CN1806175A

CN1806175A - Method of diagnosing SARS corona virus infection

Info

Publication number: CN1806175A
Application number: CN200480016339.5A
Authority: CN
Inventors: 陈燕如; 吴佩燕; 伯特拉姆·克林顿·菲尔丁; 申硕; 林成义; 洪万进; 阮一骏; 卫嘉玲
Original assignee: Agency for Science Technology and Research Singapore
Current assignee: Agency for Science Technology and Research Singapore
Priority date: 2003-06-10
Filing date: 2004-06-10
Publication date: 2006-07-19
Also published as: EP1639374A4; JP2006527382A; TW200512457A; AU2004245896A1; WO2004109289A1; EP1639374A1

Abstract

The present invention relates to methods of detecting the presence or absence of antibody to SARS coronavirus in a sample, based on the discovery that the S, M, E, N and U274 proteins of SARS coronavirus are antigenic. Such methods may be used to diagnose whether a patient has been infected by or exposed to SARS coronavirus. Antibodies directed against S, M, E, N and U274 proteins of SARS are also provided.

Description

The method of diagnosing SARS corona virus infection

The cross reference of pertinent literature

The application requires on June 10th, 2003 to submit, and application number is 60/477,059 U.S. Provisional Application No., and this application is merged in the application by reference.

Technical field

The present invention relates generally to the method for method, the especially diagnosing SARS corona virus infection of diagnosis virus infections.

Background technology

Emerging serious acute respiratory syndrome (SARS) is a kind of serious respiratory disorder with global meaning.Infected patient can show the symptom of atypical pneumonia, and early stage, the accurate differentiation of SARS and atypical pneumonia is the key of successfully controlling this hyperinfection disease.

The hyperinfection characteristic of SARS virus and high mortality can be disruptive and high cost in the world of globalization day by day.When SARS in 2003 beyond Chinese Guangdong area of origin inside the province during popular diffusion, The World Health Organization (WHO) has promptly recognized this point, WHO has issued global alarm in history the first time.Current outburst has influenced and has surpassed 8,098 people, and feeds through to during short 6 months above 29 countries and regions, and mortality ratio reaches 15%.Obviously, this disease is not the problem of a part, but has the profound influence to other department beyond the public health department.As if because in January, 2004, new case will reappear, identify rapidly and the urgency of isolating infected patient still exists.

In many countries, touch the risk that may be exposed to the virus that causes SARS in order to reduce, the people who surpasses 38 ℃ to those people that travelled in the country that is infected by SARS, with directly contacted people of SARS patient and body temperature has implemented strict Isolation Order.The initial stage of infecting to this disease carry out early diagnosis can avoid unnecessary isolation, reduce the relevant personnel stress, and help the doctor to determine suitable medical act and/or treatment.Therefore, under the prerequisite that possesses determinacy and correctness, it is vital identifying SARS patient as early as possible.

A kind of new coronavirus is accredited as the cause of disease of SARS (1-4).Coronavirus is an enveloped virus, and it comprises a 27.6-36kb long strand, just rna gene group.To the nucleotide sequence of new sars coronavirus (SARS CoV) the analysis showed that the nearly 30kb of this viral genome long (5,6), and comprised 14 potential open reading frame (ORF) (5).But, with regard to how detecting, diagnose and treat this disease, also have things to be familiar with more.Because do not have medicine or vaccine to can be used for this disease, infected patient's Rapid identification and isolation are still of crucial importance for the control of disease.

At first, when SARS was identified first, initial disease control depended on tourism and contact history, and the symptom that is showed.Afterwards, along with to the genomic evaluation of SARS-CoV, severally be devised based on the diagnostic detection of the detection of viral RNA sequence and be utilizable now by PCR.Based on these methods, WHO has revised its standard of identifying for case.

But the scheme of the existing PCR of being used for does not break away from their limitation.Although highly sensitive, such detection has intrinsic problem: all uncertain which kind of sample from the patient of global scientist and clinician (respiratory tract sample, saliva, ight soil, blood or conjunctiva liquid) provides the preparation of best replicatable rna; The RNA extracting method is remarkable, if do badly, may be prepared into the RNA preparation that can not be used for viral RNA is converted into the reverse transcription step of DNA; If must carry out replication experiment to primer with several, the overall process of then extraction, reverse transcription and PCR is consuming time.In addition, amplification method may be accompanied by false positive results, is detected as the SARS positive by PCR method as observing some in Canada in August, 2003 at first by the patient that other human corona virus infects.The pollution of PCR Lab is a problem all the time, and this can cause unnecessary isolation for SARS.

Show in the result that Singapore obtained, when using different clinical samples, the positive predictive value (PPVs) that is provided by the PCR diagnostic method changes (A.E.Ling between 56.7% to 83.8%, " SARS-the Singapore laboratory experience ", presented at theWHO Conference on SARS Research, Singapore, Republic of Singapore, 19 June 2003).

In a nearest report (7), the breadboard PCR of SARS reverse transcription (RT-PCR) scheme of two WHO to be assessed, gained finds to confirm that RT-PCR has similar shortcoming.Therefore, when obtaining negative findings, existing PCR method can not be got rid of the existence of SARS virus, can not get rid of the possibility of the recall rate because laboratory pollution leads to errors.

Comprise indirect immunofluorescence assay (IFA), the typical organization of viral isolates is cultivated and the diagnostic method of the electron microscopy of diagnostic cell culture is consuming time often or require very high technically.Therefore alternative method is very crucial for controlling this disease effectively.

Although with new coronavirus, the cause of disease that SARS-CoV is accredited as SARS makes might develop different detection methods, still will preferentially be provided for the instrument of laboratory diagnosis as WHO advises.Up to now, the standardization that still is not used in diagnosis SARS detects, no matter such detection be based on antigen also be based on antibody.To provide the useful information that overcomes this shortcoming with the standardization plate or by the test detection kit newly developed that uses a large amount of clinical samples.

Summary of the invention

On the one hand, the invention provides a kind of method that is used for detecting the antibody that whether has sars coronavirus (CoV), this method is included in is enough to make antibody capable and antigen to form under the condition of compound, is enough to make that antibody capable and antigen form the step of the time of compound for one section with the sample contact SARS CoV antigen that comprises antibody; Wherein said antigen contains the protein of a kind of S of being selected from, M, E, N, U274 and immunogenic fragments thereof; And wherein said antibody combines this sample of indication and contains SARS CoV is had specific antibody with specificity between described protein.This method can be used to detect someone and whether be infected or be exposed to SARS CoV by SARS CoV, the sample that wherein contains antibody is from detected people, and wherein said antibody combines this people of indication with specificity between described protein and infected or be exposed to SARS CoV by SARS CoV.

The present invention also provides commercial kit.On the one hand, the invention provides a kind of commercial kit that whether has the antibody of sars coronavirus (CoV) at a certain sample that is used for detecting, it comprises a kind of SARS CoV antigen, and this SARS CoV antigen comprises the protein of a kind of S of being selected from, M, E, N, U274 and immunogenic fragments thereof; And detect from the antibody of sample and the compound instrument between described antigen.

On the other hand, the invention provides a kind of whether someone has been infected or be exposed to SARS CoV by sars coronavirus (CoV) commercial kit that is used to detect, it comprises a kind of SARS CoV antigen, and this SARS CoV antigen comprises the protein of a kind of S of being selected from, M, E, N, U274 and immunogenic fragments thereof; With detect described antigen and compound instrument at the antibody of this antigen, wherein said sample is detected people's the sample that contains antibody.

On the other hand, the invention provides a kind of isolated antibody, it is at antigenicity SARSCoV albumen, and wherein said antigen SARS CoV albumen is the protein of S, M, E, N, U274 or its immunogenic fragments.

Antibody of the present invention is used to detect S, M, E, N or the U274 albumen that whether has SARS CoV in the sample.Therefore, on the other hand, the invention provides the method whether detection exists S, M, E, N or U274 SARS CoV albumen, this method comprises: be enough to make antibody capable and antigen form under the condition of compound, be enough to make that with the sample contact antibody that contains antigen antibody capable and antigen form the time of compound for one section, described antibody is at a kind of protein that is selected from S, M, E, N and U274; Wherein said antibody combines this sample of indication and contains the antigen that comprises S, M, E, N, U274 or its immunogenic fragments with specificity between described antigen.

By reading following description and accompanying drawing to specific implementations of the present invention, others of the present invention and feature are conspicuous to those skilled in the art.

Description of drawings

In the accompanying drawings, only by way of example embodiments of the present invention are described, wherein:

Fig. 1 is the synoptic diagram of forming and expressing corresponding to the structure of the ORF of structural proteins S, M, N and E and peculiar albumen (UX), and wherein X represents the coded amino acid whose number by each ORF; The ORF 1-14 that is marked is accordingly pointed out;

Fig. 2 is illustrated to be the Western engram analysis, shows the antibody that has anti-various SARS-CoV virus proteins in the patients serum;

Fig. 3 is illustrated to be to analyze and GST-U274, GST-U122, GST-N and the GST albumen of the bacterial expression that dyes with Coomassie brilliant blue R-250 (Bio-Rad) by SDS-PAGE;

Fig. 4 is graphic to be with the antibody in the antibody in anti-GST antibody, the control serum or 6 convalescent serums, the virus protein of expressing by Western engram analysis bacterial detection;

Fig. 5 is graphic to be that to be used for patient's the serum in self-infection early stage and late period anti-as one, and anti-as two with anti-human IgG, by Western trace detection N and U274;

Fig. 6 is anti-as two with anti-human IgG, anti-people IgA or anti-people IgM, to the result of the Western engram analysis of some samples among Fig. 5;

Fig. 7 is graphic to be the Chinese hamster ovary celI that utilizes stably express S albumen, by immunofluorescence method to the anti-S detection of antibodies among the patients serum;

Fig. 8 uses from infected patient's sample to analyze back (left side) or use from the collator's of health sample and analyze back (right side), separation vessel (separator) (slide) is in the photo of the fast immune chromatographic pick-up unit example that assembles of " removing " (analyzing the back) position, from having shown the chromatography line of contrast, Gst-N and Gst-U274 from top to bottom respectively;

Fig. 9 detects with ELISA, resulting OD value scatter diagram from the SARS patient of the IgG antibody of detected its SARS CoV and normal healthy controls person's serum;

Figure 10 is the serial dilution thing of expression with 7 SARS patient's samples, detects the Line Chart of the titration curve of gained through ELISA;

Figure 11 is graphic when being dilution when the reactive terminal point that relatively obtains 7 SARS patient's samples, the relation between ELISA and fast immune chromatographic detect;

Figure 12 is graphic to be the distribution that ELISA detection and fast immune chromatographic detect the number percent verification and measurement ratio aspect sample immunofluorescence titre;

Figure 13 is graphic to be the Western western blot figure of the immunoreactive protein of SARS-CoV;

Figure 14 is Western trace figure, expression be with anti-specific recombinant protein (1:SARS-positive control; 2: mouse anti S protein antiserum; 3: mouse anti nucleocapsid protein antiserum; 4: mouse anti stromatin antiserum; 5: the location of the epidemic disease proteins C reactive of the SARS-CoV that identifies of the antibody that the is produced anti-envelope protein antiserum of rabbit); With

That Figure 15 represents is serum sample (A:SARS patient; B: normal healthy controls person; C: non-SARS fever patient contrast; D: non-SARS inflammatory disease contrast; The typical immunoreactivity figure of SARS Western Western blotting E: by the false positive that ELISA identified).

Embodiment

The sequence of SARS-CoV has disclosed the ORF of 4 structural proteins, i.e. S albumen (S), memebrane protein (M), envelope protein (E) and nucleocapsid protein (N), and they are (5,6,10,11) of guarding in all coronavirus.The basic function of S albumen is mediation receptors bind and viral internalization, is a major antigen of this virus.The basic function of M and E albumen is to be responsible for the virion assembling, the N protein combination on the viral genome to form nucleocapsid.

Except these 4 kinds of total albumen, infer also from the SARS-CoV sequence several peculiar ORF that the virus protein of they and other coronavirus does not have significant sequence homology.Whether these ORF are expressed in the protein that virus replication works in the cycle is still waiting to determine.

The present invention relates to such discovery, S, M, E and the N structural proteins and a kind of predicted albumen (being referred to as U274 in this application) with unknown function of 274 amino acid longs that are SARS-CoV have antigenicity, and can be used to detect whether have anti-SARS CoV antibody in patient's body, be infected or be exposed to SARS CoV by SARS CoV to indicate this patient.

Therefore, one aspect of the present invention has provided a kind of method of using antigenicity SARS CoV Protein S, E, M, N and U274K to diagnose the SARS CoV in patient's body to infect.The one or more of this antigenic protein are expressed, and use the biological sample contact of infecting the patient who detects from pending SARS CoV.If this patient has been infected or has been exposed to SARS CoV by SARS CoV and produced anti-this viral antibody, the antibody that is present in the anti-specific SARS CoV antigenic protein that is used to diagnose in this sample will be attached on this albumen.Antibody that anti-this patient produces and the two anti-antibody of puting together with detection molecules that can be used to detect this patient's generation.

Used term SARS CoV " antigenic protein " refers to one or more in S, M, E, N or the U274 albumen of SARS CoV among the application." its immunogenic fragments " of term SARS CoV or " its antigenicity fragment " refer to SARS CoV S, M, E, N or U274 albumen have immunogenicity or an antigenic part, mean this protein fragments and will comprise one or more epi-positions, therefore can in patient's body, cause immune response.

In the practice of field of immunology, it is acceptable that the fragment of albumen or mutant are used as immunogene, because bring out the required just little immunogenicity zone (for example 8-10 amino acid) of this albumen of immune response.It is effectively effective antigen of anti-their pathogen separately, for example 11 of mouse mammary adenoma virus residue peptide (Casey﹠amp that the synthetic peptide that exposes the various weak points of antigen corresponding to the surface of pathogen has been shown; Davidson, Nucl.Acid Res. (1977) 4:1539), peptide (the Snijders et al. of 16 residues of Semliki Forest virus (Semliki forest virus), 1991.J.Gen.Virol.72:557-565), with two overlapping peptides (Langeveld et al. from 15 residues of canine parvovirus, Vaccine 12 (15): 1473-1480,1994).

Therefore, behind the instructions of reading the application, according to instruction of the present invention, it is evident that concerning those skilled in the art: the partial sequence of any among S, M, E, N or the U274 is the inner portion of full-length proteins.Such polypeptide fragment preferably is at least 12 amino acid longs.Advantageously, such polypeptide fragment is at least 15 amino acid longs, preferred at least 20,25,30,35,40,45,50 amino acid longs, more preferably at least 55,60,, 65,70,75 amino acid longs, most preferably at least 80,85,90,95,100 amino acid longs.

