"detection Kit Containing A Novel Recombinant 15-kda Polypeptide Useful for Detecting Human Infection with Bartonella Henselae" in Patent Application Approval Process
This patent application is assigned to
The following quote was obtained by the news editors from the background information supplied by the inventors: "Bartonella henselae is an intra-erythrocytic gram-negative bacterium and a causative agent for cat scratch disease in human. An estimated 28% of domestic cats are chronically infected with Bartonella henselae. Infected cats transmit the Bartonella bacteria to people from cat scratches or bites. The disease typically manifests as a regional lymphadenopathy, with some patients presenting with cutaneous lesions at the scratch sites. While cat scratch disease in most healthy individuals is self-limiting, approximately 10% of the patients may develop bacillary angiomatosis, bacillary peliosis, recurrent bacteriemia, and infective endocarditis. (See, e.g., Rochalimaea N., et al.,
"Young children and individuals with weakened immune systems are vulnerable to the Bartonella infection. In HIV-1 patients, Bartonella henselae can cause bacillary angiomatosis or peliosis hepatis which may include visceral involvement (Fournier, P. E., and
"Several diagnostic assays for Bartonella infection are presently available. These include: (i) culturing, (ii) immunofluorescence assay ('IFA'), and (iii) polymerase chain reaction ('PCR') technologies. Culturing of Bartonella bacteria from blood samples is proven to be tedious and requires an extensive period (i.e., weeks), thus making this assay sub-optimal. Because of varying experimental culture conditions, it adversely affects the reproducibility of the methodology. IFA is not a quantitative assay, and does not provide useful information relating to sensitivity and specificity. In addition, IFAs for Bartonella bacterium have technical issues with cross-reactivity with other human pathogens (e.g., Coxiella burnetii, Rickettsia rickettsii, Ehrlichia chaffeensis, and Treponema pallidum) (Cooper, M. D.,
"PCR technology utilizes the 16S rRNA gene in Bartonella bacterium to detect the presence of the pathogen. PCR has the advantage of simultaneous detection of DNA from multiple Bartonella species (e.g., Bartonella henselae, Bartonella quintana, Bartonella bacilliformis, Bartonella elizabethae, and Bartonella clarridgeiae). The problem with PCR is that it requires the presence of either the organism or its DNA in the test samples at the time of the assay in order to allow species-specific identification. Because of this limitation, PCR assay is only useful during the early stage of infection.
"Detection of antigens represents an alternative method of detecting Bartonella henselae. For example, ELISA represents a quantitative detection assay that can provide good sensitivity and specificity. Unfortunately, there are only a limited number of antigens from Bartonella bacterium that have been shown to be useful for serological detection of Bartonella. Loa et al. reported the use of 17-kDa protein from Bartonella in the development of an ELISA (Loa et al., 2007). There is a continuing need for identification of novel Bartonella bacterium antigens useful in serological detection. The present inventors have addressed this deficiency of the prior art and have successfully cloned a gene from Bartonella henselae and expressed the corresponding polypeptide. The present inventors discovered that a 15-kDa polypeptide that is extremely useful in providing an immunoassay to detect the presence of antibodies to Bartonella henselae in patient's sera. The present disclosed antigen is novel and provides an improved clinical diagnostic tool in the field of Bartonella infection."
In addition to the background information obtained for this patent application, VerticalNews journalists also obtained the inventors' summary information for this patent application: "The present invention is directed to the cloning and expression of the isolated virB7 gene in Bartonella to provide a recombinant protein of a molecular weight of 15-kDa. The present inventors discovered that the 15-kDa protein is useful in an ELISA assay in diagnostic tests for detecting Bartonella henselae.
"In one aspect, the present invention provides a recombinant 15-kDa polypeptide. In another aspect, the 15-kDa protein has an amino acid sequence as set forth in SEQ ID NO: 2.
"In another aspect, the present invention provides a composition comprising a recombinant polypeptide having an amino acid sequence as set forth in SEQ ID NO: 2 and a support. The support may be a microtiter well, polyethylene, polypropylene, or glass.
