WO2004109289A1 - Method of diagnosing sars corona virus infection - Google Patents

Method of diagnosing sars corona virus infection Download PDF

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Publication number
WO2004109289A1
WO2004109289A1 PCT/SG2004/000174 SG2004000174W WO2004109289A1 WO 2004109289 A1 WO2004109289 A1 WO 2004109289A1 SG 2004000174 W SG2004000174 W SG 2004000174W WO 2004109289 A1 WO2004109289 A1 WO 2004109289A1
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Prior art keywords
antibody
protein
antigen
sars
complex
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PCT/SG2004/000174
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English (en)
French (fr)
Inventor
Yee-Joo Tan
Phuay-Yee Goh
Burtram Clinton Fielding
Shuo Shen
Seng Gee Lim
Wan Jin Hong
Yijun Ruan
Chia Lin Wei
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Agency For Science, Technology And Research
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Application filed by Agency For Science, Technology And Research filed Critical Agency For Science, Technology And Research
Priority to AU2004245896A priority Critical patent/AU2004245896A1/en
Priority to JP2006517063A priority patent/JP2006527382A/ja
Priority to EP04736615A priority patent/EP1639374A4/en
Publication of WO2004109289A1 publication Critical patent/WO2004109289A1/en

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C07K16/1002—Coronaviridae
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/08—RNA viruses
    • G01N2333/165—Coronaviridae, e.g. avian infectious bronchitis virus

Definitions

  • the present invention relates generally to methods of diagnosing viral infection, and particularly to methods of diagnosing SARS coronavirus infection.
  • SARS Severe Acute Respiratory Syndrome
  • a novel coronavirus was identified as the etiological agent of SARS (1- 4).
  • Coronaviruses are enveloped viruses that contain a single-stranded, positive-sense RNA genome of 27.6 to 31 kb.
  • Analyses of the nucleotide sequence of the novel SARS coronavirus (SARS CoV) showed that the viral genome is nearly 30 kb in length (5, 6) and contains 14 potential open reading frames (ORFs) (5).
  • ORFs open reading frames
  • the invention provides a method for detecting the presence or absence of antibody to SARS coronavirus (CoV), the method comprising the step of contacting a SARS CoV antigen with an antibody-containing sample, for a time and under conditions sufficient for the antibody to form a complex with the antigen, wherein the antigen comprises a protein selected from the group consisting of S, M, E, N, U274 and an immunogenic fragment thereof, and wherein specific binding between the antibody and the protein indicates that the sample contains antibody specific to SARS CoN.
  • CoV SARS coronavirus
  • the method may be used to test whether a human is infected by, or has been exposed to, SARS CoV, wherein the antibody-containing sample is from the human being tested, and wherein specific binding between the antibody and the protein indicates that the human is infected by or has been exposed to SARS CoN.
  • the invention also provides commercial packages.
  • a commercial package for detecting the presence or absence of antibody to SARS coronavirus (CoV) in a sample comprising a SARS CoV antigen comprising a protein selected from the group consisting of S, M, E, ⁇ , U274, and an immunogenic fragment thereof; and means for detecting a complex between the antibody from the sample and the antigen.
  • CoV SARS coronavirus
  • a commercial package for testing whether a human is infected by or has been exposed to SARS coronavirus comprising a SARS CoV antigen comprising a protein selected from the group consisting of S, M, E, ⁇ , U274, and an immunogenic fragment thereof; and means for detecting a complex between the antigen and an antibody against the antigen, wherein the sample is an antibody-containing sample of the human being tested.
  • SARS coronavirus CoV
  • the invention provides an isolated antibody directed against an antigenic SARS CoV protein, wherein the antigenic SARS CoV protein is S, M, E, ⁇ or U274 protein or an antigenic fragment thereof.
  • the antibodies of the invention may be used to detect the presence of S, M, E, ⁇ or U274 protein of SARS CoN in a sample.
  • the invention provides a method of detecting the presence or absence of S, M, E, ⁇ or U274 SARS CoV protein, the method comprising contacting an antibody directed against a protein selected from the group consisting of S, M, E, ⁇ and U274, with an antigen-containing sample, for a time and under conditions sufficient for the antibody to form a complex with an antigen; and wherein specific binding between the antibody and the antigen indicates that the sample contains an antigen that comprises S, M, E, ⁇ , U274 or an immunogenic fragment thereof.
  • Figure 1 is a schematic depiction of the structural organization and expression of ORFs corresponding to structural proteins S, M, N, and E and unique proteins (UX), where "X" denotes the number of amino acids encoded by the respective ORF; corresponding annotated ORFs 1 to 14 are indicated;
  • Figure 2 is a Western blot analysis demonstrating the presence of antibodies against the various SARS-CoV viral proteins in patient sera;
  • Figure 3 depicts bacterially expressed GST-U274, GST-U122, GST-N, and GST proteins analyzed by SDS-PAGE and stained with Coomassie brilliant blue R-250 (Bio-Rad);
  • Figure 4 illustrates detection of bacterially expressed viral proteins by Western blot analysis with anti-GST antibody, antibodies in a control serum, or antibodies in six convalescent-phase sera;
  • Figure 5 illustrates detection of N and U274 by Western blot using sera from patients during early and late phases of infection as primary antibody and anti- human IgG as secondary antibody;
  • Figure 6 is a Western blot using some of the samples as depicted in Figure 5, using anti-human IgG, anti-human IgA, or anti-human IgM antibody as secondary antibody;
  • Figure 7 illustrates detection of anti-S antibodies in patient sera by an immunofluorescence method utilizing CHO cells stably expressing the S protein
  • Figure 8 is a photograph of examples of the assembled rapid immunochromatographic test devices with their separators (transparent tabs) at the "removed" (after assay) position, either after an assay with a sample from an infected patient (left), or after an assay with a sample from a healthy control (right), showing lines for the control, Gst-N, and Gst-U274, from top to bottom respectively;
  • Figure 9 is a scatter chart of OD values obtained with sera from both SARS patients and healthy controls tested for IgG antibody to SARS CoN, using an ELISA assay;
  • Figure 10 is a line graph showing titration curves obtained with the ELISA assay using serial dilutions of seven SARS patient samples;
  • Figure 11 illustrates the correlation between the ELISA and the rapid immunochromatography test when comparing the dilutions at which reactivity end points were obtained with seven SARS patient samples;
  • Figure 12 illustrates the distribution of percentage detection rates of the ELISA assay and the rapid immunochromatography assay in relation to sample immunofluorescence titers
  • Figure 13 illustrates Western immunoblot pattern of immunoreactive proteins of SARS-CoV
  • Figure 14 is a Western blot demonstrating the location of the immunoreactive proteins of SARS-CoV as identified with antibodies raised against specific recombinant proteins (1: SARS-positive control; 2: mouse anti-spike protein antiserum; 3: mouse anti-nucleocapsid protein antiserum; 4: mouse anti-matrix protein antiserum; 5: rabbit anti-envelop protein antiserum); and
  • Figure 15 shows representative immunoreactive patterns of the SARS Western immunoblot with serum samples (A: SARS patients; B: healthy controls; C: non-SARS fever patient controls; D: non-SARS respiratory disease controls; D: false positive identified by ELISA).
  • SARS-CoV The sequence of SARS-CoV reveals ORFs for four structural proteins, i.e., spike (S), membrane (M), envelope (E), and nucleocapsid (N), which are conserved in all coronaviruses (5, 6, 10, 11).
  • S protein plays essential roles in mediating receptor binding and internalization of the virus and is one of the major antigens of the virus.
  • M and E proteins are essential for virion assembly, and the N protein binds to the viral genome to form the nucleocapsid.
  • the present invention relates to the discovery that the S, M, E and N structural proteins of SARS CoV, as well as one of the proteins of unknown function having a predicted length of 274 amino acids (referred to herein as U274), are antigenic and may be used to detect the presence of anti-SARS CoV antibodies in a patient, indicating the patient is infected with SARS CoV, or has been exposed to SARS CoV.
  • a method of diagnosing SARS CoV infection in a patient using the antigenic SARS CoV proteins S, M, E, N and U274.
  • One or more of the antigenic proteins is expressed, and is contacted with a biological sample from a patient whom is to be tested for SARS CoV infection. If the patient is infected with SARS CoV or has been exposed to SARS CoV, and has generated antibodies against the virus, antibodies in the sample directed against the particular SARS CoV antigenic protein being used for diagnosis will bind to the protein.
  • a secondary antibody directed against the patient-generated antibodies and conjugated to a detection molecule may be used to detect the patient-generated antibodies.
  • antigenic protein of SARS CoV refers to one or more of the SARS CoV S, M, E, N or U274 protein.
  • immunogenic fragment thereof or antigenic fragment thereof refers to a portion of the SARS CoV S, M, E, N or U274 protein which is immunogenic or antigenic, meaning that the protein fragment will contain one or more epitopes, thereby being capable of eliciting an immune response in a patient.
  • polypeptide fragments preferably are at least 12 amino acids in length.