The used term of the application " homology " or " homolog " refer to a kind of protein sequence, its amino acid sequence and S, M, E, N or U274, or the amino acid sequence of its fragment is basic identical." homolog " can with S, M, E, N or U274, or its fragment has the homology at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 87%, 90%, 93%, 96% and 99%.Use sequence analysis software, Sequence Analysis SoftwarePackage of the Genetics Computer Group for example, University of WisconsinBiotechnology Center, 1710 University Avenue, Madison, WI 53705, and " homolog " measured.Amino acid sequence is carried out than making the homogeneity maximization.Can will manually be incorporated in this sequence to obtain suitable comparison at interval.In case set up best comparison, the degree of homology is by identical position can not ratio be established with respect to total positional number of amino acid in the record two sequences.

The partial sequence of any among spendable coding S, M, E, N and the U274, or has a sequence homology with it, or have the polynucleotide of 30-600 nucleotide of partial sequence homology with it, by the parameter summarized above using and use the primer with the upstream and downstream sequence pairing of 5 of this fragment ' and 3 ' terminal to extract through pcr amplification.Be used for the template polynucleotide of such amplification or the total length polynucleotide of coding total length S, M, E, N or U274, or be contained in the nucleotide in a polynucleotide potpourri such as DNA or RNA library.A kind of alternative method as the extraction unit sub-sequence, screen hybridization in the above under the described condition and use the formula that calculates Tm, the multi-disc section of 30-600 nucleotide wherein to be extracted, the deduction (600/ in base-pair polynucleotide size) that passes through that calculates is proofreaied and correct; Stringent condition is defined as hybridization temperature and is lower than Tm5-10 ℃.Wait to obtain the oligonucleotides less than the 20-30 base, the formula that calculates Tm is as follows: Tm=4 * (G+C)+2 (A+T).For example, the Tm of the fragment of 18 of 50%G+C nucleotide is approximately 54 ℃.The short sequence of the fragment of any or its homologous sequence directly obtains by chemosynthesis among S, M, E, N and the U274.

Useful polypeptide derivative, for example polypeptide fragment uses the computer-aided analysis of amino acid sequence to design.This will identify that possible surface exposes, antigenicity zone (Hugheset al, 1992.Infect.Immun.60 (9): 3497).Based on using SEQSEE program (Wishart DS, et al. " SEQSEE:a comprehensive program suite forprotein sequence analysis. " Comput Appl Biosci.1994 Apr; 10 (2): flexibility that 121-32) obtains and hydrophobic tendency product can be used to disclose potential B-and T-cell epitope to the analysis that the amino acid sequence that is contained in any of S, M, E, N or U274 carries out, and this epi-position can be used as selects the useful immunogenic fragments and the basis of mutant.This analysis will reasonably be combined by the exterior surface feature of antibody recognition probably.The possible t cell epitope that is used for HLA-A0201 MHC subclassification can be by algorithm and revealed, this has imitated a kind of method that NIH developed (Parker KC, et al. " Peptidebinding to MHC class I molecules:implications for antigenic peptideprediction. " Immunol Res 1995; 14 (1): 34-57)).

Induce the epi-position of protectiveness T cytophylaxis immune response all to exist in the total length of this polypeptide.But some epi-positions may be sheltered by the secondary of this polypeptide and tertiary structure.In order to disclose these masked epi-positions, set up big inside disappearance, this has removed a large amount of initial protein structures and masked epi-position has been come out.Such inside disappearance has produced extra advantage removing the immune determinant zone that highly makes a variation in each bacterial strain sometimes.

Have the coded polypeptide fragment of big inside disappearance and the polynucleotide of polypeptide and make up (Ausubel et al., Current Protocols in Molecular Biology, JohnWiley﹠amp by standard method; Sons Inc., 1994).The dna molecular Restriction Enzyme that such method comprises Standard PC R, inverse PCR, be cloned into is handled or by the disclosed method of Kunkel et al (Kunkel et al.Proc.Natl.Acad.Sci.USA (1985) 82:448).Be used for the composition of these methods and operation instruction thereof can from various commercial source for example Stratagene easily obtain.

The antigenic protein of total length can be used in the diagnostic method.But, preferably using the antigenicity fragment of this corresponding SARS CoV antigenic protein, this antigenic protein is not high conservative in each coronavirus, so that the antibody generation cross reaction chance of anti-other coronavirus that produces with the patient reduces to minimum.In addition, those skilled in the art can understand, it is the corresponding SARS CoV antigenic protein part of membrane spaning domain that disappearance is speculated as, or only uses a kind of soluble antigenicity fragment of this corresponding proteins matter to have advantage for the easy degree of expressing and the processing of this albumen.

Enjoyably, do not resemble some wherein coronavirus of M protein content maximum, N is the albumen of the middle content maximum of SARS-CoV (6) seemingly.This result shows that N has produced strong immune response.

Remove theoretical constraint, might be released to the blood circulation in some stage of infecting, or might can be presented by the antigen presenting cell that the cytotoxin that is used for infected cell causes death by N from SARS CoV or infected patient's cell.In addition, might can help to protect the patient to exempt from the humoral immunoresponse(HI) of SARS by N.

For N albumen, when the sequence that is committed to 18 SARS separators in the GenBank database was compared, the amino acid sequence that wherein has only two sequences to demonstrate with the Singapore's separator (SIN2774) that is used for the clone that following examples (13) put down in writing had an amino acid whose difference.Therefore, this N albumen does not demonstrate and has passed through quick sudden change, and this is its another advantage that is used for method of the present invention.

The existence of the antibody of anti-N albumen can early detect in patient's body after infecting 2 days, also can infect the back longer time and detect, and for example infects back 16 days.Immune response bring out in early days and extremely strong immune response makes N albumen be particularly useful in diagnosis SARS CoV infects.

Therefore, each preferred embodiment in, being used to detect the protein that whether has anti-SARS CoV antibody in patient's body is N albumen or its antigenicity fragment.N albumen can be the N albumen of total length; It also can be N (Δ 111-118), and this refers to 111-118 amino acids disappearance N albumen.These amino acid are corresponding to sequence FYYLGTGP, the sequence of a high conservative of finding in the homologue of coronavirus.Also can use N protein immunization originality fragment, the fragment that for example comprises or form by amino acid 69-422,120-422 or 121-422.

S albumen has formed the petal spike of finding on the surface of coronavirus (10,11).This albumen still is high immunogenicity.S albumen is a kind of large protein, and it has numerous stride diaphragm area and be high glycosylation.This S albumen is contained the hypervariable region by supposition, because these zones have appeared in the S albumen of other coronavirus (10,11).Preferred use does not contain the extracellular region of the S albumen of hypervariable region, to guarantee processing ease and to improve the possibility that the true positives of SARS CoV infected patient is diagnosed.

Therefore, in some embodiments, can use the S albumen of total length, or for example use the fragment of the S albumen comprise or to be formed by amino acid 460-480.

Examined up to now Most patients all has the antibody of the C end of anti-U274 (U274 is corresponding to ORF3, and as the note in list of references 5, X1 is as the note in list of references 6).The immunoreactivity of U274 shows that this virus protein new, uniqueness is expressed in this virus, and might be a kind of protein that has participated in the biological evolution of SARS CoV.In addition, found a potential transcription regulating nucleotide sequence in the upstream of U274 coded sequence, shown that it is first ORF (5,6) of the main subgenomic RNA of cell that infects of SARS CoV.Any albumen in U274 and the database is not enjoyed significant homology, but it demonstrates to M albumen and have similar topological structure, has 3 and strides film district and a big C-end structure territory, inside.Because the fragment of expression and purifying protein is more easy, when using U274 in the method for the invention, preferred use does not comprise the fragment of striding the film district of U274.

In addition, U274 tends to not show some low-level sequence variations in the viral isolates, to 5 separators with U274 sequence studies show that out an amino acid whose replacement (comparing) with SIN2774,15 separator with U274 sequence demonstrates two amino acid whose replacements (14).

Therefore, in some embodiments, employed antigenicity SARS CoV albumen can be total length U274, or the antigenicity fragment of the U274 that comprises or be made up of amino acid/11 34-274.

Examined up to now patient is not all to have the M of anti-SARS CoV and the antibody of E albumen.Therefore, so not useful in diagnostic method of the present invention although these albumen have antigenicity, because they seem and can not cause immune response constantly in infected individual body.In each embodiment, employed antigenicity SARS CoV albumen can be the M albumen of total length, or an antigenicity fragment of the M albumen that comprises or be made up of amino acid 98-121.In other embodiments, employed antigenicity SARS CoV albumen can be the E albumen of total length, or an antigenicity fragment of the E albumen that comprises or be made up of amino acid 38-76.

In the method for the invention, antigenic protein is expressed first, and can express with method as known in the art." expression " a kind of albumen is meant the template by RNA, normally synthetic a kind of albumen of the translation of mRNA template or polypeptide, this mRNA encode this albumen or polypeptide; And can comprise a transcription step, in this step, the RNA template transcribes from dna profiling by RNA polymerase.This albumen can be expressed in any expression system, cell for example, or in not celliferous system.

For example, this albumen can be in prokaryotic expression system, for example in bacterium such as Escherichia coli; Or at eukaryotic system, for example comprise in Saccharofnyces cerevisiae and the methanol yeast at yeast, comprise in COS1, COS7, CHO, NIH3T3 or the JEG3 cell at mammalian cell, comprise in Spodoptera frugEperda (SF9) cell at insect cell, or in vegetable cell.

This albumen can be expressed in not celliferous system; such system must comprise the required reagent of this protein expression of realization; comprise that ribosomes, tRNAs, amino acid comprise aminoacyl tRNAs, RNA template; and can further include dna profiling, RNA polymerase, RNA (ribonucleic acid) and any essential accessory factor buffering agent and the necessary salt of enzymatic activity, and can also comprise cell pyrolysis liquid.

It will be understood to those of skill in the art that and how in a kind of suitable expression system, to express this required protein or protein fragments.It is soluble being supposed to through the protein of posttranslational modification, the preferred bacterium expression system.But, for large protein, through the protein of posttranslational modification or need the protein of mRNA montage, preferred eukaryotic system, for example mammlian system.

Typically, use will the encode gene of this antigenic protein or protein fragments of standard technique as known in the art will be cloned in the expression vector, this expression vector and selected be compatible in order to the expression system of expressing this antigenic protein.This expression vector contains necessary promoter of expression and the hereditary signal in the selecteed expression system, this expression vector can contain coding one and operably is connected to the gene of the protein on the promoter compatible with this specific expression system, and can plasmid.For example, this cell can be the E.Coli cell, this expression vector can contain the gene that a coding is operably connected the interested antigenicity SARS CoV albumen on all essential regulating and controlling sequences, makes that this gene is transcribed, and this RNA is translated by the E.Coli cell mechanism.The expression of gene of coding protein of interest matter can be started by a kind of inducible promoter, make intracellular expression can as desiredly be controlled to make and express maximization, for example by making the exponential phase of protein expression and cell culture reach synchronization.

For the ease of purifying and use, this albumen can be expressed as fusion.Fusion is protein or the polypeptide that contains the antigenicity SARS CoV albumen of the N-that is fused to second peptide species or C-end.A kind of simple mode that obtains such fusion is to merge the i.e. translation of hybrid gene in the frame by polynucleotide sequence.The hybrid gene of coding fused polypeptide is inserted in the expression vector in order to transform or transfection host cell.Perhaps, the encode polynucleotide sequence of this polypeptide or polypeptide derivative is inserted in the expression vector of the polynucleotide that wherein had the second kind of peptide of encoding.This second kind of peptide can be that a kind of affinity type peptide on part or the functional groups or protein sequence of being attached to is to help purifying.For example, this second kind of peptide can be in conjunction with Ni ^{+ 2}Six histidine sequences of ion, or in conjunction with the glutathione-S-transferase of glutathione.

In case expressed, this antigenic protein can adopt the purification technique of standard to carry out purifying, and affinity column chromatography for example combines the chromatographic column purifying gst fusion protein of glutathione as use.Adopt known affinity chromatography, the fusion that contains this antigenic protein can be purified to high level, it is higher that this makes that diagnostic method is used for the sensitivity and the specificity of thick employing virus cracking liquid of serological analysis with respect to use, because the existence of the antibody of the cellular component in the antiviral lysate may cause false positive.

The antigenic protein of being expressed is used to detect the antibody whether patient has produced anti-SARS CoV albumen by contacting with SARSCoV albumen from patient's sample.By contact, can discern this antigenicity SARS CoV albumen, the antibody by the patient produced in patient's sample will be attached on this antigenic protein, form the anti-compound of antigen-one.

" specificity combines " between antibody and antigen, or antibody is meant this antibody recognition to " specificity " of antigen and optionally is attached to ability on the defined epitope that is contained in this antigen.Such combination by this antibody antigen binding site and the complementarity between the epi-position of this antigen measure.Complementarity is meant the mutual pairing of the electric charge in the binding site of the spatial disposition of chemical part and this antibody and antigen.This sample is any cell surface binding antibody that produced by the patient or sample of circulating antibody of comprising.This sample can be blood, blood plasma or serum.This sample is taken from arbitrary patient who is infected by SARS under a cloud, and can be between 2-60 after the infection days, take a sample between the 16-60 days or between 2-11 days.Term " DAI " is meant that the patient touches SARS CoV or the length of the fate after time of being infected by SARS CoV.

For example among the ELISA (enzyme linked immunosorbent assay (ELISA)), the contact of carrying out antigenicity SARS CoV albumen is to detect the antibody of S, M, E, N or U274 in immunoassays.Before with the contact of patient's sample, this antigenic protein can be immobilized on the solid support.Perhaps, this antigenic protein can be expressed in cell or at cell surface.Immunoassay is that those skilled in the art are extensively known, comprises ELISA, Western Western blotting, immunofluorescence assay and immunochromatographic measurement.

In the detectability that remains on immunoassays, have necessity patient's sample is diluted.Can carry out serial dilution to determine the optimum dilution degree of sample in a certain specific detecting pattern to this sample.For example, can sample to account for cumulative volume be that about 1: 10, about 1: 20, about 1: 40, about 1: 80, about 1: 160, about 1: 320, about 1: 640 ratio are diluted in a kind of suitable damping fluid to patient's sample.

The used term of the application " immunoassays " is meant a kind of analytical approach, its used a certain antibodies to a certain specific antigen ability as the instrument that determines whether to exist this antigen or antibody.The antibody capture immunoassays are so a kind of analyses, and a kind of antigen that is used to detect the antibody of the special pathogen in the biological sample of detected object promptly is provided.Usually, this antigen is immobilized on the solid support and can be in conjunction with the antibody in this biological sample.This antibody is provided by this biological sample.In the version that antibody capture is measured, this antigen mixes with the antibody of this biological sample, and formed antigen-antibody complex is by two anti-catching of antigen in anti-this antigen-antibody complex that is immobilized on the solid support and/or antibody.Perhaps, in solution, measure the formation of antigen-antibody complex.

