, the described monoclonal antibody of a kind of claim 1～11, it is characterized in that can be separately in conjunction with or be combined in application in the reagent of diagnosing SARS corona virus infection with polyclonal antibody.
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