  • polypeptide fragments are at least 15 amino acids, preferably at least 20, 25, 30, 35, 40, 45, 50 amino acids, more preferably at least 55, 60, 65, 70, 75 amino acids, and most preferably at least 80, 85, 90, 95, 100 amino acids in length.
  • homologous refers to a protein sequence that has substantial correspondence with the amino acid sequence of S, M, E, N or U274, or a fragment thereof.
  • the homolog may be at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 87%, 90%, 93%, 96% and 99% homologous to S, M, E, N or U274, or a fragment thereof.
  • Homology is measured using sequence analysis software such as Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, WI 53705. Amino acid sequences are aligned to maximize identity. Gaps may be artificially introduced into the sequence to attain proper alignment. Once the optimal alignment has been set up, the degree of homology is established by recording all of the positions in which the amino acids of both sequences are identical, relative to the total number of positions.
  • Polynucleotides of 30 to 600 nucleotides, as applicable, encoding partial sequences of, or sequences homologous to, or partial sequences homologous to, any one of S, M, E, N and U274 are retrieved by PCR amplification using the parameters outlined above and using primers matching the sequences upstream and downstream of the 5' and 3' ends of the fragment to be amplified.
  • the template polynucleotide for such amplification is either the full length polynucleotide that encodes full-length S, M, E, N or U274, or a polynucleotide contained in a mixture of polynucleotides such as a DNA or RNA library.
  • polypeptide derivatives are designed using computer-assisted analysis of amino acid sequences. This would identify probable surface-exposed, antigenic regions (Hughes et al., 1992. Infect. Immun. 60(9):3497). Analysis of amino acid sequences contained in any one of S, M, E, N or U274, based on the product of flexibility and hydrophobicity propensities using the program SEQSEE (Wishart DS, et al. "SEQSEE: a comprehensive program suite for protein sequence analysis.” Comput Appl Biosci.
  • Probable T-cell epitopes for HLA-A0201 MHC subclass may be revealed by an algorithm that emulates an approach developed at the NIH (Parker KC, et al. "Peptide binding to MHC class I molecules: implications for antigenic peptide prediction.” Immunol Res 1995;14(l):34-57).
  • Epitopes which induce a protective T cell-dependent immune response are present throughout the length of the polypeptide. However, some epitopes may be masked by secondary and tertiary structures of the polypeptide. To reveal such masked epitopes large internal deletions are created which remove much of the original protein structure and expose the masked epitopes. Such internal deletions sometimes effect the additional advantage of removing immunodominant regions of high variability among strains.
  • Polynucleotides encoding polypeptide fragments and polypeptides having large internal deletions are constructed using standard methods (Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons Inc., 1994). Such methods include standard PCR, inverse PCR, restriction enzyme treatment of cloned DNA molecules, or the method of Kunkel et al. (Kunkel et al. Proc. Natl. Acad. Sci. USA (1985) 82:448). Components for these methods and instructions for their use are readily available from various commercial sources such as Stratagene.
  • the full-length antigenic protein or proteins may be used in the diagnosis method. However, it is preferred to use an antigenic fragment of the relevant SARS CoV antigenic protein that is not highly conserved among coronaviruses, so as to minimize the chance of cross-reactivity with patient-generated antibodies directed against other coronaviruses. As well, it may be advantageous to delete portions of the relevant SARS CoV antigenic protein that are predicted to be transmembrane domains, or to use only a soluble antigenic fragment of the relevant protein, for ease of expression and handling of the protein, as will be understood by a skilled person.
  • N appears to be the most abundant protein in SARS-CoV (6).
  • the results presented herein indicate that N generates a strong immunoreaction.
  • N may be released from SARS CoV or from infected patient cells into the circulation at some stage of infection, or possibly that N may be presented by antigen-presenting cells for cytotoxic killing of infected cells. Furthermore, it is possible that N may contribute to the humoral immune response protecting patients against SARS.
  • N protein For the N protein, when the sequences of 18 SARS isolates deposited in the GenBank database were compared, only two of them showed a difference, at one amino acid, from that of the Singapore isolate (SIN2774) used for the clones described in the Examples below (13). Therefore, the N protein does not appear to undergo rapid mutation, which is another advantage for its use in the present method.
  • the presence of antibodies directed against N can be detected in patients as early as 2 days post-infection, as well as later in infection, for example, 16 days post-infection.
  • the early elicitation of immune response, in combination with an extremely strong immune response make N extremely useful in diagnosing SARS CoV infection.
  • the protein used to detect the presence of anti-SARS CoV antibodies in a patient is N or an antigenic fragment thereof.
  • N may be full-length N, or it may be N ( ⁇ l 11-118), which refers to N having amino acids 111 to 118 deleted. These amino acids correspond to the sequence FYYLGTGP, a highly conserved sequence found in the N homologue of coronaviruses.
  • An immunogenic fragment of N may be used, for example a fragment comprising or consisting of amino acids 69 to 422, 120 to 422, or 121 to 422.
  • S forms the petal-shaped spikes found on the surfaces of coronaviruses (10, 11). This protein is also highly immunogenic. S is a large protein, with a number of transmembrane regions, and is highly glycosylated. The S protein is predicted to contain hypervariable regions, as these regions appear in S proteins of other coronaviruses (10, 11). It may be preferred to use an extracellular region of S that does not contain a hypervariable region, to ensure ease of handling and to increase the likelihood of a properly positive diagnosis for patients infected with SARS CoV.
  • full-length S may be used, or for example, a fragment of S comprising or consisting of amino acids 460 to 480 may be used.
  • U274 corresponding to ORF 3, as annotated in reference 5, and XI in reference 6
  • the immunoreactivity of U274 indicates that this novel and unique viral protein is expressed in the virus and is likely to be a protein involved in the biogenesis of SARS-CoV.
  • a potential transcription regulating sequence was found upstream of the U274 coding sequence, suggesting that it is the first ORF for one of the major subgenomic RNAs of SARS-CoV-infected cells (5, 6).
  • U274 does not share significant homology with any protein in the database, but it appears to have a similar topology to that of M, with three transmembrane regions and a large internal C-terminal domain. Since it easier to express and purify soluble fragments of proteins, it may be preferred, when using U274 in the present method, to use a fragment that does not include the transmembrane regions of U274.
  • U274 does tend to exhibit some low level sequence variation in viral isolates, with five isolates of those studied having U274 sequences showing one amino acid substitution (when compared to SIN2774) and one isolate having a U274 sequence showing two amino acid substitutions (14).
  • the antigenic SARS CoV protein used may be full-length U274, or it may be an antigenic fragment of U274 comprising or consisting of amino acids 134 to 274.
  • the antigenic SARS CoV protein used may be full-length M, or it may be an antigenic fragment of M comprising or consisting of amino acids 98 to 121. In other embodiments, the antigenic SARS CoV protein used may be full-length E, or it may be an antigenic fragment of E comprising or consisting of amino acids 38 to 76.
  • the antigenic protein or proteins are first expressed, and may be expressed using methods known in the art. "Expressing" a protein refers to the synthesis of a protein or polypeptide by the translation of an RNA template, usually an mRNA, which encodes the protein or polypeptide and may include a transcription step in which an RNA template is transcribed by an RNA polymerase enzyme from a DNA template.
  • the protein may be expressed within in any expression system, such as a cell, or within a cell-free system.
  • the proteins may be expressed in a prokaryotic expression system, for example in bacteria such as Escherichia coli, or in a eukaryotic expression system, for example, in yeast including Saccharomyces cerevisiae or Pichia pastoris, in mammalian cells including COS1, COS7, CHO, NIH3T3, or JEG3 cells, in insect cells including Spodoptera frugiperda (SF9) cells, or in plant cells.
  • a prokaryotic expression system for example in bacteria such as Escherichia coli, or in a eukaryotic expression system, for example, in yeast including Saccharomyces cerevisiae or Pichia pastoris, in mammalian cells including COS1, COS7, CHO, NIH3T3, or JEG3 cells, in insect cells including Spodoptera frugiperda (SF9) cells, or in plant cells.
  • the proteins may be expressed in a cell-free expression system, and such a system will include the reagents necessary to effect expression of the protein, including ribosomes, tRNAs, amino acids, including amino acyl tRNAs, RNA template, and may further include DNA template, RNA polymerase, ribonucleotides, and any necessary cofactors, buffering agents and salts that are required for enzymatic activity, and may include a cell lysate.
  • a bacterial expression system may be preferred for a protein that is not post- translationally modified and is expected to be soluble.
  • a eukaryotic system for example a mammalian system, may be preferred.
  • a gene encoding the antigenic protein or protein fragment will be cloned into an expression vector that is compatible with the expression system chosen to express the antigenic protein, using standard techniques known in the art.
  • the expression vector will have the necessary promoter and genetic signals required to effect expression in the chosen system.
  • the expression vector may comprise a gene encoding the protein operably linked to a promoter that is compatible with the particular expression system, and may be a plasmid.
  • the cell may be an E. coli cell, and the expression vector may contain a gene encoding the antigenic SARS CoV protein of interest operably linked to all of the necessary regulatory sequences such that the gene is transcribed and the RNA is translated by the E. coli cellular machinery.