The scope of expection immunoassays form is included in this definition, includes but not limited to that direct immunization is measured, immunoassays and " sandwich " immunoassays indirectly.A kind of particularly preferred form is sandwich enzyme-linked immunosorbent assay (ELISA).But this does not mean that the present invention is limited to this form.Expect other form to comprise radiommunoassay (RIA), immunofluorescence assay (IFA) and other mensuration form, include but not limited to that the distortion of ELISA method will be useful in the method for the invention.Therefore, other antigen-antibody reaction form also can be used among the present invention, include but not limited to " flocculation " (promptly in the colloidal suspensions that generates on the basis that antigen-antibody complex forms), " aggegation " (cell or other material is intensive after promptly being exposed to antibody), " microparticle agglutination " (promptly under the condition that antibody exists, intensive by antigen coated particulate; Or under the condition that antigen exists, intensive by the particulate of antibody sandwich), " complement in conjunction with " (promptly in antibody-antigen-reactive method, using complement) and other method that in serology, immunology, immunocytochemistry, histochemistry and association area, is commonly used.

The detection of antibody-antigenic compound can be undertaken by several method.Movably antigen can be prepared to having mark biological example element, enzyme, fluorescence labeling or radioactivity, and uses this mark directly to detect.Perhaps, can add " two is anti-" or " the report antibody " that are labeled that identification one resists, form the compound of forming by Ag-Ab-antibody.In addition, if desired, can add the antibody that suitable report reagent is labeled with detection subsequently.If desired, can add any amount of other antibody.These antibody also can carry out mark with mark, and described mark includes but not limited to enzyme, fluorescence labeling, radioactivity or heavy metal complex.Antigen or antibody (one anti-or two anti-) can be immobilized on the solid support, but the component that is labeled can not be immobilized, because can not be with this detectable signal as the index of measuring.

Used term among the application " report reagent " is meant a kind of compound that can detect the existence that is incorporated into the antibody on the antigen.For example, report reagent can be a kind of calorimetric material, and it is attached on a kind of zymolyte.In case the color generation that antibody and antigen combination, this enzyme are worked to its substrate and caused.Other report reagent includes but not limited to the compound or the molecule of fluorescence and radioactivity.This definition also comprises uses biotin and based on the compound (for example including but not limited to compound neutravidin and streptavidin) of the avidin part as detection system.In an embodiment of the invention, the solid support of biotinylated antibody and avidin bag quilt is used among the present invention together.

Used term " solid support " is meant any solid material that can be adhered to by reagent such as antibody, antigen and other chemicals among the application.For example, in the ELISA method, micro titer plate well is equipped with solid support usually.Other example of solid support comprises slide, cover glass, pearl, particulate, Tissue Culture Flask and many other solids.

A kind of antibody kit or commercial kit that is used to detect S, M, E, N or U274, it generally includes the antigenicity SARS CoV albumen of the q.s that is enough to be used in once measuring; And be used to detect antigenicity SARS CoV albumen and from the instrument of formed compound between the antibody of patient's sample, as packaged immuno-chemical reagent.Typically, in the operation instructions of packaged immuno-chemical reagent also are included in.

Used term " packaged " can refer to use solid matrix or material among the application, reaches as glass, plastics, paper, fiber, paper tinsel and similarly antibody of the present invention can be remained on fixed constraints with interior solid matrix or material.Therefore, for example packing can be the vial that contains the pre-stage antigens of milligram quantities, or has operationally been adhered to the micro titer plate well of the pre-stage antigens of microgram amount.Perhaps, packing can comprise and is trapped within the perforated membrane or is encapsulated into antigen coated particulate in calibration tape or the test paper (dipstick) etc.Perhaps, this antigen can be by direct coated on film, calibration tape or the test paper etc. of contact sample liquid.Also there are many other possibilities, and can be easily discerned by those skilled in the art.

Operation instructions typically comprise the description reagent concentration, or at least one assay method parameter truly expressed of the relative quantity, reagent/cycle sample mixtures retention time, temperature, buffer condition etc. of reagent and sample to be mixed for example.

In preferred embodiment, kit of the present invention comprises that also a kind of mark maybe can represent the marking tools of the formation of the compound between antigenicity SARS CoV albumen and the antibody.

Used term " mark " or " labelled reagent " and the instrument (" marking tools ") that is used to detect antibody-antigenic compound are meant directly or participate in indirectly generating the molecule of detectable signal of the existence of indication compound among the application.Any mark or marking tools can be connected to by on the antibody molecule of expressed protein, peptide or a part of the present invention or be impregnated in wherein; Also can use separately; These atoms or molecule can be used alone or unite use with other reagent.These marks self are well-known in the clinical diagnosis chemistry.For example, this mark or marking tools can be a kind of enzymes, its can cutting reagent to generate coloured molecule, fluorescence molecule, immunological molecule, chemiluminescence molecule or heavy metal complex.As will be appreciated by a person skilled in the art, the exact method that detects by the signal that this mark or marking tools produced will depend on employed mark or marking tools and used specific immunoassay.

This mark or marking tools can be a kind of fluorescent labeling reagents, its chemical bond under the situation that does not make antibody or antigen sex change on antibody or the antigen to form fluorchrome (dyestuff), a kind of useful immunofluorescence tracer agent.Suitable fluorescent labeling reagent is for example isocyanate fluorescein (FIC), fluorescein isothiocynate (FITC), 5-dimethylamine-1-natpthalenesulfonyl chloride (DANSC), isothiocyanic acid tetramethyl rose-red (TRITC), Liz amine, rhodamine 8200 sulfonic acid chlorides (RB 200 SC) or the like of fluorchrome.

In preferred embodiment, this mark or marking tools are a kind of enzymes, for example horseradish peroxidase (HRP), glucose oxidase or similar enzyme.At main indication group is enzyme for example under the situation of HRP or glucose oxidase, needs extra reagent indicate receptor-ligand compound (immunoreactant) to form.The extra reagent of this HRP of being suitable for comprises hydrogen peroxide and a kind of oxidation dye precursors for example diaminobenzidine and N tetramethyl benzidine.A kind of extra reagent useful for glucose oxidase is 2,2, and-azine group-two-(3-ethyl-benzene thiazolidine-G-sulfonic acid) (ABTS).

The radioreaction element also is useful labelled reagent, also is illustrated in this application.A kind of typical radioactive label reagent is a kind of radioelement, and it produces the gamma-rays emission.Self launch gamma-ray element, for example ²⁴I, ¹²⁵I, ¹²⁸I, ¹³²I and ⁵¹On behalf of a class, Cr produce the radioelement indication group of gamma-rays radiation.Especially preferredly be ¹²⁵I.The useful marking tool of another group for example is ¹¹C, ¹⁸F, ¹⁵O and ¹³What N was such self launches the element of positron.The β emissive source for example ¹¹¹Indium or ³H is useful too.

The connection of mark, promptly the mark of peptide and albumen is well known in the art.For example, can carry out mark by containing radioisotopic amino acid by the monoclonal antibody that hybridoma produced, this amino acid is provided as a kind of component of culture medium.Put together or the especially practicality of matching technology that is coupled by the albumen that the functional group that is activated carries out.

Method of the present invention and kit also can comprise a kind of " specificity combinating reagent " of independent packaging, and it can optionally be attached to antibody of the present invention or antigen or comprise on certain isotopic compound, but itself are not antigen of the present invention or antibody.Typical bond is two anti-molecules, for example anti-people's antibody, complement protein or its fragment, S.aureus albumin A or the like.Preferably, when antibody and antigen existed with the part of compound, this specific bond combined with antibody or antigen.

In preferred embodiment, specificity combinating reagent is labeled.But when method of the present invention or kit comprised unlabelled specificity combinating reagent, this reagent typically was used as the reagent of amplification instrument or amplifying signal.In these embodiments, when described amplification instrument was incorporated on the compound, the specificity combinating reagent that is labeled can be specifically in conjunction with this amplification instrument.

Kit of the present invention can be used in " ELISA " form the amount with the antibody that detects anti-S, M, E, N or U274 in liquid sample or extract." ELISA " is meant enzyme linked immunosorbent assay (ELISA) as previously discussed, and it has been used and has been attached to a solid phase, and antibody on enzyme-antigen or the enzyme-antibody conjugates or antigen, to detect the amount of existing antigen in the sample.

In many embodiments, antigenicity SARS CoV albumen can be adhered to a formation one solid support on the solid matrix.Well known to a person skilled in the art other absorption mode that is applicable to albumen and peptide although also can use, reagent typically is fixed on the solid matrix by absorption from an aqueous medium.

Useful solid matrix is well known in the art.Such raw material is water miscible and comprises the crosslinked glucosan that closes that this glucosan can be available from Pharmacia Fine Chemicals (Piscataway, the product of trade mark SEPHADEX N.J.); Agarose; Diameter is the polyacrylamide of about 1 micron-5 millimeters polystyrene, Polyvinylchloride, glue connection, based on the thin plate of nitrocellulose or nylon for example thin slice, thin bar or flat plate; Test tube, flat board or for example by polystyrene or the prepared micro titer plate well of Polyvinylchloride.

The immunoreagent of the described any diagnostic system of the application can be dissolved in the solution, with liquid dispersant or be essentially dry powder for example the form of freeze-drying be provided.The instrument of wherein instigating is a kind of enzyme, and the substrate of this enzyme also is provided with the form of independent packaging.Solid support, microtiter plate for example described above and a kind of or many middle damping fluid also can be included in the form of independent packaging in the diagnostic system of the present invention.

Preferably, antigenicity SARS CoV is coated or be adsorbed on the surface of a matrix.Contact interested sample with antigenicity SARS CoV and may reside in any antibodies of the antigen in this sample to this antigen.With antigen/sample contact Anti-Human's antibody and this Anti-Human's antibodies to the antibody that is incorporated into antigenicity SARS CoV albumen.

Employed two anti-can in any and examined patient animal body not of the same race, generations.Therefore can use mouse anti human antibody, or this two anti-can be anti-people's antibody of rabbit or mountain goat anti-human antibody.Described anti-people's antibody can for example anti-human IgG, IgM or IgA antibody.

Described anti-people's antibody can carry out mark with mark.Described mark is any material object that can be puted together or be attached on this anti-people's antibody, and can detect by analytical technology.Described mark can be puted together contact described anti-people's antibody with antigen/sample before or be attached on this anti-people's antibody.

The detection of mark is whether described people's antibody is present in the indication in the sample.If detect less than described mark, illustrate that so described people's antibody is not present in this sample.Because the existence of people's antibody in sample of described anti-antigenicity SARS CoV albumen is to be infected causedly by SARS CoV by hypothesis, therefore whether having described people's antibody is someone Ei SARSCoV virus infections or once be exposed to SARS CoV virus whether of indication.

In one embodiment, described mark can be that a kind of energy passes through to analyze detected chemical part, and described chemical part is puted together or is attached on described anti-people's antibody.In this embodiment, described chemical part was puted together contact described anti-people's antibody with antigen/sample before usually or is attached on described anti-people's antibody.

In another embodiment, described mark can be the another kind of antibody or the set of having puted together other antibody of a chemical part on it, and described chemical part can detect by analytical approach.In this embodiment, described another kind of antibody or puted together on it chemical part other antibody set contact described anti-people's antibody with antigen/sample after, be incorporated on described anti-people's antibody usually.

One preferred embodiment in, this method further comprise step (b) and (c) between a washing step, more preferably between per two steps of this method, comprise a washing step.Preferably, wash with the wash solution that contains damping fluid, described damping fluid for example is the phosphate buffer that contains 1% the normal serum of having an appointment, and described normal serum is from the animal species that has wherein generated the antibody of having puted together described chemical part on it.In this wash solution, also can comprise a kind of emulsifying agent.

Multiple can be detected by analytical technology and chemical part that can be conjugated on a kind of antibody can be used in the described mark.For example, this chemical part can be epipolic or radioactive (referring to Fuller S.A., Evelegh M.J.﹠amp; Hurrell J.G.R. (2000) Conjugatesof Enzymes to Antibodies.In:Current Protocols in Molecular Biology. (Eds.Ausubel E.M., Brent R., Kingston R.E., Moore D.D., Seidman J.G., Smith J.A.﹠amp; Struhl K.) John Wiley﹠amp; Sons, Inc.Vol.2, pp.11.1.1-11.1.7 and Sambrook J., Fritsch E.F.﹠amp; Maniatis T. (1989) Molecular cloning.Alaboratory manual.2nd edn.Cold Spring Harbor Laboratory Press.ColdSpring Harbor, NY, both are merged in the present invention by reference at disclosed content).An example that can be used for chemical part of the present invention is an alkaline phosphatase.Depend on used specific technology or chemical part analytical technology being used to detect between the described mark necessary further processing

The analytical technology that can be used as detection method is widely known by the people in the art.For example colorimetric technology, electrophoretic techniques and radioactive label technology (referring to Sambrook J., Fritsch E.F.﹠amp; Maniatis T. (1989) Molecular cloning.A laboratory manual.2nd edn.Cold Spring Harbor Laboratory Press.Cold Spring Harbor, NY, its disclosed content is incorporated among the present invention by reference).

The colorimetric technology is normally preferred, also can use spectrophotometer or naked eyes to confirm to indicate the change color of positive or negative testing result.One of ordinary skill in the art would recognize that other technology of the detection method that can be used as among the present invention.

Kit of the present invention may further include the instrument that is used to detect described mark, or such instrument can separate independent packaging from kit.

By measuring the antibody that whether has S, M, E, N or U274, can detect whether there is or existed SARS CoV among a certain experimenter.Therefore, method of the present invention not only can be used to detect acute infection (having virus and antibody simultaneously); Can also be used to detect and only have antibody and do not exist in the situation of detectable virus, for example the experimenter who recovers from SARS CoV.

Method of the present invention and kit for example are used for SARS CoV and infect the Routine Test Lab detection that detects by using widely; And do not carrying out under the situation of immunity inoculation, this detection can detect asymptomatic SARS CoV carrier to obtain that SARS CoV is infected the true estimation of popular situation.

In a specific embodiment, immunoassays are ELISA.Antigenic protein GST-N (Δ 111-118), and alternatively, antigenic protein GST-U274 (134-274) is immobilized on the assay plate.With the serum of the antigenic protein contact that is immobilized, wash, blockade, wash described assay plate according to above-described standard ELISA technology from the patient.If the patients serum picks up from the patient (between infection back 2-11 days) who is in early infection, the Anti-Human IgA or the Anti-Human IgM two that put together with horseradish peroxidase resist the existence that are used to detect the anti-compound of antigen-one.Be in the patient that late period or convalescence infect (infect back 10-60 days between) if the patients serum picks up from, the Anti-Human IgG that puts together with horseradish peroxidase two is anti-to be used to detect existing of the anti-compound of antigen-one.The benzidine is colour reaction in the dark with the reagent tetramethyl, and on the dull and stereotyped reader of ELISA in OD 450nm place reading.