"The present invention also provides for an isolated polynucleotide encoding the 15-kDa polypeptide of the present invention. In one aspect, the present invention provides an isolated polynucleotide encoding a polypeptide as set forth in SEQ ID NO: 2.
"In another aspect, the present invention provides an isolated polynucleotide having a nucleotide sequence as set forth in SEQ ID NO: 1.
"In another aspect, the present invention provides an expression vector comprising the isolated polynucleotide of the invention, operably linked to one or more expression control sequences. In an embodiment, the expression control sequences comprise a lac promoter. In another embodiment, the vector is an expression vector in E. coli.
"The present invention also provides for a recombinant host cell containing the isolated polynucleotide and vector of the present invention.
"In another aspect, the invention provides a method of preparing the recombinant polypeptide of the present invention, which comprises the steps of (a) introducing an isolated polynucleotide into a host cell, said isolated polynucleotide having a nucleotide sequence as set forth in SEQ ID NO: 1; (b) growing said host cell in a culture under suitable conditions to permit production of said recombinant polypeptide; and © isolating said recombinant polypeptide.
"In another aspect, the present invention provides a method of detecting the presence of an antibody against Bartonella henselae in a biological sample of a mammal, comprising the steps of: (a) immobilizing a recombinant polypeptide of the present invention onto a surface; (b) contacting said recombinant polypeptide with a biological sample under conditions that allow formation of an antibody-antigen complex; and © detecting the formation of said antibody-antigen complex, wherein the presence of said antibody-antigen complex is indicative of the presence of said antibody against Bartonella henselae in said biological sample.
"In another aspect, the present invention provides a method of diagnosing an infection of Bartonella henselae in a mammal, comprising the steps of: (a) obtaining a biological sample from a mammal suspected of having a Bartonella henselae infection; (b) immobilizing a recombinant polypeptide of the current invention onto a surface; © contacting said recombinant polypeptide with said biological sample, under conditions that allow formation of antibody-antigen complex; and (d) detecting said antibody-antigen complex, wherein said detected antibody-antigen complex is indicative of the presence of the antibody against Bartonella henselae in said biological sample.
"In yet another aspect, the present invention provides an article of manufacture or a kit comprising a recombinant polypeptide of the present invention, and an instruction indicating the use of said recombinant polypeptide in detecting the presence of an antibody against Bartonella henselae.
BRIEF DESCRIPTION OF THE DRAWINGS
"FIG. 1 depicts the genomic organization of Bartonella henselae virB operon. Open reading frames of various virulence proteins are represented by arrows. The gene (locus tag BH13310) encoding Bartonella henselae 15-kDa antigen is present in the virB7 operon.
"FIG. 2 depicts the nucleotide sequence of the full-length 15-kDa gene (SEQ ID NO: 1) in Bartonella henselae, previously deposited into the NCBI GenBank (GeneID: 2865504).
"FIG. 3 depicts the amino acid sequence of the 15-kDa polypeptide (SEQ ID NO: 2) in Bartonella henselae, previously deposited into the NCBI GenBank (Accession # YP.sub.--034056).
"FIG. 4a depicts the transmembrane topology for the15-kDa protein in Bartonella henselae using the Phobius online tool (http://phobius.sbc.su.se). According to this model, the 15-kDa protein contains a single transmembrane domain spanning the region from amino acid residues 37 to 55 of SEQ ID NO: 2.
"FIG. 4b depicts the predicted topology for the 15-kDa protein within the cellular membrane using the SOSUI (classification and secondary structure prediction system for membrane proteins) online tool (http://bp.nuap.nagoya-u.ac.jp/sosui). According to this model, the predicted topology of the 15-kDa (virB7) protein indicates the presence of a single transmembrane helix spanning the region from residues 37-55 of SEQ ID NO: 2.
"FIG. 5 illustrates PCR amplification of the 15-kDa gene from Bartonella henselae
"FIG. 6 is a schematic representation of the recombinant pET30 vector containing the 15-kDa gene in-frame of the T7 promoter and His-tag sequences.