  • the expression of the gene encoding the protein of interest may be driven by an inducible promoter such that the expression within the cell may be controlled as desired, so as to maximize expression, for example by synchronizing protein expression with logarithmic growth phase of the cell culture.
  • a fusion protein is a protein or polypeptide that contains an antigenic SARS CoV protein fused at the N- or C-terminal end to a second polypeptide.
  • a simple way to obtain such a fusion polypeptide is by translation of an in-frame fusion of the polynucleotide sequences, i.e., a hybrid gene.
  • the hybrid gene encoding the fusion polypeptide is inserted into an expression vector which is used to transform or transfect a host cell.
  • the polynucleotide sequence encoding the polypeptide or polypeptide derivative is inserted into an expression vector in which the polynucleotide encoding the second peptide is already present.
  • the second peptide may be an affinity peptide or protein sequence that binds to a ligand or functional group, to assist in purification.
  • the second peptide may be a six-histidine sequence which binds to Ni +2 ions, or it may be glutathione-S-transferase (GST) which binds to glutathione.
  • the antigenic protein may be purified, using standard purification techniques, such as affinity column chromatography, for example, using a chromatography column having bound glutathione to purify a GST fusion protein.
  • affinity column chromatography for example, using a chromatography column having bound glutathione to purify a GST fusion protein.
  • affinity column chromatography a fusion protein containing the antigenic protein may be purified to a high degree, which results in a diagnosis method having better sensitivity and specificity when compared with the use of coarse viral lysates for serological assays, since the presence of antibodies against cellular components in viral lysates can result in false positivity.
  • the expressed antigenic protein is used to determine if the patient has generated antibodies directed against SARS CoV by contacting the protein with a sample from the patient. Upon contact, the patient-generated antibodies in the patient sample that can recognize the antigenic SARS CoV protein will bind to the antigenic protein, forming an antigen-primary antibody complex.
  • Specific binding between an antibody and an antigen, or “specificity” of an antibody for an antigen refers to the ability of the antibody to recognise and selectively bind to a particular epitope contained within the antigen. Such binding is determined by complementarity between the antigen-binding sites of the antibody and the epitope of the antigen. Complementarity refers to a reciprocal pairing of the spatial arrangement of chemical moieties and charges within the binding sites of each of the antibody and antigen.
  • the sample is any sample that contains patient-generated antibodies, either as cell-surface bound antibodies or as circulating antibodies.
  • the sample may be blood, plasma, or serum.
  • the sample is taken from any patient suspected of being infected with SARS, and may be taken between 2 and 60 days post-infection, between 16 and 60 days post-infection or between 2 and 11 days post-infection.
  • days post-infection refers to the length of time in days following the time at which it is suspected that the patient came into contact with or contracted SARS CoV.
  • an antigenic SARS CoV protein is done in the context of an immunoassay, such as ELISA (enzyme-linked immunosorbent assay), for detection of antibodies to S, M, E, N or U274.
  • the antigenic protein may be immobilized on a solid support prior to contacting with the patient sample. Alternatively, the antigenic protein may be expressed in a cell or at a cell surface.
  • Immunoassay techniques are generally known to a skilled person and include ELISAs, Western immunoblots, immunofluorescence assays and immunochromatography assays.
  • the patient sample may be diluted in a suitable buffer in a ratio of sample to total volume of about 1:10, about 1:20, about 1:40, about 1:80, about 1:160, about 1:320, about 1:640.
  • immunoassay refers to an analytical method that uses the ability of an antibody to bind a particular antigen as the means for determining the presence of the antigen or antibody.
  • An antibody-capture immunoassay is an assay that provides an antigen which is used to detect antibodies against a particular pathogen in a biological sample of a test subject.
  • the antigen is immobilized on a support and is capable of binding an antibody in a biological sample.
  • the antibody is provided by the biological sample.
  • the antigen is mixed with the antibody in the biological sample and the antigen-antibody complex thus formed is captured by a second antibody against the antigen or antibody or both in the antigen-antibody complex which is immobilized on a support.
  • the formation of the antigen- antibody complex is measured in solution.
  • immunoassay formats include but not limited to direct immunoassays, indirect immunoassays, and "sandwich” immunoassays.
  • a particularly preferred format is a sandwich enzyme-linked immunosorbent assay (ELISA).
  • ELISA sandwich enzyme-linked immunosorbent assay
  • RIA radioimmunoassays
  • IF A immunofluorescent assays
  • other assay formats including, but not limited to, variations on the ELISA method will be useful in the method of the present invention.
  • antigen-antibody reaction formats may be used in the present invention, including but not limited to "flocculation” (ie., a colloidal suspension produced upon the formation of antigen- antibody complexes), "agglutination” (i.e., clumping of cells or other substances upon exposure to antibody), "particle agglutination” (i.e., clumping of particles coated with antigen in the presence of antibody or the clumping of particles coated with antibody in the presence of antigen); “complement fixation” (ie., the use of complement in an antibody-antigen reaction method), and other methods commonly used in serology, immunology, immunocytochemistry, histochernistry, and related fields.
  • flocculation ie., a colloidal suspension produced upon the formation of antigen- antibody complexes
  • agglutination i.e., clumping of cells or other substances upon exposure to antibody
  • particle agglutination i.e., clumping of particles coated
  • Detection of an antibody-antigen complex can be performed by several methods.
  • the mobile antigen may be prepared with a label such as biotin, an enzyme, a fluorescent marker, or radioactivity, and may be detected directly using this label.
  • a labelled "secondary antibody” or “reporter antibody” which recognizes the primary antibody may be added, forming a complex comprised of antigen-antibody-antibody.
  • appropriate reporter reagents are then added to detect the labelled antibody. Any number of additional antibodies may be added as desired.
  • These antibodies may also be labelled with a marker, including, but not limited to an enzyme, fluorescent marker, radioactivity, or a heavy metal complex. Either the antigen or the antibody (primary or secondary) may be immobilized on a solid support, but the labelled component cannot be immobilized because the detectable signal is precluded from being a measure of binding.
  • reporter reagent is used in reference to compounds which are capable of detecting the presence of antibody bound to antigen.
  • a reporter reagent may be a calorimetric substance which is attached to an enzymatic substrate. Upon binding of antibody and antigen, the enzyme acts on its substrate and causes the production of a colour.
  • Other reporter reagents include, but are not limited to fluorogenic and radioactive compounds or molecules.
  • This definition also encompasses the use of biotin and avidin-based compounds (e.g., including compounds but not limited to neutravidin and streptavidin) as part of the detection system.
  • biotinylated antibodies may be used in the present invention in conjunction with avidin-coated solid support.
  • solid support is used in reference to any solid material to which reagents such as antibodies, antigens, and other compounds may be attached.
  • reagents such as antibodies, antigens, and other compounds
  • solid supports include microscope slides, coverslips, beads, particles, cell culture flasks, as well as many other items.
  • a kit, or a commercial package, for detecting antibodies to S, M, E, N or U274 generally comprises, in an amount sufficient for at least one assay, an antigenic SARS CoV protein and means for detecting a complex between the antigenic SARS CoV protein and the antibody from the patient sample, as packaged immunochemical reagents. Instructions for use of a packaged immunochemical reagent are also typically included.
  • a package can refer to the use of a solid matrix or material such as glass, plastic, paper, fiber, foil and the like capable of holding within fixed limits an antibody of this invention.
  • a package can be a glass vial used to contain milligram quantities of a contemplated antigen or it can be a microtiter plate well to which microgram quantities of a contemplated antigen has been operatively affixed.
  • a package could include antigen-coated microparticles entrapped within a porous membrane or embedded in a test strip or dipstick, etc.
  • the antigen can be directly coated onto a membrane, test strip or dipstick, etc. which contacts the sample fluid.
  • Instructions for use typically include a tangible expression describing the reagent concentration or at least one assay method parameter such as the relative amounts of reagent and sample to be admixed, maintenance time periods for reagent/sample admixtures, temperature, buffer conditions and the like.
  • kits of the present invention further includes a label or indicating means capable of signaling the formation of a complex between the antigenic SARS CoV protein and the antibody.
  • label or "labelling agent” and means for detecting the antibody-antigen complex
  • indicating means refer to molecules that are either directly or indirectly involved in the production of a detectable signal to indicate the presence of a complex.
  • Any label or indicating means can be linked to or incorporated in an expressed protein, peptide, or antibody molecule that is part of the present invention, or used separately, and those atoms or molecules can be used alone or in conjunction with additional reagents.
  • labels are themselves well known in clinical diagnostic chemistry.
  • the label or indicating means may be an enzyme that cleaves a reagent to produce a coloured molecule, a coloured molecule, a fluorescent molecule, a radioactive molecule, a chemiluminescent molecule or a heavy metal complex.
  • the precise method of detecting the signal produced by the label or indicating means will depend on the label or indicating means used and the particular immunoassay technique used, as will be understood by a skilled person.
  • the label or indicating means can be a fluorescent labeling agent that chemically binds to antibodies or antigens without denaturing them to form a fluorochrome (dye) that is a useful immunofluorescent tracer.