ELISA method of the present invention has dual specificity, produces a spot of false positive testing result, and highly sensitive, produce a spot of false negative testing result, especially when use GST-N (Δ 111-118) and Anti-Human IgG two are anti-.In addition, the minimum OD cutoff value that is used to read color reaction can be adjusted.Therefore, when using in low-risk area, the positive predictive value (PPV) of setting ELISA is that 100% higher cutoff value 0.45 may be suitable so that false positive results minimizes.Similarly, when using ELISA in the high risk area, it may be suitable setting sensitivity raising and the higher cutoff value 0.25 of negative predictive value (NPV).

In another specific embodiment, described immunoassays are that fast immune chromatographic detects.Antigenic protein is by the independent immobilization discontinuous row to the nitrocellulose filter, preferred GST-N (Δ 111-118) and GST-U274 (134-274).Independently chromatographic band comprises at the one end and has puted together the two anti-of colloid gold particle, has a separation vessel one in position and stops described two anti-early stage moving.The pad that carries reagent separates from nitrocellulose filter.In case added the patients serum to described film, a kind of release liquid is added on the chromatographic band, and described separation vessel is removed so that the two anti-compounds that resist with the antigen that has formed on nitrocellulose filter-one that move into contact.Typically, owing to contain the formation that the antigen-one of the colloid gold particle of being puted together resists-two compounds that resist, positive test symbol can display in 2-15 minute

In case the chromatographic apparatus for preparing has in advance been arranged, has only needed small number of devices just can carry out described fast immune chromatographic and detect.Identical with above-mentioned ELISA, described fast immune chromatographic detection sensitivity is high and have a specificity.Two kinds of results that detect gained have relatively observed and have had 99.6% good consistance between the two, and the kappa statistical value is 1.00; And good correlativity, the R of reactive terminal point ²Be 0.988.Therefore, it is a kind of detection simply fast that described fast immune chromatographic detects, and not needing to carry out special training could use; And provide the diagnostic result similar to ELISA.

Employed antigenic protein also can be applied to the fast immune chromatographic detection in above-mentioned ELISA, and can be used as two kinds of different certification marks.In essence, the indication that can not only provide for the single patient diagnosis is provided fast immune chromatographic newly developed; And because in single test, can detect the reactivity of each antigenic protein, this detection can also provide the details of mass diagnosis, this can provide the valuable epidemiology information of propagating about specific virus variation body in the crowd, this is because U274 albumen is compared with N albumen and had bigger variability.

In another specific embodiment, described immune detection is the Western Western blotting.Usually, use standard technique as known in the art that described antigenic protein is transferred on the film.Identical with the fast immune chromatographic detection, in same detection, every kind of antigenic protein can be split in the discontinuous band, makes and can detect the immune response of patient to specific antigen albumen simultaneously.Contain antigenic protein in the film property testing bar, preferred (Δ 111-118) and GST-U274 (134-274).By the Western engram technology of standard, put together two anti-described film is surveyed of antibody with antibody patients serum and anti-people.As above-mentioned, described two anti-select according to the time point between the infection period of gathering patient's sample.Described two anti-can being conjugated on the alkaline phosphatase are carried out color reaction with 5-bromo-4-chloro-3-indolylphosphate and nitroblue tetrazolium then and are detected.

Above-mentioned Western Western blotting is more consuming time and be not easy to be adjusted to the high flux that is fit to a large amount of patients.But it is quite useful as validation test, to confirm the result by another diagnostic method gained.

On the other hand, the present invention also provides a kind of antibody of anti-antigenicity SARS CoV albumen.Peculiar region S, M, E, N or the U274 of the preferably anti-SARS CoV of described antibody are to prevent antibody and cross reaction from the antigen of other coronavirus.

Antibody of the present invention is polyclone or monoclonal antibody, and it has specificity to the epi-position that is contained in a kind of antigenicity SARS CoV albumen among S, M, E, N or the U274.Monospecific antibody can be recombinant antibodies, for example chimera (for example the variable region by mouse source makes up with people's constant region), humanized antibody (the hypermutation zone of the constant main chain of human immunoglobulin(HIg) and animal for example mouse originate structure) and/or single-chain antibody.Polyclone and monospecific antibody can exist with the form of immunoglobulin (Ig), for example F (ab) ' 2 or Fab fragment.Antibody of the present invention is any isotype, as IgG or IgA; Polyclonal antibody is the single isotype or the potpourri of isotype.

Producing S, M, N, E or the U274 albumen of SARS CoV or the antibody of its homolog or its fragment produces by the composition immunity mammal that contains described protein, homolog or fragment.Such antibody can be monoclonal or polyclonal antibody.The method of manufacture order clone and polyclonal antibody is well-known in the art.Such method can be referring to " Antibodies; a Laboratory Manual, Cold Spring Harbor Laboratory, Eds.E.Harlow and D.Lane (1988); with D.E.Yelton et al., 1981.Ann.Rev.Biochem.50:657-680.For monoclonal antibody, referring to Kohler﹠amp; Milstein (1975) Nature 256:495-497.

With the preparation of the immunological testing of standard with identify the S to SARS CoV of the present invention, M, N, E or U274 albumen or its homolog or antibody that its fragment produced, for example Western engram analysis, dot blot analysis or ELISA (referring to, Coligan et al., Current Protocolsin Immunology (1994), John Wiley﹠amp; Sons, Inc.New York, NY).Whether described antibody exists antigenicity SARS CoV Protein S, M, N, E or U27 in being used to imitate originally with detection sample biological example in the diagnostic method.Described antibody also is used in the affinity chromatography S, M, N, E or U27 albumen or its homolog or its fragment in order to purifying SARS CoV.

Those skilled in the art will easily understand immune complex between the composition and S, M, N, E or U27 albumen or its homolog or its fragment of sample; Or between antibody of the present invention and its target antigen, no matter use that intersegmental formation of S, M, N, E or U27 albumen or its homolog or its sheet; And before detecting described immune complex, remove unconjugated material.Can understand, when antibody of the present invention is used to Screening Samples, when whether having SARS CoV polypeptide as gastric juice extractive or slicer to detect, for example whether to have the antibody of anti--S, M, N, E or U27 in the blood sample be useful to antigenicity SARS CoV protein reagent for detecting sample.

In brief,, the body cell and the myeloma cell of host animal tool antibody producing potentiality, the antigen immune of using by oneself are merged by conventional method, form the hybrid cell of two kinds of cells for the preparation monoclonal antibody.Body cell can be from the peripheral blood of spleen, lymph node and transgenic animals.The myeloma cell who can be used for preparing hybrid cell comprises mouse myeloma cell line for example MPCII-45.6TGI.7, NSI-Ag4/1, SP2/0-Agl4, X63-Ag8.653, P3-NS-1-Ag-4-1, P.sub.3X 63Ag8U.sub.1, OF and S194/5XX0.BU.1; Rat cell system comprises 210.RCY3.Agl.2.3; Clone comprises U-226AR and GM1500GTGA1.2; And mouse-people is hybridized myeloma cell line (Hammerling, etal. (editors), Monoclonal Antibodies and T-cell Hybridomas IN:J.L.Turk (editor) Research Monographs in Immunology, Vol.3, Elsevier/NorthHolland Biomedical Press, New York (1981)).

With a kind of selective medium for example the HAT nutrient culture media body cell-myeloma cell's hybrid cell is implanted in the various kinds of cell.Selective medium makes and the hybrid cell that produces antibody can be detected from other unwanted hybrid fused cell.Selective medium also can prevent non-fusion myeloma cell's growth, otherwise this non-fusions myeloma cell will continue to divide indefinitely, because the myeloma cell lacks the generation cell necessary enzyme hereditary information of growing.Comprise from somatic bone-marrow-derived lymphocyte and to produce the cell necessary enzyme hereditary information of growing, but lack " infinite multiplication " characteristic of myeloma cell, therefore in selective medium, keep the very short time.Therefore, have only those body cells that successfully merge with the myeloma cell in selective medium, to grow.The cell of Rong Heing will not killed by HAT or selective medium.

A kind of screening technique is used to detect the antibody that derives from potential anti--S, M, E, N or the U274 that are grown in hybrid cell in the multiple hole.Use multiple hole to prevent the excessive of each hybrid knurl length.The screening technique that is used to screen the antibody of potential anti--S, M, E, N or U274 comprise enzyme immunoassay (EIA), radiommunoassay, plaque mensuration, cytotoxic assay, spot immune in conjunction with measure, fluorescence-activated cell sorting (FACS) (FACS) and other carry outer in conjunction with mensuration.

Detection is retained in the nutrient culture media for the hybrid cell of the antibody positive of anti--S, M, E, N or U274, and can be had specific monoclonal antibody to produce to S, M, E, N or U274 by the clone.The animal that perhaps required hybrid cell can be expelled to and provide body cell and myeloma cell carries out initial fusion in the animal body of compatibility in a organized way.Injected animal development has generated the knurl of secretion by the monoclonal antibody specific of hybrid cell generation.

By the immunoglobulin purification method of routine monoclonal antibody that selecteed hybrid cell is secreted from cell culture medium rightly purifying come out for example albumin A-SEPHAROSE hydroxyapatite, gel electrophoresis, dialysis or affinity chromatography.

The antibody that produces according to the present invention can be used to detect the existence of antigenicity SARS CoV albumen or its antigenicity fragment, be included in the diagnostic method, whether evaluation exists specific antigenic protein in patient's sample or in available from patient's viral isolates, and the recombinant antigen SARS CoV albumen or its antigenicity fragment that detect purifying.This antibody also can be used in the purification technique of standard, for example immunoprecipitation.According to the known technology in this area, antibody provided by the present invention can be used in the immunoassays, as above-mentioned catching-ELISA, Western Western blotting or immunofluorescence assay.

Therefore, the invention provides the method for a kind of use antibody test antigenicity of the present invention SARSCoV albumen, especially S, M, E, N or U274 or an antigenicity fragment.Be enough to make antibody capable and antigen form under the condition of compound, be enough to make that antibody capable and antigen form the step of the time of compound for one section with the sample contact SARS CoV antigen that comprises antibody; With anti-contact sample a period of time of anti-antigenicity SARS CoV albumen, especially S, M, E, N or U274 or an antigenicity fragment.If described antigenic protein is present in the described sample, described one anti-will being attached on the described antigenic protein, form the anti-compound of antigen-one, detect this compound by above-mentioned the whole bag of tricks then, for example anti-described one anti-two anti-by using, this two anti-can being conjugated on the detection molecules.

Whole documents that this paper quoted are intactly incorporated into the present invention by reference.

Embodiment

Embodiment 1:SARS CoV albumen is as the purposes of diagnostic flag

Material and method

Material: unless otherwise indicated, in this research used whole reagent all available from Sigma (St.Louis, Mo.).All clone all available from American type culture collection (Manassas, Va.) and in 37 ℃ of cultivations at 5%CO ₂, in the Eagle nutrient culture media modified of Dulbecco ' s, wrap this nutrient culture media comprise 1g glucose/liter, the sodium bicarbonate of 2mM L-glutamine, 1.5g/liter, the plain and 5% fetal bovine serum (HyClone of blue or green enzyme of chain enzyme/ml, the 100U of 0.1mM nonessential amino acid, 0.1mg, South Logan, Utah).

The structure of plasmid:, be pXJ40HA for carrying out the employed carrier of mark at the N of albumen end with a hemagglutinin (HA) epi-position (15) in order in mammalian cell, to carry out transient expression; For carrying out the employed carrier of mark with three hemagglutinin epi-positions at the C of albumen end is pXJ40-3-HA.For the expression of glutathione S-transferase (GST) fusion in bacterium, (Amersham PharmaciaBiotech, Uppsala is in reading frame Sweden) to pGEX-4T-1 with gene clone with GST.For the stable transfection of S in Chinese hamster ovary celI, described in GB 2314332, the C end is cloned in the pMMTC carrier by the total length S structure of green fluorescent protein (GFP) mark.Employed all structures are listed in the table 1 in this research.

Table 1 is used for the plasmid of this research

Plasmid	Nucleotide position among the ORF	Amino acid position	The amino acid sum
Plasmid	Nucleotide position among the ORF	Amino acid position	The amino acid sum	pXJ40HA-U274 pXJ40-E-3’HA pXJ40-M-3’HA pXJ40-U122-3’HA pXJ40-N pMMTC-S-GFP pGEX-4T1-U274 pGEX-4T1-U122 pGEX-4T1-N	400-822 1-228 1-663 1-366 203-1266 1-3765 400-822 46-333 357-1266	134-274 1-76 1-221 1-122 69-422 1-1255 134-274 16-111 120-422	141 76 221 122 254 1,255 141 96 303

SARS-CoV 2003VA2774, a kind of separator from the SARS patient of Singapore is used to this research.In order to clone different genes, with Qiagen viral RNA kit (Valencia, Calif.) extract RNA from the Vero E6 cell culture supernatant that SARS-CoV-infects, described supernatant is gathered in the crops at least when described culture shows 75% cytopathic effect.Use Superscript II ^TMRNA enzyme H-reverse transcriptase (Gibco BRL, Gaithersburg, Md.) and Oligonucleolide primers (dT) carry out reverse transcription.Use HotSta ^TMPolymerase (Qiagen), Titanium ^TMThe Taq archaeal dna polymerase (ClontechLaboratories Inc., Palo Alto, Calif.) or high precision Taq polymerase (RocheMolecular Biochemicals, Indianapolis, Ind.).In some cases, use overlapping cDNA (the separator SIN2774 that Genome Inst of Singapore Nat U. provides instead; The number of obtaining: AY283798) as template.All used in this research primers are synthetic by Genset SingaporeBiotech (Singapore).The dna sequencing that the centralab of the sequence of employed all constructions by molecule and cell biological institute carries out in this research determines that order-checking is by using PE Applied Biosystems (Foster City, Taq DyeDeoxy Calif.) ^TMStopping cycle sequencing kit and robotization dna sequencing instrument (373 type) carries out with dideoxy.

The transient transfection of mammalian cell: Transient transfection experi Effectene ^TMTransfection agents (Qiagen) carries out the transient transfection experiment according to the scheme of manufacturer.Typically ,～10 ⁶Individual COS-7 cell is placed in the plate of a 6cm diameter and adsorbs 4h at least.Each dull and stereotyped DNA that uses the 1-2 microgram places 14h at least with described cell before with phosphate buffer (PBS) washed cell, direct staining in Laemmli ' s lauryl sodium sulfate (SDS) damping fluid, and be used for the Western engram analysis.