"FIG. 7 illustrates PCR amplification of the recombinant 15-kDa gene in pET30 vector following transformation into NovaBlue E. coli. Ten colonies were randomly selected from NovaBlue E. coli grown on LB plates and amplified by PCR.
"FIG. 8 illustrates PCR amplification of the recombinant 15-kDa gene in pET30 vector following transformation into BL21 (DE3) E. coli. Ten colonies were randomly selected from BL21 (DE3) E. coli grown on LB plates and amplified by PCR.
"FIG. 9 illustrates the work-flow for IPTG induction and SDS-PAGE analysis of the recombinant 15-kDa protein.
"FIG. 10 depicts a Coomassie-blue stained SDS-PAGE gel of IPTG-induced 15-kDa protein. Recombinant 15-kDa in BL21 E. coli was grown in LB supplemented with glucose prior to the addition of IPTG as described in the text. Cells were incubated in LB with IPTG for the indicated times (2.0-3.5 hours). The arrows point to an induced protein band of expected size (.about.21-kDa).
"FIG. 11 depicts a Coomassie-blue stained SDS-PAGE gel of IPTG-induced 15-kDa protein. Recombinant 15-kDa in BL21 E. coli was grown in LB supplemented with glucose prior to the addition of IPTG as described in the text. Cells were incubated in LB with IPTG for the indicated time (3.5 hours). The arrows point to an induced protein band of expected size (.about.21-kDa). No induction is observed in the control lane (i.e., 'No IPTG').
"FIG. 12 is a flow chart depicting the purification protocol for soluble and insoluble (inclusion body) fractions of the E. coli cells expressing recombinant 15-kDa protein.
"FIG. 13 depicts Western detection of the purified recombinant 15-kDa protein (semi-dry blotted onto nitrocellulose membrane) using a monoclonal antibody directed against the 6.times.His-tag. The antibody cross-reacts with a protein band of .about.21-kDa (i.e.,15-kDa+6-kDa for 6.times.His-tag and associated sequences). The antibody also cross-reacts with a protein band of .about.12-kDa.
"FIG. 14 depicts a Coomassie blue-stained SDS-PAGE gel showing the protein fractions obtained following nickel resin (Ni-NTA) purification of 15-kDa. Lysis denaturing buffer containing 15-kDa protein under urea denaturing conditions was eluted using Ni-NTA. FT=Flow-through, W1=Wash 1 (pH 6.3), W2=Wash 2 (pH 6.3), E1-E5=Elution 1-5, (pH 5.9), E6-E10=Elution 6-10, pH 4.5. Majority of the 15-kDa protein (shown by arrows in E6, E7) elutes in these two fractions.
"FIG. 15 depicts a Coomassie blue-stained SDS-PAGE gel showing eight separate preparations (P1-P8) of the recombinant 15-kDa protein. Elution fractions 6 and 7 from each of the eight preparations were combined into a single tube and analyzed by SDS-PAGE.
"FIG. 16 illustrates ELISA data using recombinant 15-kDa protein to coat ELISA plates. Testing was carried out on four IFA positive (HHP=High high positive, High positive, Mid-positive, and Low-positive) and four (4) IFA negative patient sera samples.
"FIG. 17 illustrates IgG ELISA data using recombinant 15-kDa protein to coat ELISA plates. Testing was carried out on ten IFA positive (HHP=High high positive, High positive, Mid-positive, and Low-positive) and ten (10) IFA negative patient sera samples.
"FIG. 18 illustrates IgM ELISA data using recombinant 15-kDa protein to coat ELISA plates. Testing was carried out on two (2) IFA positive and two (2) IFA negative patient sera samples."
URL and more information on this patent application, see: Hoey, John G.; Huang,
Keywords for this news article include: Antibodies, Antigens, Peptides, Immunology, Amino Acids, Escherichia, Blood Proteins, Immunoglobulins, Biological Factors, Enterobacteriaceae, Gammaproteobacteria, Medical Diagnostic Laboratories Llc.
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