  • Suitable fluorescent labeling agents are fluorochromes such as fluorescein isocyanate (FIC), fluorescein isothiocyante (FITC), 5-dimethylamine-l-natpthalenesulfonyl chloride (DANSC), tetramethylrhodamine isothiocyanate (TRITC), lissamine, rhodamine 8200 sulphonyl chloride (RB 200 SC) and the like.
  • fluorochromes such as fluorescein isocyanate (FIC), fluorescein isothiocyante (FITC), 5-dimethylamine-l-natpthalenesulfonyl chloride (DANSC), tetramethylrhodamine isothio
  • the label or indicating means is an enzyme, such as horseradish peroxidase (HRP), glucose oxidase, or the like.
  • HRP horseradish peroxidase
  • glucose oxidase or the like.
  • additional reagents are required to indicate that a receptor-ligand complex (immunoreactant) has formed.
  • additional reagents for HRP include hydrogen peroxide and an oxidation dye precursor such as diaminobenzidine or tetramethylbenzidine.
  • An additional reagent useful with glucose oxidase is 2,2,-azino- di-(3-ethyl-benzthiazoline-G-sulfonic acid) (ABTS).
  • Radioactive elements are also useful labeling agents and are used illustratively herein.
  • An exemplary radiolabeling agent is a radioactive element that produces gamma ray emissions. Elements which themselves emit gamma rays, such as 124 I, I 5 I, 128 1, 132 I and 51 Cr represent one class of gamma ray emission-producing radioactive element indicating groups. Particularly preferred is 125 I.
  • Another group of useful labeling means are those elements such as n C, 18 F, 15 O and 13 N which themselves emit positrons. Also useful is a beta emitter, such as m indium or 3 H.
  • labeling of peptides and proteins is well known in the art.
  • monoclonal antibodies produced by a hybridoma can be labeled by metabolic incorporation of radioisotope-containing amino acids provided as a component in the culture medium.
  • the techniques of protein conjugation or coupling through activated functional groups are particularly applicable.
  • the specific binding agent is labeled.
  • the agent is typically used as an amplifying means or reagent to amplify the signal.
  • the labeled specific binding agent is capable of specifically binding the amplifying means when the amplifying means is bound to a complex.
  • kits of the present invention can be used in an "ELISA" format to detect the quantity of anti-S, M, E, N or U274 antibody in a fluid sample or extract.
  • ELISA refers to an enzyme linked immunosorbent assay such as those discussed above, which employ an antibody or antigen bound to a solid phase and an enzyme- antigen or enzyme-antibody conjugate to detect and quantify the amount of an antigen present in a sample.
  • an antigenic SARS CoV protein can be affixed to a solid matrix to form a solid support.
  • a reagent is typically affixed to a solid matrix by adsorption from an aqueous medium although other modes of affixation applicable to proteins and peptides well known to those skilled in the art, can be used.
  • Useful solid matrices are also well known in the art. Such materials are water insoluble and include the crosslinked dextran available under the trademark SEPHADEX from Pharmacia Fine Chemicals (Piscataway, N.J.); agarose; polystyrene beads about 1 micron to about 5 millimeters in diameter polyvinyl chloride, polystyrene, crosslinked polyacrylamide, nitrocellulose- or nylon-based webs such as sheets, strips or paddles; or tubes, plates or the wells of a microtiter plate such as those made from polystyrene or polyvinylchloride.
  • SEPHADEX crosslinked dextran available under the trademark SEPHADEX from Pharmacia Fine Chemicals (Piscataway, N.J.)
  • agarose polystyrene beads about 1 micron to about 5 millimeters in diameter polyvinyl chloride, polystyrene, crosslinked polyacrylamide, nitrocellulose- or nylon-based webs such as sheets, strips or paddles
  • the immunoreagents of any diagnostic system described herein can be provided in solution, as a liquid dispersion or as a substantially dry powder, e.g., in lyophilized form.
  • the indicating means is an enzyme
  • the enzyme's substrate can also be provided in a separate package.
  • a solid support such as the above- described microtiter plate and one or more buffers can also be included as separately packaged elements in the diagnostic assay systems of this invention.
  • the antigenic SARS CoV protein is coated or adsorbed on to the surface of a substrate.
  • a sample of interest is contacted with the antigenic SARS CoV protein and any antibodies to the antigen which may be present in the sample bind to the antigen.
  • Anti-human antibodies are contacted with the antigen/sample and bind to the antibodies that are bound to the antigenic SARS CoV protein.
  • the secondary antibody used may be generated in any animal that is not the same species as the patient being tested.
  • mouse anti-human antibodies may be used, or the secondary antibody may be rabbit anti-human or goat anti-human.
  • the anti-human antibodies may be, for example, directed against human IgG, IgM or IgA antibodies.
  • the anti-human antibodies may be labelled with a label.
  • the label is any entity that is capable of being conjugated or bound to the anti-human antibody and that is capable of being detected by an analytical technique.
  • the label may be conjugated or bound to the anti-human antibody prior to or after contacting the anti- human antibody with the antigen/sample.
  • Detection of the label is an indication that the human antibody is present in the sample. If the label cannot be detected, then this is an indication that the human antibody is not in the sample. Since the presence, in the sample, of the human antibody to the antigenic SARS CoV protein is presumed to arise from SARS CoV infection, the presence or absence of the human antibody is an indication of whether the human is, or has been, exposed to the SARS CoV virus.
  • the label may be a chemical moiety capable of being detected by an analytical technique, the chemical moiety being conjugated to the anti- human antibody.
  • the chemical moiety is generally conjugated to the anti-human antibody before the anti-human antibody is contacted with the antigen/sample.
  • the label may be another antibody or collection of other antibodies having conjugated thereto a chemical moiety that is capable of being detected by an analytical technique.
  • the other antibody or collection of other antibodies having the chemical moiety conjugated thereto is generally bound to the anti-human antibody after the anti-human antibody is contacted with the antigen/sample.
  • the method further comprises a washing step between steps (b) and (c), and more preferably between each of the steps of the method. Washing is preferably accomplished using a washing solution comprising a buffer, such as phosphate buffered saline solution containing about 1% normal serum from the animal species in which the antibody to which the chemical moiety is conjugated was prepared. An emulsifier may also be present in the washing solution.
  • a buffer such as phosphate buffered saline solution containing about 1% normal serum from the animal species in which the antibody to which the chemical moiety is conjugated was prepared.
  • An emulsifier may also be present in the washing solution.
  • a variety of chemical moieties capable of being detected by an analytical technique and capable of being conjugated to an antibody may be used in the label.
  • the chemical moiety may be capable of fluorescence or radioactivity (see Fuller S.A., Evelegh M.J. & Hurrell J.G.R. (2000) Conjugates of Enzymes to Antibodies. In: Current Protocols in Molecular Biology. (Eds. Ausubel F.M., Brent R., guitarist R.E., Moore D.D., Seidman J.G., Smith J.A. & Struhl K.) John Wiley & Sons, Inc. Vol. 2, pp. 11.1.1-11.1.7, and, Sambrook J., Fritsch E.F.
  • Analytical techniques useful as detection methods are generally known in the art. For example, colorimetric, electrophoretic and radio-labelling techniques may be used (see Sambrook J., Fritsch E.F. & Maniatis T. (1989) Molecular cloning. A laboratory manual. 2nd edn. Cold Spring Harbor Laboratory Press. Cold Spring Harbor, NY, the disclosure of which is hereby incorporated by reference).
  • Colorimetric techniques are generally preferred and may employ spectroscopic or visual verification of a colour change indicating a positive or negative test result.
  • spectroscopic or visual verification of a colour change indicating a positive or negative test result One skilled in the art will appreciate that other techniques may be employed as detection methods in the present invention.
  • the kit of the present invention may further comprise means for detecting the label, or such means may be separate from the kit.
  • the presence or past presence of SARS CoV in a subject may be determined.
  • the method of the present invention may be used to detect not only acute infection (one in which both the virus and antibody are present), but may also be used in cases wherein the antibody is present but there is no detectable virus, such as a subject that has recovered from SARS CoV infection.
  • the method and kit of the present invention may be used for field application such as a routine laboratory test for detection of SARS CoV infection, and where vaccination is not performed the test can detect asymptomatic SARS CoV carriers to obtain a realistic estimate of the prevalence of SARS CoV infection.
  • the immunoassay is an ELISA.
  • Antigenic protein GST-N ( ⁇ lll-118) and, optionally, antigenic protein GST-U274 (134-274) is immobilized onto the assay plate. Serum from a patient is contacted with the immobilised antigenic protein, and the assay plate is washed, blocked and washed in accordance with standard ELISA techniques described above. If the patient serum is collected from a patient in early phase infection (between 2 and 11 days post- infection), anti-human IgA or anti-human IgM secondary antibody, both conjugated with horseradish peroxidase, is used to detect the presence of the antigen-primary antibody complex.
  • anti-human IgG secondary antibody conjugated with horseradish peroxidase is used.
  • a colour reaction is developed in the dark using the reagent tetramethylbenzidine, and is read in an ELISA plate reader at OD 450 nm.
  • the ELISA method of the present invention is both specific, yielding low numbers of false positive tests, and sensitive, yielding low numbers of false negative tests, particularly when using GST-N( ⁇ 111-118) and when anti-human IgG secondary antibody is used. Furthermore, the minimum OD cutoff value for reading of the colour reaction may be adjusted.