The expression of gst fusion protein: the exponential growth culture (optical density at 600nm place is～0.7) of Escherichia coli (BL21/DE3) cell that contains the pGEX-4T-1 expression structure is induced with synthetic fusion by dosing 1mM isopropyl ss-D-thiogalactoside (IPTG), thereafter with above-mentioned cell in 37 ℃ of regrowth 4h, or at 30 ℃ of regrowth 12h.Harvesting, and it is resuspended among the PBS that contains 0.5%Triton X-100 and 1mM phenylmethylsulfonyl fluoride, (Misonix Inc., Farmingdale N.Y.) carry out ultrasonic degradation to use processor for ultrasonic wave then.Use glutathione (GSH)-Ago-Gel pearl (Amersham Pharmacia Biotech) from lysate, to be purified into gst fusion protein then.Go out albumen with 10mM reduced glutathione among the 50mM Tris-HCl (pH9.2) and 0.1%SDS from described pearl wash-out; Use Coomassie Plus ^TM(Pierce, Rockford Ill.) measure protein concentration to measure kit.Protein is also separated in the SDS-polyacrylamide gel, and is used for 0.25% Coomassie brilliant blue R-250 (Bio-Rad, Hercules, Calif.) dyeing of 45% methyl alcohol and 10% acetate.

Western engram analysis: for the Western engram analysis, the gst fusion protein of about 105 transfected COS-7 cells or 50ng is separated in the SDS-polyacrylamide gel, and is transferred on the nitrocellulose Hybond C film (Amersham PharmaciaBiotech).With 5% skimmed milk power this film of blockading.Detection for HA-mark or gst fusion protein, with anti--HA polyclone or anti--GST monoclonal antibody (Santa CruzBiotechnology, Santa Cruz, Calif.) spend the night in 4 ℃ of described films of incubation, and with fully washing of PBST (PBS that contains 0.05%Tween 20), continue to resist (Pierce) in room temperature incubation 1h with two of suitable horseradish peroxidase (HRP)-put together.With PBST film is fully washed then, signal is detected by the chemoluminescence method (Pierce) that strengthens.For the patients serum, at first use 0.5%Triton X-100 and 0.1mg of RNA enzyme (Sigma)/ml to handle each sample, then with PBST containing 1% dilution 1: the 150-1: 500 that contains 1% skimmed milk power.In behind the room temperature incubation 1-3h or after 4 ℃ of temperature spend the night, described film is puted together immunoglobulin G (IgG) (Santa CruzBiochemicals), IgA or IgM (Zymed aboratories Inc. in room temperature with Anti-Human HRP-, San Fran-isco, Calif.) antibody incubation continues with above-mentioned detection.All two anti-ly all use with 1: 2000 dilution.Because finite quantity from the patients serum of 7 pairs of serum, the Western trace and the GST of the potpourri of 3 kinds of gst fusion protein N, U274 and U122 carry out electrophoresis in single groove SDS-10% polyacrylamide gel electrophoresis (PAGE) gel, and are transferred on the nitrocellulose filter.Described film is cut into the wide bar of about 0.8mm, each bar is surveyed with the dilute serum of 300-400 microlitre.For the serum of later time point, with described with the dilution serum in room temperature incubation 1.5h.For the serum of time point early, must be with described serum in room temperature with described overnight incubation to detect any signal.Described two resist in room temperature and are hatched.When the screening great amount of samples, also use this method.

The S-GFP fusion in Chinese hamster ovary celI expression and immunofluorescence analysis to detect the anti--S-immunocompetence in patient's serum: according to the scheme of manufacturer, with DMRIE-C reagent (Gibco BRL) with the pMMTC-S-GFP transfection CHO cell.At Geneticin ^TMSelect transfected about 1 week of cell in (Gibco BRL).Then at Zeiss microscope (CarlZeiss Vision GmbH, Hallbergmoss, Germany) following analysis of cells, and at the ZnSO that contains 100 μ M ₄Nutrient culture media in pick and the strongest clone of culture expression signal.Zn ²⁺Ion has improved expression of gene by pMMTC carrier (referring to GB 2314332).From flat board, remove cell with 0.04%EDTA, this cell inoculation to black teflon Menzel diagnosis microslide (Merck), and is dried up, then in～20 ℃ of fixing 1h in acetone.Use then serum with the dilutability of 1: 20,1: 40,1: 80 and 1: 160 (in PBS) in 37 ℃ of this cell of incubation 1.5h, continue with fluorescein isothiocynate or rhodamine (Rh)-Anti-Human IgG (Sigma) that puts together with 1: 20 dilutability in 37 ℃ of incubation 1.5h.As the Anti-Human IgG that uses Rh-to put together, it is diluted among the PBS; When Anti-Human IgG, the IgM (Sigma) that use FITC-to put together or IgA (Dako A/S, Glostrup is in the time of Denmark), it is diluted in 0.05% Evans blue solution (Fluka, Buchs, Switerland) in, the described Evans blue solution GFP fluorescence that transfected cell sends of having blockaded.Observe microslide down at Zeiss microscope (Carl Zeiss VisionGmbH) then, and press following score: +++, very strong is painted; ++, medium colorant; With+, weak painted.

The patient who infects from SARS-CoV-gathers serum: 6 serum samples (coming patient 1-6), and except obtaining from all passing through plasmapheresis (plasmapheresis) patient 3 the sample.Following patient is implemented plasmapheresis (i) be determined in advance as according to the standard of The World Health Organization (WHO) that SARS-CoV infects; (ii) at least 6 weeks on the cycle of recovery; (iii) asymptomatic but be ready to participate in this research.Gather blood from patient 3, and serum separated be used for test.All participants provide the written commitment book and have obtained the permission of ethics committee of Tan Tock Seng hospital.Second group of serum is made up of 7 pairs of samples, the patient that described sample collection is accepted for medical treatment from Tan Tock Seng hospital or Singapore General hospital.As the standard of WHO was defined, these patients were accepted for medical treatment at the initial stage of a disease, accepted for medical treatment and the date of taking a blood sample is shown in the table 2.First group of sample (group A) gathered after morbidity in 2-11 days, and second group of sample (group B) gathered after morbidity in 16-5 days.

Table 2: the description of gathering the patient's who infects from 7 SARS CoV serum

The patient	Sample	Date of the onset	Date collected	Fate after the morbidity
The patient	Sample	Date of the onset	Date collected	Fate after the morbidity	D2 D3 D4	2A 2B 3A 3B 4A 4B	2003.3.18 2003.3.18 2003.3.17 2003.3.17 2003.3.16 2003.3.16	2003.3.20 2003.4.15 2003.3.20 2003.4.17 2003.3.20 2003.5.31	2 28 3 31 4 48

D5 D8 D9 D11

5A 5B 8A 8B 9A 9B 11A 11B

2003.4.2 2003.4.2 2003.3.19 2003.3.19 2003.3.9 2003.3.9 2003.2.24 2003.2.24

2003.4.7 2003.4.29 2003.3.27 2003.4.4 2003.3.18 2003.5.2 2003.3.7 2003.4.3

5 27 8 16 9 54 11 38

Last group serum is available from 61 possible SARS patients that leave hospital from Tan Tock Seng hospital.For the some of them patient, gather serum (morbidity back 3-4 week) once leaving hospital, and most patient is accepted for medical treatment (describing in detail referring to table 3) in the back of leaving hospital again above 3 weeks (the morbidity back surpassed for 6 weeks).Wherein 1 patient (patient P3L) gathered serum sample (3 first quarter moons) in back 111 days in morbidity.Come other patients' (P7 and P8) sample to obtain by plasmapheresis.Control group serum obtains 99 serum samples available from Sigma from the healthy contributor who agrees donations.These healthy contributors (i) are not suffered from SARS by diagnosis, and not under a cloud suffer from SARS, or do not have contacted being in to carry out the people that SARS isolates; (ii) last week do not have heating, flu-like symptom, have a running nose or have a sore throat in donations; (iii) do not use immunodepressant; (iv) there is not tangible medical conditions; (v) less than the experience of since in November, 2002, having traveled in the SARS infected zone.

The result

The IgG detection of antibodies of the antiviral protein of the SARS-CoV genome encoding in possible SARS patient's the serum: can be used as diagnostic antigen by which kind of virus protein of SARS-CoV genome encoding and to develop and detect the determination of serology that SARS-CoV infects in order to assess, the inventor is cloned into 3 kinds in 4 kinds of structural proteins (E, M and N) in the expression vector.Because the size of furcella (S) albumen big (about 4.7kbp) and a large amount of glycosylations are carried out separate analysis with immunofluorescence method described as follows to it.In addition, also comprised two kinds of peculiar albumen of SARS-CoV (U274 and U122) in this research, below will be known as UX, wherein " X " represents the predicted amino acid number of protein.In this research, the applicant has only used the terminal hydrophilic area of the C-of U274, owing to have the dissolubility that 3 potential membrane spaning domains and these hydrophobic regions may have influence on reorganization U274 albumen at its N-end.The position of structural proteins and peculiar albumen, and the ORF number of their correspondences as specified by Marra and colleague (5), are shown among Fig. 1.

The protein of this group HA-mark is expressed by transient transfection in the COS-7 cell, use from 3 bit recovery phase patients serum (Fig. 2, patient 1-3) by Western engram analysis analyzing total protein dissolution product with whether determine these serum have any anti-this by the antibody of expressed

protein.Patient

1 and 2 is implemented plasmapheresis, and the product of plasmapheresis is used in the Western engram analysis; For patient 3, serum is used for analyzing.

Whole 3 patients have the antibody of anti-N albumen (aa 69-422), but not anti-other structural proteins, the antibody of M and E.What is interesting is that the terminal hydrophilic area of the C-of U274 (aa134-274) is the serum by

patient

1 and 3 but not patient 2 serum is detected (Fig. 2) also.Control group serum does not detect N, U274 or any other albumen.Also do not detect U122 with in above-mentioned 3 kinds of serum any, this shows that it may not expressed, or it is not a kind of structural proteins, or it does not have enough antigenicities.

Because the protein of preparation bacterial expression is simpler, more cheap, thus can carry out large-scale production, the applicant next with the formal representation of gst fusion protein N albumen and U274, and detected their reactivities to the patients serum.Coomassie dyeing shows GST-N (aa120-422) and GST-U274 (aa 134-274) purity＞90% (chart 3) after carrying out single step purification with GSDH-Ago-Gel pearl.GST-N used herein (aa 120-422) albumen lacks the motif (FYYLGTGP who is found in high conservative in all coronavirus (10) that contains of N albumen; The amino acid/11 1-118 of SARS-CoV N) N-end portion, therefore with the chance of the antibody generation cross reaction of anti-other coronavirus still less.From the bacterial cultures of a 400ml, can obtain the GST-N of about 10mg and the GST-U274 of 2mg respectively.GST-U122 (aa 16-111 lacks the signal peptide and the hydrophobicity C end of N-end) and GST are also expressed and are used as contrast.With regard to the mammal expressed protein, survey gst fusion protein (Fig. 2) with 3 kinds of identical patients serums and a kind of control serum.Consistent with protein expressed in the COS-7 cell, GST-N is clearly detected by whole 3 kinds of patients serums, and GST-U274 is only detected (Fig. 4) by the serum from patient 1 and 3.Whole 3 kinds of patients serums do not make GST-U122 and GST demonstrate any background, and there are not non-specific binding (Fig. 4) in control group serum and arbitrary albumen.Serum from other 3 bit recovery phase patients (patient 4-6) is also detected, and they possess specificity to GST-N and GST-U274 both, but GST-U122 or GST are not possessed specificity (Fig. 4)

The N in 7 pairs of serum that the time point of two infection obtains and IgG, IgM and the IgA distribution of antibody figure of U274 albumen: one group of also detected its reactivity of the paired sera from 7 patients to GST-N and GST-U274.These serum obtain at two time points that infect, and one is 2-11 days (group A) after morbidity, and another is 16-54 days (group B) (tables 2) after morbidity.For all 7 patients, the sampling at least 8 days after very first time point sample sampling of the sample of second time point.As shown in Figure 5, anti--anti-GST-N albumen of IgG antibody is present in the serum (group B) of whole 7 patients' second time point, but is not present in the serum of early stage time point (group A).For two groups of serum, with serum (1: 150) the incubation 1.5h of film, resist (the Anti-Human IgG that HRP-puts together) in room temperature incubation 1h with two then with dilution in room temperature.In addition, the serum of whole 7 second time points also contains the antibody of anti-GST-U274, though be in lower level (Fig. 5, group B) at the antibody with respect to anti-GST-N.

Repeat the experiment of serum (group A), but spend the night in room temperature incubation spot with diluted serum to early stage time point, rather than 1.5h; And use 3 kinds different two anti-, anti--IgG, anti--IgM and anti--IgA; Each the results are shown in Fig. 6.For IgG, have only sample 9A and 11A to show some reactivities to GST-N.For IgA, although 7 schedule of samples reveal the reactivity to GST-N, and the signal of sample 9A and 11A is strong especially.For IgM, whole 7 samples demonstrate the low-level reactivity to GST-N.Because the different number of days after the sample collection idiopathy of every patient's early stage time point, gained result show IgM and the IgA antibody that early just has anti-N albumen to fall ill back 2 days (patient D2, sample 2A).Falling ill occurred the IgA antibody horizontal of very high anti-N albumen in back 9 days, and IgM and IgG antibody also can be detected (Fig. 6, sample 9A and 11A).With regard to GST-U274, found that after morbidity it is to IgA (sample 5A, 8A, 9A and 11A) with to some reactivities of IgM (sample 11A) in 5-11 days.What is interesting is that when using IgM, sample 11A (falling ill back 11 days) observes the great signal to GST-U274; For the early stage sample 11B of response, its signal also very strong (Fig. 5 and 6) to GST-U274.

(IgG) the immunoreactive sensitivity and the specific detection of anti-N and U274 albumen: in order to detect immunoreactive sensitivity and the specificity to N and U274, the applicant has obtained other 6 samples and has tested the existence of the IgG of its anti-N and U274 albumen by engram analysis from the possible SARS patient who leaves hospital.As shown in table 3, sample is taken from 31-111 days after the morbidity.The legend of table 3: ^aMeasure by the Western engram analysis; Observe by radioautograph +++, ++ ,+, very strong, medium and weak signal represented respectively;-, there is not signal to be observed; ^bPass through determination of immunofluorescence method, the Chinese hamster ovary celI of wherein stably expressing S albumen carries out incubation with diluted patients serum (1: 40), observe with FITC-at the Zeiss microscopically subsequently and put together Anti-Human IgG antibody-microslide and by following scoring: +++, very strong is painted; ++, medium colorant; +, weak painted.Detected 100 serum samples at identical dilutability, all do not demonstrated any painted county Party committee from healthy contributor; ^cThe fate that sample obtains after the invention of stipulating obtains; ^dContactee (patient 868 and 873) from two health is also detected; ^eThe sample that S obtains by plasmapheresis; ^fTime point in late period from a serum in 7 pairs of serum described in the table 2.

Table 3: the reactivity of the serum sample of gathering from 74 possible SARS patients

Patient's numbering	Reactivity to N albumen ^a	Reactivity to U274 albumen ^a	Reactivity to S albumen ^b	Fate after the morbidity ^c
Patient's numbering	Reactivity to N albumen ^a	Reactivity to U274 albumen ^a	Reactivity to S albumen ^b	Fate after the morbidity ^c	174	+++	++	+	49
318	+++	-	+	53	174	+++	++	+	49
318	+++	-	+	53	350	+++	++	+	62
358	+++	+	+	60	350	+++	++	+	62
358	+++	+	+	60	377	+++	+	+	61
387	+++	-	+	63	377	+++	+	+	61
387	+++	-	+	63	432	+++	++	+++	42
442	+++	+	++	63	432	+++	++	+++	42
442	+++	+	++	63	487	+++	+	++	54
492	+++	+	+	31	487	+++	+	++	54
492	+++	+	+	31	526	+++	+	+	63
541	+++	+	++	58	526	+++	+	+	63
541	+++	+	++	58	546	+++	++	++	62
561	+++	++	+	57	546	+++	++	++	62
561	+++	++	+	57	566	+++	+	++	59
571	+++	+	+	57	566	+++	+	++	59
571	+++	+	+	57	576	+++	+	++	63
581	+++	-	++	53	576	+++	+	++	63
581	+++	-	++	53	586	+++	++	++	38
596	+++	-	+	39	586	+++	++	++	38
596	+++	-	+	39	603	+++	++	++	67
321	+++	++	+	50	603	+++	++	++	67
321	+++	++	+	50	633	+++	-	+++	39
638	+++	+	+	60	633	+++	-	+++	39
638	+++	+	+	60	644	+++	+	++	41
672	+++	+	+	53	644	+++	+	++	41
672	+++	+	+	53	677	+++	++	++	59
682	+++	-	+	58	677	+++	++	++	59
682	+++	-	+	58	687	+++	+	++	61
696	+++	-	+	41	687	+++	+	++	61
696	+++	-	+	41	701	+++	++	++	40

706	+++	+	++	39
706	+++	+	++	39	711	+++	-	+	54
716	+++	-	++	40	711	+++	-	+	54
716	+++	-	++	40	726	+++	+	++	34
734	+++	-	+	41	726	+++	+	++	34
734	+++	-	+	41	739	+++	-	++	49
744	+++	+	++	68	739	+++	-	++	49
744	+++	+	++	68	749	+++	+++	++	59
759	+++	+	+	42	749	+++	+++	++	59
759	+++	+	+	42	764	+++	+	++	43
769	+++	+	++	64	764	+++	+	++	43
769	+++	+	++	64	774	+++	+	++	62
784	+++	-	+++	38	774	+++	+	++	62
784	+++	-	+++	38	789	+++	-	++	55
793	+++	+	+	48	789	+++	-	++	55
793	+++	+	+	48	798	+++	-	++	44
803	+++	-	++	40	798	+++	-	++	44
803	+++	-	++	40	823	+++	+	++	37
840	+++	++	+++	43	823	+++	+	++	37
840	+++	++	+++	43	845	+++	++	++	36
859	+++	+++	++	55	845	+++	++	++	36
859	+++	+++	++	55	868 ^d	-	-	-	Inapplicable
873 ^d	-	-	-	Inapplicable	868 ^d	-	-	-	Inapplicable
873 ^d	-	-	-	Inapplicable	878	+++	++	+++	58
883	+++	+	++	56	878	+++	++	+++	58
883	+++	+	++	56	888	+++	+	++	33
893	+++	++	+++	41	888	+++	+	++	33
893	+++	++	+++	41	4153	+++	-	++	41
4209	+++	-	++	21	4153	+++	-	++	41
4209	+++	-	++	21	P3L	+++	++	+	111
P1 ^e	+++	++	++	＞42	P3L	+++	++	+	111
P1 ^e	+++	++	++	＞42	P2 ^e	+++	-	+	＞42
P3 ^e	+++	++	+	＞42	P2 ^e	+++	-	+	＞42
P3 ^e	+++	++	+	＞42	P4 ^e	+++	++	+	＞42

P5 ^e	+++	++	+	＞42
P5 ^e	+++	++	+	＞42	P6 ^e	+++	++	+	＞42
P7 ^e	+++	+	+	＞42	P6 ^e	+++	++	+	＞42
P7 ^e	+++	+	+	＞42	P8 ^e	+++	++	++	＞42
2B ^f	+++	+	++	28	P8 ^e	+++	++	++	＞42
2B ^f	+++	+	++	28	3B ^f	+++	++	+++	31
4B ^f	+++	+	+	48	3B ^f	+++	++	+++	31
4B ^f	+++	+	+	48	5B ^f	+++	+	+++	27
8B ^f	+++	+	+++	16	5B ^f	+++	+	+++	27
8B ^f	+++	+	+++	16	9B ^f	+++	+	++	54
11B ^f	+++	++	++	38	9B ^f	+++	+	++	54

Used two patients' (P7 and P8) plasmapheresis product and from other 59 patients' serum.All samples all demonstrate the strong immunoreactivity (100%) to N albumen, and 44 (72%) in 61 samples are the U274 positives (table 3).Usually, the signal that observed of U274 albumen is a lot of a little less than than the signal that N albumen observed.Exception is the sample from patient 749 and 859, and it is to the signal equality strength of U274 and N albumen.In addition 99 samples from healthy contributor are also detected, wherein do not have sample to demonstrate any immunoreactivity to N or U274.In addition, from two possible excessively SARS patients of intimate contact once but the sample that does not show the patient (patient 868 and 873) of any clinical symptoms does not demonstrate any immunoreactivity (table 3) to N or U274 albumen yet.Therefore, the immunoreactivity of anti-N albumen is highly sensitive (81 samples from possible SARS patient are found to be 100%) and have specificity.

Detect the IgG antibody of the S albumen of the anti-SARS-CoV in the convalescent serum by immunofluorescence technique: because the large scale and the high glycosylation of S albumen, it is favourable not expressing this albumen in bacterium at mammalian cell, might make the conformation and/or the glycosylation antagonist that can depend on S albumen detect subsequently.Be used in the C-end by GFP total length S Stability Analysis of Structures transfection CHO cell.After selecting with antibody, obtained to express the clone pond of the S albumen of the level of signifiance, by GFP fluorescence indicated (Fig. 7).These Chinese hamster ovary celIs are used to the immunofluorescence dyeing method to be determined at the IgG antibody of the S albumen that whether has anti-SARS-CoV in the convalescent serum.In brief, use the acetone fixed cell, carry out incubation with diluted patients serum then, the Anti-Human IgG antibody of puting together with Rh-carries out incubation subsequently.

As shown in Figure 7,, all contain anti-S protein I gG antibody, when using control group serum, do not observe signal from the serum of patient 1-6 according to the detection of immunofluorescence.All remaining other samples are tested similarly, the Anti-Human IgG antibody that replaces FITC-to put together except the Anti-Human IgG antibody of puting together with Rh-under the situation about existing at high concentration moral prestige Evans blue solution (0.5%).This is because judge that cell is painted than judging that cell is painted much easier by Rh by FITC, and the Evans blue solution of this concentration be enough to blockade whole GFP albumen (data not shown) in all transfected cells.

As shown in table 3, when 1: 40 dilutability, 100% convalescent serum (74 samples) has demonstrated the immunocompetence of anti-S albumen, does not demonstrate any signal from 100 samples of healthy contributor at identical dilutability.The minimum dilutability of testing is 1: 20, but in a few sample demonstration weak signal of this concentration from healthy contributor; Therefore, be used to comparison from 1: 40 dilution result.In higher dilutability (1: 80 and 1: 160), as desired, most patients serums observe more weak signal.Especially, for sample 432,633,784,840,878,893,3B, 5B and 8B, in 1: 40 dilutability observation road to very strong signal (+++), along with increasing signal, progressively weakens dilutability, promptly 1: 80 dilutability observe medium signal (++, the dilutability at 1: 160 observes weak signal (+).Also in the same way 7 early stage time point samples (table 2, group A) are analyzed, but use IgG respectively, IgM and IgA two be anti-, but wherein do not demonstrate any reactivity (dilutability) (data not shown) at 1: 40.

Embodiment 2:ELISA and immunochromatographic measurement

Material and method

Recombinant protein: be used to obtain described in the material and method such as above-mentioned embodiment 1 of recombinant protein.For a research, use Superdex ^TMS-200 HR10/30 post is further purified Gst-U274 in AKTA liquid chromatography (FPLC) system of quick albumen (Amersham).Employed damping fluid contains 20mM Tris-HCl (pH7.5), 100mM NaCl, 6M urea and 1mM beta-mercaptoethanol, and flow velocity is 0.5ml/min, collects the fragment of 1ml.Fragment 12 and 13 is combined together and carry out dialysed overnight and exchange buffering liquid 3 times at least with phosphate buffer (PBS).

Serum sample: 74 convalescent serum samples are gathered the patient who accepts for medical treatment from Tan Tock Seng hospital or Singapore General hospital.Obtain 91 control group serum from the local contributor of health, these contributors are in Singapore's molecule and the work of RESEARCH ON CELL-BIOLOGY institute.All samples all pass through to be agreed to be gathered, and patient's sample picked up from 16-65 days that symptom takes place.In addition, from BioClinical Partners, Inc. has bought 119 serum from healthy contributor, and (Franklin Mass.) is included in this research as extra normal healthy controls.

ELISA: before bag is by plate, Gst-N and GtT-U27 albumen 4 is diluted in carbonate buffer solution (pH9.6) to final concentration in advance is respectively 0.1 and 0.15 microgram/ml.Described plate prepares as described in the list of references 16.In brief, by be incubated overnight in room temperature (16-18h), with protein mixture with the volume bag in 100 microlitres/hole by 96-hole polystyrene microtiter plate (Immuno 1B; Themo Labsystem, Franklin, Mass.).Wash this plate 5 times with PBS-Tween 80 (PBST), with 200 microlitres (every hole) based on the dilution of Tris in the room temperature nonspecific binding site 1h that blockades.Making an addition to/the 10 microlitre serum of 200 microlitres based on the dilution (respectively containing 1% bovine serum albumin(BSA) [BSA] and skimmed milk power) of Tris before, the further described plate of washing 5 times again.Subsequently, in 37 ℃ of described plates of incubation 30 minutes, continue with PBST washing 6 times.Dose Anti-Human's immunoglobulin G (IgG, 1: 500 dilution) of horseradish peroxidase-put together with the amount in 100 microlitres/hole, this potpourri in 37 ℃ by incubation 30 minutes.In PBST, wash described plate 6 times then, add zymolyte tetramethyl benzidine (TMB) with the amount in 100 microlitres/hole and continue colour generation.In 37 ℃ of incubations after 15 minutes, 100 microlitre 1N HCl cessation reactions are added in every hole in the dark.With 620nm reference optical filter, measure optical density (OD) in the 450nm place.

Fast immune chromatographic is measured: the immunochromatographic measurement device based on film is made up of chromatoplate, separation vessel and absorption layer, and they are placed in the box entirely, as described in the US patent 6,316,205; Before in being mounted to described device, prepare chromatoplate in advance through slight modification according to the program that is described in detail in the document.Briefly, with BioDot (Irvine, Calif.) Gst-N and Gst-U274 recombinant antigen are sprayed at average pore size with concentration 0.1 and 0.15mg/ml respectively is that (Whatman, Ann Arbor is in two independent row Mich.) for 8 microns nitrocellulose filter to spraying machine.With dry 10 minutes of described film, then it is immersed in one and blockaded in the damping fluid 1 minute, described damping fluid is by the StabilCoat that contains 6.7% ^TMMilli-Q pure water, 0.05%Triton X-100 and 0.5% casein form.The film of being blockaded in 37 ℃ of dryings is 60 minutes then, is fixed to then on the film holder.Carry the pad of reagent with the preparation of porous infiltration matrix.Described porous infiltration matrix is smeared with goat-anti-human IgG antibody, and the colloid gold particle that described antibody diameter is 25-30nm carries out mark.In the described pad that carries reagent of 37 ℃ of dryings two hours, then it is combined in the described device then.The chromatography card makes according to following, is about to porous that 0.1%Triton X-100 the handled matrix that seeps water and is fixed to an end of nitrocellulose filter, the pad that carries reagent is fixed to the other end of same film holder.Be cut into about 4/56mm then ²The sheet of size.When the first half of the described box of sealing, place an absorption layer in the latter half of described box, subsequently the chromatography sheet of 1 unit is put into to be assembled into determinator.In addition, also prepared reagent and discharged lavation buffer solution with the Milli-Q pure water that contains 50mMNaH2PO4,300mM NaCl and 0.1% sodium dodecylsulphonate (pH8.0).

When detecting, the undiluted serum sample of 25 microlitres is added in the sample window of described determinator.Make described sample be displaced sideways and cover an one of described film.When sample touched indicator in the view window in about 3 seconds, 3 reagent discharged lavation buffer solutions and are added in the damping fluid window to discharge the goat-anti-human IgG antibody of colloid gold label.Spur jag then and remove separation vessel, make the chromatography composition contact with absorption layer.Make the complete traversing chromatography sheet of crossing of goat-anti-human IgG antibody of described colloid gold label then.Typically, the result can read by view window in 2-15 minute.Negative structure is indicated by control line only occurring; And positive findings, control line and one or two detection lines all appear at (Fig. 8) in the view window.

Statistical study: the kappa statistical value is used to measure the conforming degree of the result between new fast measuring and the new ELISA.Kappa statistical value＞0.75,0.40-0.75 and＜0.40 represent that respectively consistance is fine, consistance good and consistance poor (17).To the data analysis of ELISA, any cutoff value of selected 0.45, but confirm with δ value, this will provide other indication of optimal zone of positive colony and negative light between all.

The result

ELISA: when detecting with 1: 20 sample dilution, ELISA detects the IgG antibody of SARS-CoV at the convalescence sample from SARS patient of 100% (n=74), and mean+SD OD value is 2.32+0.54 (table 4 and Fig. 9).On the contrary, detect, be initially positive sample and be found from only find 1 from healthy contributor's 210 control group serum by this, mean+SD OD value be 0.054 ± 0.05 (P＜0.001) (Fig. 9).Therefore this detection provides 98.7% PPV and 100% negative predictive value (NPV) (table 4).In addition, it is 5.4 that ELISA provides positive δ value, and negative δ value is 3.6, has demonstrated between the positive and the negative findings well but to distinguish.In addition, as shown in soil 10, when with this new ELISA method when 7 samples from SARS patient obtain titration curve, the reactivity terminating point is found according to different samples from＞1: in 40 to 1: 640 the dilution range.