  • the immunoassay is a rapid immunochromatography test.
  • the antigenic proteins are independently immobilized in discrete lines on a nitrocellulose membrane, preferably GST-N ( ⁇ l 11-118) and GST-U274 (134-274).
  • a separate chromatography strip contains secondary antibody conjugated with gold colloidal particles at one end, with a separator in place to prevent early migration of the secondary antibody.
  • the reagent-bearing pad is separated from a , and is separated from the nitrocellulose membrane.
  • a releasing buffer is added to the chromatography strip, and the separator is removed to allow the secondary antibody to migrate into contact with the antigen-primary antibody complex, which has formed on the nitrocellulose membrane.
  • a positive test result can be visualized typically in 2 to 15 minutes by the appearance of a colour line due to the formation of antigen-primary antibody-secondary antibody complex containing conjugated gold particles.
  • the rapid immunochromatography test requires little equipment to perform once the ready-made chromatography device is in hand.
  • the rapid immunochromatographic test is both sensitive and specific. In comparing results obtained with the two assays, an excellent agreement of 99.6% between the two was observed, with a kappa statistic of 1.00, and an excellent correlation, with an R 2 of 0.988 in relation to reactivity end.
  • the rapid immunochromatographic test is a simple and rapid test that needs no special training to use, and provides diagnostic performance similar to that of an ELISA.
  • the same antigenic proteins used in the above-described ELISA may also be applied to the rapid immunochromatographic test, and may be applied separately as two different testing markers.
  • the newly developed rapid immunochromatographic test could provide not only indications for individual patient diagnosis, but because of the ability to test reactivity to individual antigenic proteins in a single test, this test may also provide population diagnosis details that may provide valuable epidemiological information for the spread of particular virus variants across a population, due to the greater variation of the U274 protein in comparison to the N protein.
  • the immunoassay is a Western immunoblot.
  • the antigenic proteins are transferred onto a membrane using standard techniques known in the art. As with the rapid immunochromatography test, individual antigenic proteins can be separated into discrete bands for use in the same test, allowing for detection of patient immune response to particular antigenic proteins at the same time.
  • a strip of membrane containing antigenic proteins preferably GST- N ( ⁇ l 11-118) and GST-U274 (134-274).
  • the probing of the membrane with patient serum and with secondary anti-human conjugated antibody are performed by standard Western blotting techniques.
  • the secondary antibody is chosen based on the time point during infection at which the patient sample is collected.
  • the secondary antibody may be conjugated to the enzyme alkaline phosphatase, and the detection is then performed by developing a colour reaction using 5-bromo-4-chloro- 3-indolyl-phosphate and nitroblue tetrazolium.
  • the present invention also provides an antibody directed against the antigenic SARS CoN proteins.
  • the antibody preferably is directed to regions of S, M, E, ⁇ or U274 that are unique to SARS CoV, to prevent cross-reactivity of the antibodies with antigens from other coronaviruses.
  • An antibody of the invention is either a polyclonal or monoclonal antibody having specificity toward an epitope contained in one of the antigenic SARS CoV proteins S, M, E, N or U274.
  • Monospecific antibodies may be recombinant, e.g., chimeric (e.g., constituted by a variable region of murine origin associated with a human constant region), humanized (a human immunoglobulin constant backbone together with hypervariable region of animal, e.g., murine, origin), and/or single chain. Both polyclonal and monospecific antibodies may also be in the form of immunoglobulin fragments, e.g., F(ab)'2 or Fab fragments.
  • the antibodies of the invention are of any isotype, e.g., IgG or IgA, and polyclonal antibodies are of a single isotype or a mixture of isotypes.
  • Antibodies against the S, M, N, E or U274 proteins of SARS CoV, or homologs or fragments thereof, are generated by immunization of a mammal with a composition comprising said protein, homolog or fragment.
  • Such antibodies may be polyclonal or monoclonal. Methods to produce polyclonal or monoclonal antibodies are well known in the art. For a review, see “Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, Eds. E. Harlow and D. Lane (1988), and D.E. Yelton et al., 1981. Ann. Rev. Biochem. 50:657-680. For monoclonal antibodies, see Kohler & Milstein (1975) Nature 256:495-497.
  • U274 proteins of SARS CoV, or homologs or fragments thereof are produced and identified using standard immunological assays, e.g., Western blot analysis, dot blot assay, or ELISA (see, e.g., Coligan et al., Current Protocols in Immunology (1994) John Wiley & Sons, Inc., New York, NY).
  • the antibodies are used in diagnostic methods to detect the presence of antigenic SARS CoV protein S, M, E, N or U274 in a sample, such as a biological sample.
  • the antibodies are also used in affinity chromatography for purifying S, M, N, E or U274 proteins of SARS CoV, or homologs or fragments thereof.
  • an immune complex is formed between a component of the sample and the S, M, E, N or U274 protein or homologs or fragments thereof, or between the antibody of the invention and its target antigen, being S, M, E, N or U274 protein or homologs or fragments thereof, whichever is used, and that any unbound material is removed prior to detecting the immune complex.
  • the antigenic SARS CoV protein reagents are useful for detecting the presence of anti-S, M, E, N or U274 antibodies in a sample, e.g., a blood sample, while an antibody of the invention is used for screening a sample, such as a gastric extract or biopsy, for the presence of SARS CoV polypeptides.
  • somatic cells from a host animal immunized with antigen, with potential for producing antibody are fused with myeloma cells, forming a hybridoma of two cells by conventional protocol.
  • Somatic cells may be derived from the spleen, lymph node, and peripheral blood of transgenic mammals.
  • Myeloma cells which may be used for the production of hybridomas include murine myeloma cell lines such as MPCII-45.6TGI.7, NSI-Ag4/l, SP2/0- Agl4, X63-Ag8.653, P3-NS-l-Ag-4-l, P.sub.3 X63Ag8U.sub.l, OF, and S194/5XX0.BU.1; rat cell lines including 210.RCY3.Agl.2.3; cell lines including U- 226AR and GM1500GTGA1.2; and mouse-human hetero yeloma cell lines (Hammerling, et al. (editors), Monoclonal Antibodies and T-cell Hybridomas IN: J. L. Turk (editor) Research Monographs in Immunology, Vol. 3, Elsevier/North Holland Biomedical Press, New York (1981)).
  • murine myeloma cell lines such as MPCII-45.6TGI.
  • Somatic cell-myeloma cell hybrids are plated in multiple wells with a selective medium, such as HAT medium.
  • Selective media allow for the detection of antibodyproducing hybridomas over other undesirable fused-cell hybrids.
  • Selective media also prevent growth of unfused myeloma cells which would otherwise continue to divide indefinitely, since myeloma cells lack genetic information necessary to generate enzymes for cell growth.
  • B lymphocytes derived from somatic cells contain genetic information necessary for generating enzymes for cell growth but lack the "immortal" qualities of myeloma cells, and thus, last for a short time in selective media. Therefore, only those somatic cells which have successfully fused with myeloma cells grow in the selective medium. The infused cells were killed off by the HAT or selective medium.
  • a screening method is used to examine for potential anti-S, M, E, N or
  • U274 antibodies derived from hybridomas grown in the multiple wells Multiple wells are used in order to prevent individual hybridomas from overgrowing others. Screening methods used to examine for potential anti-S, M, E, N or U274 antibodies include enzyme immunoassays, radioimmunoassays, plaque assays, cytotoxicity assays, dot immunobinding assays, fluorescence activated cell sorting (FACS), and other in vitro binding assays.
  • enzyme immunoassays include enzyme immunoassays, radioimmunoassays, plaque assays, cytotoxicity assays, dot immunobinding assays, fluorescence activated cell sorting (FACS), and other in vitro binding assays.
  • Hybridomas whicb test positive for anti-S, M, E, N or U274 antibody are maintained in culture and may be cloned in order to produce monoclonal antibodies specific for S, M, E, N or U274 .
  • desired hybridomas can be injected into a histocompatible animal of the type used to provide the somatic and myeloma cells for the original fusion. The injected animal develops tumors secreting the specific monoclonal antibody produced by the hybridoma.
  • the monoclonal antibodies secreted by the selected hybridoma cells are suitably purified from cell culture medium or ascites fluid by conventional immunoglobulin purification procedures such as, for example, protein A- SEPHAROSE hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.
  • the antibodies produce according to the present invention can be used to detect the presence of an antigenic SARS CoV protein or antigenic fragment thereof, including in diagnostic methods, identifying the presence of the particular antigenic protein in a patient sample or in a viral isolate obtained from a patient, as well as detecting purified recombinant antigenic SARS CoV proteins or antigenic fragments thereof.
  • the antibodies may also be used in standard purification techniques, for example, immunoprecipitation.
  • the antibodies provided by the invention may be used in immunoassays in accordance with known techniques, for example, capture ELISAs, Western immunoblots, or immunofluorescence assays, as described above.
  • an antigenic SARS CoV protein particularly S, M, E, N or U274, or an antigenic fragment thereof, using an antibody of the invention.