Table 4: the result of single marking

The mensuration form	SARS number positive/sum	Patient's sensitivity %	Healthy number positive/sum	Contrast sensitivity %	PPV(％)	NPV(％)
The mensuration form	SARS number positive/sum	Patient's sensitivity %	Healthy number positive/sum	Contrast sensitivity %	PPV(％)	NPV(％)	ELISA (combining form) ^aFast detecting Gst-N Gst-U274 combining form	74/24 42/42 36/42 42/42	100 100 85.7 100	1/210 2/210 0/210 2/210	99.5 99.0 100 99.0	98.7 95.3 100 95.3	100 100 97.2 100

^aGst-N adds Gst-U274.

Fast immune chromatographic is measured: when the sample with not diluted detects, be used for the Gst-N of fast detecting and Gst-U274 albumen respectively with from meeting WHO for the IgG antibody (42/42) of 100% in the SARS patient's of the standard of SARS the serum and 85.7% (36/42) IgG antibody response (table 4).Therefore, the complete recall rate of this new assay method is 100% (42/42) (table 4).In this fast measuring, have only Gst-N albumen to be found and cross reaction takes place from 2 parts in the serum of normal healthy controls with 210 parts.Therefore, in tested colony, the specificity that this mensuration demonstrates, PPV and NPV are respectively 99.0,95.3 and 100% (table 4).When the result with the result of this fast detecting and ELISA compares, 99.6% splendid consistance is arranged between the two, the kappa statistical value is 1.00 (tables 5).In addition, during to the reactivity terminal point of ELISA and this fast measuring (being in the dilutability in the scope from 1: 8 to 1: 128), finding has good correlativity (R2=0.988) between the two, (Figure 11) with 7 identical patient's samples.

Table 5: the consistance between immunochromatographic measurement and ELISA measure

Fast measuring	The ELISA number of results		Consistance (%)	The Kappa statistics ^a
	The ELISA number of results				Positive	Negative
	Positive negative	43 0			Positive	Negative	1 208	99.6 100	1.00

^aKappa statistical value 〉=fabulous the consistance of 0.75 expression, 0.40-0.75 represents good consistance,＜0.40 expression consistance poor (11).

The assessment of embodiment 3:ELISA and immunochromatographic measurement

Measure

Material and method

Serum sample: the serum sample collection is suspect to be the patient of SARS and 3 patients that acute disease ground district hospital accepts for medical treatment in Hong Kong clinically from the definition (18) according to WHO between on March 18th, 2003 and May 24.Detect (9) from 227 parts of serum samples altogether of these patients with IF and measure, and be confirmed to be and have IF titre＞1: 10-1: 2560 dilutabilitys.Simultaneously, 385 parts of 385 parts of serum samples from healthy contributor gathering from area, Hong Kong are used as contrast.In addition, (Franklin, 1066 parts of serum that derive from healthy contributor MA) are included in this research as extra normal healthy controls available from BioClinical Partner Inc..(GLD Singapore) also is used the file serum sample from each research formerly for control disease Genelabs Diagnostics; This comprised 50 increments this, they are respectively from patient's (being confirmed to be dengue fever) of the heating relevant with non--SARS or suffer from the patient of the respiratory disorder (being confirmed to be pulmonary tuberculosis) that non--SARS is correlated with.

Immunofluorescence assay: press and prepare and implement the IF detection described in the foregoing description 1.In brief, the smear of the Vero cell that preparation SARS CoV-infects was fixed in the acetone 10 minutes, and is housed in subzero 80 ℃ before use.Before use, determine the cell smear of the SARS CoV-infection of 60-70% with high titre positive control serum sample.Patient's sample adds on the described smear since 1: 10 with continuous dual dilutability, and in 37 ℃ of incubations 30 minutes.In the PBS washed twice, the goat Anti-Human IgG that uses marked by fluorescein isothiocyanate then is in 37 ℃ of described smears of incubation 30 minutes again with described smear.If fluorescence intensity equals or when being better than weak positive control in this research, a certain sample is marked positive.

ELISA: described in above-mentioned embodiment 2, Singapore Genelabs Diagnostics PteLtd proposes to use the ELISA of two kinds of recombinant proteins (Gst-N and Gst-U274).This mensuration is carried out in strict accordance with instructions.In brief, elisa plate has been added the serum of 10 microlitres, with the dilution that contains BSA and skimmed milk power in 200 microlitres/hole based on Tris, and in 37 ℃ of incubations 30 minutes, then with PBST washing 6 times.Add goat-Anti-Human IgG (1: 500 dilution) that HRP-puts together with 100 microlitres/hole, and in 37 ℃ of incubations 30 minutes again.Washing described plate 6 times in PBST then, and the zymolyte TMB (tetramethyl benzidine) that adds 100 microlitres/hole develops the color.Incubation added the 1N HCl cessation reaction in 100 microlitres/hole after 15 minutes in the dark.Optimal light density (OD) with 620nm reference optical filter, is measured optical density (OD) in the 450nm place.

Fast immune chromatographic is measured: once more, and according to the program described in the embodiment 2, at the pick-up unit of Singapore Genelabs Diagnostics Pte Ltd preparation based on film.This device is made up of chromatography strip, separation vessel and absorption layer, and they are all as above-mentioned being placed in the box.

According to the disclosed content of Guan et al (19,20), two kinds of SARS specificity recombinant antibodies will be deposited: Gst-N and Gst-U274 on the described chromatography strip respectively.If contain the antibody of anti-SARS-CoV, detect sample and will be attached to immobilized recombinant and/or formed immune complex, when micelle is discharged by reagent and lavation buffer solution when discharging, described compound can pass through Anti-Human IgG label fixed collaurum detection (19).

This mensuration is carried out (19) in strict accordance with the instructions that manufacturer provided.In brief, the serum sample of 25 microlitres is added to make described sample be displaced sideways and cover an one of film in the view window as a result in the described sample aperture.When the moistening forward position of serum sample touches blue indicator line in the view window, 3 reagent are discharged and lavation buffer solution adds in second hole.The pulling separation vessel adds 1 reagent release and lavation buffer solution in sample aperture again until feeling resistance.

Typically, these results can be in 2-15 minute carry out reading in by view window, but records in the time of 15 minutes all.When only control line occurring, a certain sample is marked negative; But when in view window, observing control line and one or two detection lines, certain sample marked positive (19).

Statistical study: the δ value of measuring with standard deviation unit is defined as the average OD of sample colony from cutoff value than rate variance, and it also is suitable for confirming cutoff value.The Kappa statistical value is used to measure the conforming degree of the result between new fast measuring and the new ELISA.Kappa statistical value＞0.75,0.40-0.75 and＜0.40 represent that respectively consistance is fine, consistance good and consistance poor (17).

The result:

The assessment of ELISA and affirmation: as shown in table 6,3 cutoff values at OD 0.45, OD 0.30 and OD 0.25 place are used to assess the result of ELISA.The setting cutoff value is OD0.45, and ELISA produces 100% best specificity (385/385), but overall sensitivity low be 60% (136/227) (table 6).But, when cutoff value is turned down to OD0.3 or OD0.25, effect improved.Cutoff value be OD0.25 than the ELISA that is set at OD0.45, the recall rate of the sample relevant with SARS is wanted high nearly 12% (table 6) on the whole.Positive δ value is increased to 0.53 from 0.10.Replenish in the detection at one, also 1066 increments from healthy contributor are originally detected, averaging of income OD is 0.0432 ± 0.0745.OD0.25 is found and equals average OD ± 3SD; And OD0.45 equals average OD ± 5SD.OD 0.25 produces to each normal healthy controls of replenishing with OD 0.45 these two cutoff values and suffers from respiratory disorder or the non--similar specificity of SARS disease of patient contrast of heating: 98% and 99.2%, 100% and 100% and 92% and 98% (table 6)

Table 6:

Set OD0.25 when cutoff value, ELISA can not only go out the IgG antibody of the anti-SARS-CoV of convalescence in late period sample with 95% high-sensitivity detection, can also detect the IgG antibody of anti-SARS-CoV of the sample of acute infection.Detect the IgG antibody of anti-SARS-CoV at 58%, 70% and 75% of the SARS patient's who gathers after clinical symptoms takes place 1-10 days, 11-20 days and 21-30 days sample.The normal healthy controls that detects in identical test site still keeps high to 99.5% (383/385) (table 6) in the specificity that cutoff value obtained of OD0.25.The whole PPV that this mensuration therefore provides is 98.8%, NPV is 85.7%.With R ²=0.9342 IF that carries out in identical hole detects resulting IF titre and is found consistent with ELISA result (Figure 12).

The assessment that fast immune chromatographic is measured: when same group of clinical sample being detected, obtained the result similar with ELISA with fast immune chromatographic.Fast measuring is 70.5% (160/227) to the whole recall rate of the relevant sample of SARS, and its specificity is 97.7% (376/385) (table 7).To the recall rate of 4 groups of samples of gathering after clinical symptoms takes place 1-10 days, 11-20 days, 21-30 days and surpass 30 days SARS patient be respectively 55%, 68%, 8l% and 79% (table 7).Because two kinds of recombinant proteins have been used in this detection, they are respectively by the representative of the band of two gained, and the result of two kinds of albumen is also analyzed.Detect separately the antibody of the anti-SARS-CoV of Gst-N albumen, by the combination of two kinds of albumen, 157 in 160 samples are found be positive (table 7).In addition, this Gst-N albumen shows less cross reactivity, only with from wherein 1 reaction in 385 samples of normal healthy controls.On the contrary, Gst-U274 only is 8% (18/227) to the whole recall rate of the relevant sample of SARS, but accounts for the overwhelming majority (8/385) from the nonspecific reaction of the sample of normal healthy controls.Enjoyably, 3 samples that only detect without Gst-N albumen with Gst-U274 are early stage patients's (table 7) of 1-20 days that gather from being in after clinical symptoms takes place.PPV and NPV that therefore this mensuration demonstrate examined colony are respectively 94.7% and 84.9% (table 7).Once more in one replenish to be detected, with the non-SARS disease of patient control group of suffering from breathing problem or heating to this fast measure only further assess and find with 50 examined samples of each group in 3 or 4 sample generation cross reactions (table 7).When comparing fast measuring and ELISA access node fruit, both have the consistance 92.5% of splendid whole recall rate, and the kappa statistical value is 0.81 (table 8).The result of this mensuration be found with R ²=0.9182 carries out IF detects resulting immunofluorescence (IF) titre and is found consistent with ELISA result (Figure 12) in identical hole.

Table 7: fast immune chromatographic is determined at the effect in the IgG antibody that detects anti-SARS

Table 8: the consistance between immunochromatography and ELISA measure

Fast measuring	ELISA detects		Consistance (%)	The Kappa statistical value ^a
	ELISA detects				Positive	Negative
	Positive negative	144 21			Positive	Negative	25 422	85.2％ 95.3％	0.81

Embodiment 4: the Western trace that is used as the affirmation mensuration of SARS diagnosis

Material and method

Serum sample: after agreeing, the SARS patient who accepts for medical treatment from Tan Tock Seng hospital or Singapore General hospital gathers 40 convalescent serum samples.(Franklin, 50 serum from healthy contributor Mass.) also are included in this research as normal healthy controls available from BioClinicalPartner Inc..For non--SARS disease contrast, the Genelabs Diagnostics that from previous research, preserves (Genelabs Diagnostics, Singapore) serum sample, and they are to obtain have been used before the SARS outburst.These comprise 50 samples, and they are respectively from patient's (being confirmed to be dengue fever) of the heating relevant with non--SARS or suffer from the patient of the relevant respiratory disorder (being confirmed to be pulmonary tuberculosis) of non--SARS.In addition, being accredited as from 1066 healthy contributors that screen in the research formerly (Guan et al. submission) is that false-positive 18 samples also are used to this research.

SARS-CoV virolysis product and recombinant protein: SARS-CoV virolysis product is available from ZeptoMetrix Corporation (Buffalo, New York), and they are available from through saccharose gradient purifying and the lysate through containing Triton X-100 (0.5%) (KCl, 0.6M) the Vero cell that is infected by SARS CoV after handling.

As above-mentioned preparation recombinant protein.In brief, all protein form with the GST-fusion in E.coli is expressed, but has only GST-N to carry out purifying with GSH-Ago-Gel pearl (Amersham Pharmacia).For GST-S, GST-M and GST-E albumen, washing and resuspended and final electrophoresis in the 10%PAGE-SDS gel by to albumen separate each the insoluble protein in the bead.Downcut the gel strips that contains each gst fusion protein then, and carry out wash-out with Mini transfer printing unit (BioRad).With anti--GST monoclonal antibody (Santa Cruz Biotechnology, Santa Cruz, Calif.) in the Western trace, detect the fusion of gained, compare the concentration that estimates them by BSA standard (Shen et al. submission) with the PAGE gel of Coomassie blue stain.

Mouse and rabbit anti-serum: it is described to press document 21, produces specific antisera with each recombinant protein inoculation mouse or rabbit.(MO) each the recombinant protein 50 microgram subcutaneous vaccination BALB/c mouse in produce spike protein, nucleocapsid protein or memebrane protein are had specific mouse-anti serum for Sigma, St Louis with being emulsified in complete Freund's complete adjuvant.And the coating recombinant protein is had specific rabbit anti-serum with the purifying protein 1mg subcutaneous vaccination New Zealand white rabbit generation that also is emulsified in the complete Freund's complete adjuvant.(MO) the per two weekly interval booster immunization animals of each purifying protein in are 1 time for Sigma, St Louis, 8-16 time altogether with the complete Freund's complete adjuvant of being emulsified in of same amount.Last 1 immunity is collected after 10 days from the serum of the animal of quilt immunity, and adsorbs to reduce the non-specific binding of pair cell albumen with mammalian cell cultures.

SDS-polyacrylamide gel electrophoresis (PAGE) and transfer printing: on 11% separating gel with 3.5% lamination gel separation SARS CoV virolysis product.Especially, will in water-bath, boil 5 minutes in the 70 microgram SARS CoV virolysis products that 800 microlitres are followed the tracks of by the bromophenol blue of 3.2%SDS, 0.5M Tris (pH6.8), 32% glycerine, 3.2% 2 mercapto ethanol and 0.05% in the sex change damping fluid that dyestuff forms.For determining molecular weight, every kind of treated lysate sample of 50 microlitres and color molecule mark are added in each hole on the same SDS-PAGE gel (8mm is wide).For other immunoblotting assay, treated sample (800 microlitre) is added in the preprepared hole (130mm is wide) and carries out electrophoresis under a steady current and arrives the bottom until following the tracks of dyestuff.Separated protein slot type equipment (Hoeffer, San Francisco, Calif.) in by electrotransfer to nitrocellulose filter (Whatman, Gerbershausen, Germany) on.After the transfer, use Autoslot ^TM(Genelabs Diagnostics Singapore), further deposits described film with goat Anti-Human IgG (1.6 micrograms/film are added contrast as sample) and GST-N recombinant protein (4.3ng/ film) to machinery.With described film incubation 45 minutes in the hybridization solution that contains 5% skimmed milk power, in containing the PBS of 0.5%Tween-20, carry out rinsing afterwards then.Subsequently, described film placed down in room temperature (RT) carried out drying in 20 minutes, be cut into the bar of 3mm then and be stored at 2-8 ℃ standby.