  • a sample is contacted with a primary antibody directed against an antigenic SARS CoV protein, particularly S, M, E, N or U274 protein or an antigenic fragment thereof, for a time and under conditions sufficient for the antibody to form a complex with an antigen.
  • the primary antibody will bind to the antigenic protein, forming an antigen-primary antibody complex, which is then detected by the various methods described above, for example by using a secondary antibody directed against the primary antibody, which may be conjugated to a detection molecule.
  • Example 1 Use of SARS CoV proteins as diagnostic markers
  • Plasmids For transient expression in mammalian cells, the vectors used were pXJ40HA, for tagging proteins at the N terminus with one hemagglutinin (HA) epitope (15), and pXJ40-3_HA, for tagging proteins with three HA epitopes at the C terminus.
  • GST glutathione S-transferase
  • RNAs were extracted by use of a Qiagen viral RNA kit (Valencia, Calif.) from a SARS-CoV- infected Vero E6 cell culture supernatant harvested when the cultures showed at least 75% cytopathic effects. Reverse transcription was performed with Superscript IITM Rnase H " reverse transcriptase (Gibco BRL, Gaithersburg, Md.) and an oligo(dT) primer.
  • PCRs were performed with either HotStarTM polymerase (Qiagen), TitaniumTM Taq DNA polymerase (Clontech Laboratories Inc., Palo Alto, Calif), or High FidelityTM Taq polymerase (Roche Molecular Biochemicals, Indianapolis, Ind.).
  • HotStarTM polymerase Qiagen
  • TitaniumTM Taq DNA polymerase Clontech Laboratories Inc., Palo Alto, Calif
  • High FidelityTM Taq polymerase Roche Molecular Biochemicals, Indianapolis, Ind.
  • overlapping cDNAs provided by the Genome Institute of Singapore (isolate SIN2774; accession no. AY283798) were used as templates instead. All of the primers used for this study were synthesized by Genset Singapore Biotech (Singapore).
  • Transient transfection of mammalian cells Transient transfection experi-ments were performed with EffecteneTM transfection reagent (Qiagen) according to the manufacturer's protocol. Typically, ⁇ 10 6 COS-7 cells were plated on a 6 cm diameter dish and allowed to attach for at least 4 h. 1 to 2 ⁇ g of DNA was used per plate, and the cells were left for at least 14 h before the cells were washed with phosphate-buffered saline (PBS), lysed directly in Laemmli's sodium dodecyl sulfate (SDS) buffer, and used for Western blot analysis.
  • PBS phosphate-buffered saline
  • SDS Laemmli's sodium dodecyl sulfate
  • [00130] Expression of GST fusion proteins Exponentially growing cultures (optical density at 600 nm of ⁇ 0.7) of Escherichia coli (BL21/DE3) cells harboring the pGEX-4T-l expression constructs were induced to synthesize fusion proteins by the addition of 1 mM isopropyl- ⁇ -D-thiogalactopyranoside (IPTG), after which the cells were allowed to grow for another 4 h at 37°C or 12 h at 30°C.
  • IPTG isopropyl- ⁇ -D-thiogalactopyranoside
  • the membranes were incubated with either an anti-HA polyclonal or anti-GST monoclonal antibody (Santa Cruz Biotechnology, Santa Cruz, Calif.) overnight at 4°C and washed extensively with PBST (PBS containing 0.05% Tween 20), followed by incubation with an appropriate horseradish peroxidase (HRP)-conjugated secondary antibody (Pierce) for 1 h at room temperature.
  • PBST horseradish peroxidase
  • Pierce horseradish peroxidase-conjugated secondary antibody
  • each sample was first treated with 0.5% Triton X-100 and 0.1 mg of RNase (Sigma)/ml and then diluted 1:150 to 1:500 with PBST containing 1% nonfat dry milk. After incubation for 1 to 3 h at room temperature or overnight at 4°C, the membranes were incubated with an anti-human HRP-conjugated immunoglobulin G (IgG) (Santa Cruz Biochemicals), IgA, or IgM (Zymed Laboratories Inc., San Fran-cisco, Calif.) antibody for 1 h at room temperature, followed by detection as described above. All secondary antibodies were used at a 1:2000 dilution.
  • IgG anti-human HRP-conjugated immunoglobulin G
  • IgA anti-human HRP-conjugated immunoglobulin G
  • IgM Zymed Laboratories Inc., San Fran-cisco, Calif.
  • CHO cells were transfected with pMMTC-S-GFP by use of DMRIE-C reagent (Gibco BRL) according to the manufacturer's protocol. Transfected cells were selected in GeneticinTM (Gibco BRL) for ⁇ 1 week. Cells were then analyzed under a Zeiss microscope (Carl Zeiss Vision GmbH, Hallbergmoss, Germany), and the clone with the strongest expression signal was picked and grown in medium containing 100 ⁇ M ZnSO 4 . Zn 2+ ions increase the expression of genes by the pMMTC vector (see GB 2314332) .
  • Cells were dislodged from the plates with 0.04% EDTA, seeded onto black Teflon Menzel diagnostic slides (Merck), and blown dry, followed by fixing in acetone at ⁇ 20°C for 1 h. Then the cells were incubated with sera at dilutions of 1:20, 1 :40, 1 :80, and 1 : 160 (in PBS) for 1.5 h at 37°C, followed by incubation with a fluorescein isothiocyanate (FITC)- or rhodamine (Rh)-conjugated anti-human IgG (Sigma) at a 1:20 dilution for 1.5 h at 37°C.
  • FITC fluorescein isothiocyanate
  • Rh rhodamine
  • Rh-conjugated anti-human IgG When Rh-conjugated anti-human IgG was used, it was diluted in PBS, and when FITC-conjugated anti-human IgG, IgM (Sigma), or IgA (Dako A/S, Glostrup, Denmark) was used, it was diluted in 0.05% Evans Blue solution (Fluka, Buchs, Switerland), which blocks GFP fluorescence from transfected cells. Slides were then viewed under a Zeiss microscope (Carl Zeiss Vision GmbH) and scored as follows: +++, very strong staining; ++, moderate staining; and +, weak staining.
  • SARS-CoV genome in the sera of probable SARS patients In order to assess which of the viral proteins encoded by the SARS-CoV genome may be exploited as diagnostic anti-gens for the development of a serological assay to detect SARS-CoV infection, we cloned three of the four structural proteins (E, M, and N) into expression vectors. Due to the large size ( ⁇ 4.7 kbp) and heavy glycosylation of the spike (S) protein, it was analyzed separately by an immunofluorescence method as described below. In addition, two of the SARS-CoV unique proteins (U274 and U122) were also included in this study, and they will be referred to hereafter as UX, where "X" stands for the number of predicted amino acids of the proteins.
  • UX stands for the number of predicted amino acids of the proteins.
  • This panel of HA-tagged proteins was expressed in COS-7 cells by transient transfection, and total protein lysates were analyzed by Western blot analysis with sera from three convalescent patients ( Figure 2, patients 1 to 3) to determine if these sera had any antibodies against the expressed proteins. Plasmapheresis was performed on patients 1 and 2, and the plasmapheresis products were used in Western blot analysis, while for patient 3, serum was used for the analysis.
  • the GST-N (aa 120 to 422) protein used here lacked the N-terminal part of the N protein, which contains a highly conserved motif (FYYLGTGP; aa 111 to 118 of SARS-CoV N) found in all coronaviruses (10), so there would be less chance of cross-reactivity with antibodies against other coronaviruses. From a 400-ml bacterial culture, ⁇ 10 mg of GST-N and ⁇ 2 mg of GST-U274 could be obtained, respectively.
  • GST-U122 (aa 16 to 111, lacking the signal peptide at the N terminus and the hydrophobic C terminus) and GST were also expressed and used as controls.
  • GST fusion proteins were probed with the same three patient sera and one control se-rum, as for the mammalian expressed proteins (Figure 2). Consistent with the proteins expressed in COS-7 cells, GST-N was clearly detected by all three patient sera and GST-U274 was detected only by the sera from patients 1 and 3 ( Figure 4). Neither GST-U122 nor GST showed any background with all three patient sera, and there was no nonspecific binding of the control serum to any of the proteins ( Figure 4). Sera from another three convalescent patients (patients 4 to 6) were also tested, and they were reactive specifically towards both GST-N and GST-U274, but not to GST-U122 or GST ( Figure 4).
  • the membranes were incubated with the diluted sera (1:150) for 1.5 h at room temperature, followed by incubation with the secondary antibody (HRP-conjugated anti-human IgG) for 1 h at room temperature.
  • the secondary antibody HRP-conjugated anti-human IgG
  • all seven of the late time point sera also contained antibodies against GST-U274, albeit at a lower level than that for GST-N ( Figure 5, set B).
  • CHO cells were used for an immu-nofluorescence staining method to determine if there were IgG antibodies against the SARS-CoN S protein in convalescent-phase sera. Briefly, the cells were fixed with acetone and then incubated with diluted patient sera, followed by incubation with an Rh-conjugated anti-human IgG antibody.
  • Example 2 ELISA and Immunochromatographic Assays
  • Recombinant proteins The materials and methods used for obtaining the recombinant proteins are as described for Example 1 above.
  • the Gst-U274 protein was further purified with a SuperdexTM S-200 HR10/30 column on an AKTA fast protein liquid chromatography (FPLC) system (Amersham).
  • the buffer used contained 20 mM Tris-HCl (pH 7.5), 100 mM NaCl, 6 M urea, and 1 mM ⁇ - mercaptoethanol; the flow rate was 0.5 ml/min; and fractions of 1 ml were collected. Fractions 12 and 13 were combined and dialyzed against phosphate-buffered saline (PBS) overnight with at least three changes of buffer.
  • PBS phosphate-buffered saline
  • Serum specimens Seventy-four convalescent-phase serum samples were collected from SARS patients admitted to the Tan Tock Seng Hospital or the Singapore General Hospital. Ninety-one control serum samples were obtained from healthy local donors who worked at the Institute of Molecular and Cell Biology, Singapore, Republic of Singapore. All specimens were collected with consent, and patient samples were collected at 16 to 65 days from the onset of symptoms. In addition, 119 sera from healthy donors purchased from BioClinical Partners, Inc. (Franklin, Mass.), were included in the study as additional healthy controls.
  • ELISA The Gst-N and GstT-U274 proteins were pre-diluted in carbonate buffer (pH 9.6) at final concentrations of 0.1 and 0.15 ⁇ g/ml, respectively, prior to plate coating.
  • the plates were prepared as described (16). Briefly, the 96-well polystyrene microtiter plates (Immuno IB; Themo Labsystem, Franklin, Mass.) were coated with the protein mixtures at a volume of 100 ⁇ l per well by incubation overnight (16 to 18 h) at room temperature.
  • the plates were washed five times with PBS-Tween 80 (PBST), and nonspecific binding sites were blocked with 200 ⁇ l (per well) of a Tris-based diluent for 1 h at room temperature. The plates were further washed another five times before 10 ⁇ l of serum in 200 ⁇ l of Tris-based diluent (containing 1% each bovine serum albumin [BSA] and skim milk powder) was added. Subsequently, the plates were incubated for 30 min at 37°C, followed by six washes with PBST.
  • PBST PBS-Tween 80
  • Horseradish peroxidase-conjugated goat anti-human immunoglobulin G (IgG; 1:500 dilution) was added at 100 ⁇ l per well, and this mixture was incubated for 30 min at 37°C. The plates were then washed six times in PBST and color development proceeded with the addition of the enzyme substrate tetramethylbenzidine (TMB) at 100 ⁇ l per well. After a 15-min incubation in the dark at 37°C, the reaction was stopped by adding 100 ⁇ l of 1 N HC1 per well. The optical densities (OD) were measured at 450 run with a 620-nm reference filter.
  • TMB tetramethylbenzidine
  • Rapid immunochromatographic test The membrane-based immunochromatographic test device consisted of a chromatography strip, a separator, and an absorbent pad, all housed in a cassette as described previously in US Patent No. 6,316,205, and the chromatography strip was prepared separately according to the procedure detailed in that reference with slight modification before assembly into the device. Briefly, a nitrocellulose membrane with an average pore size of 8 ⁇ m (Whatman, Ann Arbor, Mich.) was sprayed with the Gst-N and the Gst-U274 recombinant antigens in two separate lines, at concentrations of 0.1 and 0.15 mg/ml, respectively, with a BioDot (Irvine, Calif.) spraying machine.
  • the membrane was dried for 10 min before being immersed for 1 min in a blocking buffer consisting of Milli-Q purified water with 6.7% StabilCoatTM (SurModics, Inc.), 0.05% Triton X- 100, and 0.5% casein.
  • the blocked membrane was then dried at 37°C for 60 min before being affixed to a membrane backing.
  • the reagent-bearing pad was prepared using a porous matrix.
  • the porous matrix was sprayed with goat anti-human IgG antibodies that were labeled with colloidal gold particles 25 to 30 nm in diameter. This reagent-bearing pad was then dried at 37°C for 2 h prior to incorporation into the device.
  • a chromatographic card was prepared by affixing a 0.1% Triton X-100- treated porous matrix to one end of the nitrocellulose strip and the reagent-bearing pad to the other end on the same membrane backing. This assembly was then cut into a strip approximately 4 by 56 mm 2 in size.
  • An assay device was assembled by placing an absorbent pad at the bottom half of the cassette and then a separator, followed by one unit of the chromatographic strip before closing the top half of the cassette.
  • a reagent-releasing wash buffer was also prepared with Milli-Q purified water with 50 mM NaH2PO4, 300 mM NaCl, and 0.1% sodium dodecyl sulfate (pH 8.0).
  • Rapid immunochromatographic test When tested with undiluted samples, the Gst-N and the Gst-U274 proteins used in the rapid test reacted to IgG antibodies in 100% (42 of 42) and 85.7% (36 of 42), respectively, of the sera from SARS patients who met the WHO criteria for SARS (Table 4). Thus, the overall detection rate for the new test was 100% (42 of 42) (Table 4). Only the Gst-N protein in the rapid test was found to cross-react with 2 of the 210 sera from the healthy controls. The test was therefore shown to have specificity, PPV, and NPV of 99.0, 95.3, and 100%, respectively, with the tested populations (Table 4).
  • Serum specimens Serum specimens were collected from patients who presented with clinical suspected SARS according to the WHO definition (18) and who were admitted to three acute regional hospitals in Hong Kong between 18 March and 24 May 2003. A total of 227 serum samples from these patients was tested using an IF test (9) and confirmed to have IF titers of >1: 10 to 1:2560 dilution. In the meantime, 385 serum samples from healthy donors collected locally in Hong Kong were used as controls. In addition, 1066 sera from healthy donors purchased from BioClinical Partner Inc. (Franklin, MA) were included in the study as additional healthy controls.
  • Immunofluorescent test The IF test was prepared and carried out as described above in Example 1. Briefly, smears of SARS CoN-infected Vero cells were prepared, fixed in acetone for 10 min, and stored at -80°C before use. Batches of smears with 60 to 70% SARS CoV infected cells were confirmed with a high titer positive control serum samples before use. Patient samples prepared in serial twofold dilutions starting with 1:10 were added to the smears and incubated for 30 min at 37°C.
  • the smears were washed two times in PBS before a further incubation for 30 min at 37°C with a goat anti-human IgG labeled with fluorescein isothiocyanate. A sample was scored as positive if the fluorescent intensity was equal to or higher than that of a weakly positive control included in the study.
  • ELISA The ELISA was produced by Genelabs Diagnostics Pte Ltd in
  • the plates were then washed six times in PBST and allowed a color development with the addition of lOO ⁇ l per well of enzyme substrate TMB (tetramethylbenzidine). After a 15-min incubation in the dark at 37 °C, the reaction was stopped by adding lOO ⁇ l per well of IN HC1. The optical densities (OD) were measured at 450nm with a 620nm reference filter.
  • TMB tetramethylbenzidine
  • Rapid Immunochromatographic Test Again, the membrane-based test device was produced at Genelabs Diagnostics Pte Ltd in Singapore following the procedure previously described for Example 2. The device consisted of a chromatography strip, a separator and an absorbent pad all housed in a cassette as described.
  • the chromatography strip was deposited with two SARS-specific recombinant proteins: Gst-N and Gst-U274 separately following a detailed disclosure of Guan et al (19, 20).
  • Test samples if contained antibody to SARS-CoN would bound to the either or both of the immobilized recombinants and the immunocomplex formed can be detected by the immobilized colloidal gold labelled with anti-human IgG when the latter was released by a reagent-releasing and washing buffer (19).
  • the assays were also carried out following strictly the instruction provided by the manufacturer (19). Briefly, a 25 ⁇ l of serum sample was added to the sample well and allowed to migrate laterally to cover a portion of the membrane in the result- viewing window. Three drops of the reagent-releasing and washing buffer were added to the second well when the serum sample-wetting front reached the blue indicator line in the viewing window. The separator tab was pulled until resistance was felt and an additional drop of the reagent-releasing and washing buffer was then added to the sample well.
  • results can be read in typically 2-15 min through the viewing window but were all recorded at 15 min. A sample was scored as negative when only the control line appeared but positive when the control line and either or both of the test lines were seen in the viewing window (19).
  • OD 0.25 was found to be equivalent to the mean OD+3SD, whereas OD 0.45 to the mean OD+5SD.
  • the two cutoff values of OD 0.25 and OD 0.45 produced similar high specificities of 98% vs. 99.2%, 100% vs. 100% and 92% vs. 98% with the respective supplemental healthy control and the disease controls groups of non-SARS patients who suffered respiratory illness or fever (Table 6).
  • the ELISA detected IgG antibodies to SARS-CoN with not only late stage convalescent samples with a high sensitivity of 95% but also acute specimens. It detected IgG antibodies to SARS-CoN in 58%, 70% and 75% of the samples collected from SARS patients 1 to 10 days, 11 to 20 days and 21 to 30 days after the onset of clinical symptoms.
  • the specificity obtained with the cutoff value of OD 0.25 remained to be high at 99.5% (383 of 385) with the healthy control group tested at the same testing site (Table 6).