The Western Western blotting is measured and analyzed: the Western Western blotting is measured and carry out all incubation step on the platform that room temperature is being shaken.When detecting, the film bar be placed on automatic hybridization incubation dish (Genelabs Diagnostics, Singapore) in and every be immersed in 1ml based in the lavation buffer solution of Tris 5 minutes.Damping fluid is siphoned away, be respectively applied for 10 microlitre serum in the liquid of blockading of the 1ml that contains 5% milk powder subsequently described film bar incubation 1.After siphoning away damping fluid, wash described film bar 3 times with the lavation buffer solution of 1ml/ bar, each washing was soaked 5 minutes.With 1ml by the conjugate (Kirkegaard﹠amp of the goat Anti-Human IgG of alkali phosphatase enzyme mark; Perry Laboratory Inc., Gaithersburg, MD) the described film bar of incubation 1 hour again, described conjugate is 1: 1000 a dilution in the damping fluid of blockading.Behind the incubation, described conjugate is absorbed once more, the film bar is washed 3 times again.Subsequently, use 5-bromo-4-chloro-3-indolylphosphate (BCIP) and the described film bar of nitroblue tetrazolium (NBT) substrate solution incubation 15 minutes.Brightness by the band on described is carried out subjective analysis to the protein band of gained as a result.

For the mensuration of carrying out with mouse or rabbit anti-serum, because their titre difference, each serum uses a specific dilution.Especially, 1: 1,000 dilution is used to mouse-anti-S antiserum and rabbit resists-the E antiserum, and 1: 500 dilution is used to mouse-anti M antiserum, and 1: 100,000 dilution was used to mouse-anti-N antiserum.But the conjugate that is used to detect anti--mouse of separately sero-fast alkali phosphatase enzyme mark or anti--rabbit antibody uses with 1: 5000 identical dilution.

The result

Identify the immunoreactive protein of SARS CoV by the Western immunoblotting assay: the apparent molecular weight of immunoreactive protein is estimated (Figure 13) by inferring molecular weight with respect to the logarithmic curve chart of the electrophoretic mobility of standard protein.When sample discrete when comprising with strong SARS positive control (P8), diffusion and that cluster is measured, immunoreactive protein appearss as the band on the Western blotting, and they locate (Figure 13) about 150ka, 97kDa (three parts), 45kDa (a pair of), 28kDa (three parts) and 24kDa (diffusion) greatly.When detecting with caused mouse of specificity recombinant protein or rabbit anti-serum, 150kDa, 45kDa and 24kDa albumen are found respectively and spike protein (S), nucleocapsid protein (N) and matrix (M) albumen relevant (Figure 14).In addition, a kind of not with the reaction of strong SARS positive sample but the protein relevant with coating (E) albumen also resisted by rabbit-the E antiserum is positioned about 10kDa place (Figure 14).

Different protein labelings and Western exempt from the result of trace: be specific to from SARS patient's sample or from the different binding pattern (Figure 15) of the band style of the sample of control group when using not on the same group sample from SARS patient or health or disease control group to carry out Western blotting when detecting, observing.The analysis showed that of band style, the most normal appearance of protein band (Nh and N1) of the two kind about 45kDas relevant with the N albumen of SARS CoV is with all sample reactions (Figure 15, table 9) from SARS patient.But although these two kinds of albumen are higher with the band brightness from SARS patient's sample, Nh and N1 both have more non-specific, also with sample reaction (Figure 15) from health or disease control group.For example, cross reaction (table 10) takes place with 64%, 68% and 52% of the sample that contrasts from health, breathing problem or heating respectively in Nh albumen.In fact, 15 (83%) reaction in the protein that these are relevant with N and 18 samples, this formerly is detected as false positive (table 10) by ELISA.On the contrary, S, M and GST-N recombinant protein with from 78%, 75% and 100% in 40 samples of SARS patient reactions, and do not comprise by ELISA and be accredited as false-positive sample generation cross reaction (table 10) with any contrast.Other high immunoreactive protein comprises that 97kDa albumen and 28kDa albumen react respectively with from 78% and 75% of SARA patient's sample; And only with a small amount of control reaction, cross reacting rate is 2%-6% (table 10).In addition, at the fixed band of having found a treaty 60kDa in the research but not occurred of organizing with the strong SARS positive control that is used for the molecular weight estimation.Part reaction (11-34%) in this 60kDa albumen and all examined samples, and no matter they are from SARS patient or health or disease control group (table 10).On the other hand, the human serum sample that demonstrates being detected in any the research of the E albumen of being identified by rabbit anti-serum does not have immunoreactivity (table 10).

Table 9:Western exempts from the sensitivity and the specificity of trace

The reaction pattern EMI53.1 of the immunoreactive protein in the table 10:Western Western blotting

The further analysis of the band style of immunoreactive protein has disclosed and has been used to distinguish from SARS patient's sample with from the useful standard of the sample of control group.Be configured to testing result when being interpreted as the condition precedent of the SARS positive when detecting N albumen (Nh and N1) and S albumen simultaneously, find that Western blotting has 78% sensitivity and 100 specificity (table 9) for 4 control groups.Similarly, when N albumen (still Nh and N1) when being used for result's explanation with 97kDa, 28kDa, M or GST-N albumen respectively, find that Western blotting mensuration has 78%, 75%, 75% and 100% sensitivity respectively, has kept 100% specificity (table 9) simultaneously.But owing to lack sensitivity, 60kDa and E albumen are found in the effect little (table 9) in any combination.Use N albumen and at least a specific proteins to comprise that the joint interpretation standard of S, 97kDa, 28kDa and M albumen provides 95% kit sensitivity and 100% specificity (table 11).The GST-N recombinant protein is additionally joined in the above-mentioned criteria for interpretation, sensitivity is increased to 100% and do not change specificity (table 11).

Table 11 is based on the sensitivity and the specificity of the Western Western blotting of established standards

It will be understood by those skilled in the art that and to carry out multiple modification the illustrative embodiments of being put down in writing among the application.As claim limited, all such modifications were also further comprised within the scope of the invention.

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Claims

1. one kind is used for the method whether detection exists the antibody of sars coronavirus (CoV), this method is included in is enough to make antibody and antigen to form under the condition of compound, SARSCoV antigen is contacted one section with the sample that contains antibody be enough to make that antibody and antigen form the step of the time of compound, and wherein said antigen comprises the protein of a kind of S of being selected from, M, E, N, U274 and immunogenic fragments thereof; And wherein said antibody combines this sample of indication and contains SARS CoV is had specific antibody with specificity between described protein.

2. the method for claim 1, it is used to detect the people and whether has been infected or be exposed to SARS CoV by SARS CoV, the wherein said sample that contains antibody is from detected people, and wherein said antibody combines this people of indication with specificity between described protein and infected or be exposed to SARS CoV by SARSCoV.

3. claim 1 or 2 method, wherein said antigen comprises the protein of a kind of M of being selected from, E, N, U274 and immunogenic fragments thereof.

4. claim 1 or 2 method, wherein said antigen comprises:

The 46-480 amino acids of S albumen,

The N albumen that the 111-118 amino acids is lacked, or

The 134-274 amino acids of U274.

5. the method for claim 3, wherein said antigen comprises:

The N albumen that the 111-118 amino acids is lacked, or

The 134-274 amino acids of U274.

6. each method among the claim 1-3, wherein said antigen comprises N or U274 or its immunogenic fragments.

7. each method among the claim 1-6, wherein said protein merges in a heterologous protein.

8. the method for claim 7, wherein said heterologous protein is glutathione S-transferase (GST).

9. each method among the claim 1-8, wherein said antigen is immobilized.

10. each method among the claim 3-9, wherein said sample is a serum.

11. the method for claim 10, wherein said serum were gathered between 2-60 after the infection days from people that infected by SARS CoV or that be exposed to SARS CoV.

12. the method for claim 10, wherein said serum were gathered between 2-11 after the infection days from people that infected by SARS CoV or that be exposed to SARS CoV.

13. the method for claim 10, wherein said serum were gathered between 16-60 after the infection days from people that infected by SARS CoV or that be exposed to SARS CoV.

14. each method among the claim 1-13 also comprises the step that detects formed compound between described antibody and the described antigen.

15. comprising with a kind of anti--homotype antibody at people's antibody, the method for claim 14, the step that wherein detects described compound contact described compound.

16. the method for claim 12 also comprises by contacting compound to detect the step of described compound with a kind of Anti-Human IgA antibody or Anti-Human IgM antibody.

17. the method for claim 13 also comprises by contacting compound to detect the step of described compound with a kind of Anti-Human IgG antibody.

18. comprising with enzyme, coloured dyestuff, fluorescent dye, chemiluminescent molecule, the mode that contains the molecule of radioactive atom or contain the molecule of heavy metal, each method among the claim 14-17, the step that wherein detects compound make described compound colour developing.

19. the method for claim 18, wherein said enzyme are alkaline phosphatase or horseradish peroxidase; Wherein said fluorescent dye is FITC or rhodamine; The wherein said molecule that contains heavy metal is a colloidal gold composite.

20. the isolated antibody at antigenicity SARS CoV albumen, wherein said antigenicity SARS CoV albumen are S, M, E, N or U274 albumen or its antigenicity fragment.

21. the antibody of claim 20, wherein this antibody is at M, E, N or U274 albumen or its antigenicity fragment.

22. the antibody of claim 20, wherein said antibody is anti-:

The 46-480 amino acids of S albumen,

The N albumen that the 111-118 amino acids is lacked, or

The 134-274 amino acids of U274.

23. the method for claim 21, wherein said antibody is anti-:

The N albumen that the 111-118 amino acids is lacked, or

The 134-274 amino acids of U274.

24. each antibody among the claim 20-23, wherein said antibody is polyclonal antibody.

25. each antibody among the claim 20-23, wherein said antibody is monoclonal antibody.

26. one kind is used for detecting the commercial kit whether sample exists the antibody of sars coronavirus (CoV), described kit comprises:

(a) a kind of SARS CoV antigen, this SARS CoV antigen comprises the protein of a kind of S of being selected from, M, E, N, U274 and immunogenic fragments thereof; With

(b) be used to detect from the antibody of described sample and the instrument of the compound between described antigen.

27. one kind is used to detect whether someone has been infected or be exposed to SARS CoV by sars coronavirus (CoV) commercial kit, described kit comprises:

(b) be used to detect described antigen and at the instrument of the compound between the antibody of this antigen, wherein said sample is detected people's the sample that contains antibody.

28. the kit of claim 26 or 27, wherein said antigen comprises the protein of a kind of M of being selected from, E, N, U274 and immunogenic fragments thereof.

29. the kit of claim 26 or 27, wherein said antigen comprises:

The 46-480 amino acids of S albumen,

The N albumen that the 111-118 amino acids is lacked, or

The 134-274 amino acids of U274.

30. the kit of claim 28, wherein said antigen comprises:

The N albumen that the 111-118 amino acids is lacked, or

The 134-274 amino acids of U274.

31. the kit of claim 26 or 27, wherein said antigen comprise N or U274 or its immunogenic fragments.

32. each kit among the claim 26-31, wherein said protein merges in a heterologous protein.

33. the kit of claim 32, wherein said heterologous protein are glutathione S-transferase (GST).

34. each kit among the claim 26-33, wherein said antigen is immobilized.

35. each kit among the claim 26-34, wherein said sample is a serum.

36. the kit of claim 35, wherein said serum were gathered between 2-60 after the infection days from people that infected by SARS CoV or that be exposed to SARS CoV.

37. the kit of claim 36, wherein said serum were gathered between 2-11 after the infection days from people that infected by SARS CoV or that be exposed to SARS CoV.

38. the kit of claim 36, wherein said serum were gathered between 16-60 after the infection days from people that infected by SARS CoV or that be exposed to SARS CoV.

39. each kit among the claim 26-38, the instrument that wherein is used to detect described compound comprises the anti--homotype antibody at people's antibody.

40. the kit of claim 37, the instrument that wherein is used to detect described compound comprises Anti-Human IgA antibody or Anti-Human IgM antibody.

41. the kit of claim 38, the instrument that wherein is used to detect described compound comprises Anti-Human IgG antibody.

Be selected from enzyme, coloured dyestuff, fluorescent dye, chemiluminescent molecule, contain the molecule of radioactive atom and contain the colour developing instrument of the molecule of heavy metal 42. each kit among the claim 39-41, the instrument that wherein is used to detect compound further comprise.

43. the kit of claim 42, wherein said enzyme are alkaline phosphatase or horseradish peroxidase; Wherein said fluorescent dye is FITC or rhodamine; The wherein said molecule that contains heavy metal is a colloidal gold composite.

44. whether a detection exists the method for S, M, E, N or U274SARS CoV albumen, this method is included in is enough to make antibody and antigen to form under the condition of compound, and will contact one section at the antibody of a kind of protein that is selected from S, M, E, N and U274 with the sample that contains antigen be enough to make that antibody and antigen form the time of compound; And wherein said antibody combines this sample of indication and contains the antigen that comprises S, M, E, N, U274 or its immunogenic fragments with specificity between described antigen.

45. whether a detection exists the method for M, E, N or U274 SARS CoV albumen, this method is included in is enough to make antibody and antigen to form under the condition of compound, and will contact one section at the antibody of a kind of albumen that is selected from M, E, N and U274 with the sample that contains antigen be enough to make that antibody and antigen form the time of compound; And wherein said antibody combines this sample of indication and contains the antigen that comprises M, E, N, U274 or its immunogenic fragments with specificity between described antigen.

46. the method for claim 44 or 45 further comprises the step that detects the compound between described antibody and the described antigen.

47. the method for claim 46, it is Western Western blotting, ELISA, catch ELISA or immunofluorescence assay.

48. comprising with enzyme, coloured dyestuff, fluorescent dye, chemiluminescent molecule, the mode that contains the molecule of radioactive atom or contain the molecule of heavy metal, the method for claim 46 or 47, the step that wherein detects described compound make described compound colour developing.

49. the method for claim 48, wherein said enzyme are alkaline phosphatase or horseradish peroxidase; Wherein said fluorescent dye is FITC or rhodamine; The wherein said molecule that contains heavy metal is a colloidal gold composite.