  • the test thus provided an overall PPV of 98.8% and ⁇ PV of 85.7%.
  • the test was therefore showed to have PPV and NPV of 94.7% and 84.9% respectively with the tested populations (Table 7).
  • the rapid test was further evaluated with disease control groups of non-SARS patients who suffered respiratory illness or fever and found to cross-react to only 3 of 50 or 4 of 50 of the tested samples in the respective groups (Table 7).
  • the rapid test and the ELISA gave an excellent overall agreement of 92.5% with a kappa statistic of 0.81 (Table 8).
  • Example 4 Western Blot as confirmatory test for diagnosis of SARS
  • Serum specimens Forty convalescent serum specimens were collected with consent from SARS patients who were admitted to the Tan Tock Seng Hospital or the Singapore General Hospital. Fifty sera from healthy donors, purchased from BioClinical Partner Inc. (Franklin, Mass.) were also included in the study as healthy controls. For the non-SARS disease controls, archived Genelabs Diagnostics (Genelabs Diagnostics, Singapore) serum samples from previous studies were used and they were obtained prior to the SARS outbreak. These included 50 samples each from patients who had non-SARS related fever (confirmed as dengue fever) or suffered non-SARS related respiratory illness (confirmed as tuberculosis). In addition, 18 samples identified as false positives from screening 1066 healthy donors in a previous study (Guan et al. submitted) were also included in the present study.
  • SARS-Co V viral lysate and recombinant proteins The SARS-CoV viral lysate was purchased from ZeptoMetrix Corporation (Buffalo, New York) and they were obtained from SARS CoV-infected Vero cells after sucrose gradient purification and treatment with a disruption buffer (KCl, 0.6M) containing a Triton X- 100 (0.5%).
  • the recombinant proteins were prepared as described above. Briefly, all the proteins were expressed as GST-fusion proteins in E. coli but only the GST-N was purified using GSH-sepharose beads (Amersham Pharmacia). For the GST-S, the GST-M and the GST-E proteins, the separation of the respective insoluble proteins in pellet was carried out by washing and re-suspension of the proteins and eventually by electrophoresis in 10% PAGE-SDS gels. Gel strips containing the respective GST- fusion proteins were then cut and subjected to the elution using Mini Trans-BlotTM cell (BioRad).
  • the resulting fusion proteins were detected in Western Blot using an anti-GST monoclonal antibody (Santa Cruz Biotechnology, Santa Cruz, Calif.) and their concentrations were estimated by comparing with BSA standards in Coomassie blue-stained PAGE gel (Shen et al, submitted).
  • Mouse and Rabbit antisera Specific antisera were raised by inoculating mice or rabbits as described elsewhere (21) using the respective recombinant proteins.
  • the mouse antisera specific to the spike, the nucleocapsid, or the membrane proteins were generated by subcutaneous inoculation of BALB/c mice with 50 ⁇ g of the respective recombinant proteins emulsified in complete Freund's adjuvant (Sigma, St Louis, MO).
  • complete Freund's adjuvant Sigma, St Louis, MO
  • the rabbit antiserum specific to the envelope recombinant protein was generated by subcutaneous inoculation of New Zealand white rabbits with 1 mg of the purified protein again emulsified in complete Freund's adjuvant.
  • the animals were boosted at 2 weeks interval for 8 to 16 times with the same amount of the respective purified proteins but emulsified in incomplete Freund's adjuvant (Sigma, St. Louis, MO). Sera from the immunized animals were harvested 10 days after the last immunization and were adsorbed with mammalian cell cultures to reduce unspecific binding to cellular proteins.
  • Separation of the SARS-CoV viral lysate was performed on 11% separating gels with a 3.5%o stacking gel.
  • 70 ⁇ g of the SARS-CoV viral lysate in 800 ⁇ l of denaturing buffer of 3.2% SDS, 0.5 M Tris (pH6.8), 32% glycerol, 3.2% 2- mercaptoethanol and 0.05% bromophenol blue tracking dye was boiled for 5 min in a water bath.
  • 50 ⁇ l each of the treated lysate sample and the rainbow molecular markers were applied to separate wells (8 mm width) on the same SDS-PAGE gel.
  • the treated sample 800 ⁇ l was applied to the preparative well (130 mm width) and electrophoresed at a constant current until the tracking dye reached the bottom.
  • the separated proteins were electro-transferred in a tank apparatus (Hoeffer, San Francisco, Calif.) to a nitrocellulose membrane (Whatman, Gerbershausen, Germany). After the transfer, the membranes were further deposited with goat anti-human IgG (1.6 ⁇ g per membrane, as a sample addition control) and the GST-N recombinant protein (4.3 ng per membrane) using the AutoslotTM machine (Genelabs Diagnostics, Singapore).
  • the membrane was then incubated for 45 min in a blotting solution containing 5% nonfat milk powder before being rinsed for 30 min in PBS containing 0.5%> Tween-20. Subsequently, the membrane was left to dry at room temperature (RT) for 20 min prior to being cut into 3 mm strips and stored at 2-8°C until used.
  • RT room temperature
  • the membrane strips were further incubated for 1 hour with 1ml of a conjugate of goat anti-human IgG labelled with alkaline phosphatase (Kirkegaard & Perry Laboratory Inc., Gaithersburg, MD) at a dilution of 1:1000 in the blocking buffer. After the incubation, the conjugate was removed again by aspiration and the membrane strips were washed another 3 times. This was followed by a 15-min incubation of the membrane strips with a substrate solution of 5-bromo-4-chloro-3- indolyl-phosphate (BCIP) and nitroblue tetrazolium (NBT). The resultant protein bands were analyzed subjectively by the intensity of the bands on the strips.
  • BCIP 5-bromo-4-chloro-3- indolyl-phosphate
  • NBT nitroblue tetrazolium
  • Immunoblot analysis The apparent molecular weights of the immunoreactive proteins were estimated by extrapolating a plot of the logarithm of the molecular weights versus the electrophoretic mobilities of standard proteins ( Figure 13).
  • the 150-kDa, the 45-kDa and the 24-kDa proteins were found to be associated with the spike (S), the nucleocapsid (N) and the matrix (M) proteins respectively ( Figure 14).
  • a protein not reactive with the strong SARS-positive sample but associated with the envelope (E) protein was also located at approximately 10-kDa by the rabbit anti-E antiserum ( Figure 14).
  • the Nh protein cross-reacted to 64%, 68% and 52% of the samples from the healthy, the respiratory illness and the fever controls respectively (Table 10).
  • these N related proteins reacted to 15 out of the 18 samples (83%) previously found to be false positive by an ELISA (Table 10).
  • the S, the M and the GST-N recombinant protein reacted to 78%, 75% and 100% of the 40 samples from the SARS patients without any cross-reactivity with any of the controls including those identified as false positive by an ELISA (Table 10).
  • ZCURNE_CoN a new system to recognize protein coding genes in coronavirus genomes, and its applications in analyzing SARS-CoN genomes. Biochem. Biophys. Res. Commun. 307:382-388.
  • SARS Severe acute resiratory syndrome

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AT17386U1 (de) * 2020-04-15 2022-02-15 Protzek Ges Fuer Biomedizinische Technik Gmbh Vorrichtung zur gezielten Prüfung auf eine erfolgte Infektion mit dem Coronavirus-SARS-CoV-2
DE102020110335B3 (de) 2020-04-15 2021-07-08 Protzek Gesellschaft für Biomedizinische Technik GmbH Vorrichtung und Verfahren zur gezielten Prüfung auf eine erfolgte Infektion mit dem Coronavirus-SARS-CoV-2
CN111521818A (zh) * 2020-04-27 2020-08-11 深圳海博生物技术有限公司 特异性IgA在评价COVID-19患病风险、患病严重程度以及预后评估中的应用
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CN112114139A (zh) * 2020-09-11 2020-12-22 博奥赛斯（天津）生物科技有限公司 一种新型冠状病毒IgM-IgA-IgG抗体胶体金检测试剂盒
KR20220052645A (ko) * 2020-10-21 2022-04-28 재단법인 바이오나노헬스가드연구단 스트렙트아비딘 결합 펩타이드가 태그된 중증급성호흡기증후군 코로나바이러스 특이적 항원 및 이의 용도
KR102437114B1 (ko) 2020-10-21 2022-08-26 재단법인 바이오나노헬스가드연구단 스트렙트아비딘 결합 펩타이드가 태그된 중증급성호흡기증후군 코로나바이러스 특이적 항원 및 이의 용도
CN112798529A (zh) * 2021-01-04 2021-05-14 中国工程物理研究院激光聚变研究中心 一种基于增强拉曼光谱和神经网络的新型冠状病毒检测方法及系统
CN113092414A (zh) * 2021-04-06 2021-07-09 中国人民大学 一种SARS-CoV-2抗体的检测方法
CN113092414B (zh) * 2021-04-06 2023-03-10 中国人民大学 一种SARS-CoV-2抗体的检测方法
CN113219174A (zh) * 2021-04-14 2021-08-06 中国科学院西北高原生物研究所 一种新型冠状病毒s抗原检测试剂盒及其使用方法

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