# Patent

## Title
Recombinant antigens for the detection of Coxiella burnetii

## Abstract

The invention relates to a method for the detection of prior exposure to Coxiella. burnetii infection by antibody-based assays using recombinant, immunodominant C. burnetii polypeptides. The invention also relates to the design of biotin or His-tagged C. burnetii proteins useful in the antibody-based assays as standardized antigen reagents.

## Background
CROSS-REFERENCES TO RELATED APPLICATIONS
This application claims priority to provisional application Ser. No. 60/668,591, filed Apr. 6, 2005, now abandoned.


SEQUENCE LISTING
The application contains a Sequence Listing in paper form and a computer readable copy. I hereby state that the information recorded in computer readable form is identical to the written sequence listing.
BACKGROUND OF THE INVENTION
1. Field of the Invention
The inventive subject matter relates to a method of diagnosing Q fever and detecting exposure to Coxiella burnetii using specific immunodominant C. burnetti directly as antigen in antibody based assays such as enzyme-linked immunosorbent assays or western blot or the specific immundominant C. burnetti proteins fused to an upstream biotinylated peptide.
2. Description of the Related Art
Coxiella burnetii is the causative agent of the disease Q fever and is found worldwide (1,2). The genus Coxiella is composed of the single species, C. burnetii, short, rod-shaped bacteria, 0.2 to 0.4 μm in diameter and 0.4 to 1.0 μm in length (1, 3). C. burnetii is typically maintained as an inapparent infection in domesticated animals, such as cattle, sheep and goats and is found in a number of tick species. The organism persists in feces, urine, milk and tissues so that fomites and infective aerosols are capable of easily forming. Disease occurs among farm workers in close proximity with the animals or their products. Transmission, therefore, is generally by inhalation of infected aerosols or even by the ingestion of raw milk that is infected with the bacteria. Additionally, the organism is highly infectious and is considered a potential biowarfare agent (4).
C. burnetii displays an antigenic phase variation, unlike other rickettsial species. In nature, the bacteria expresses phase I antigen, which is a polysaccharide component of the Coxiella lipopolysaccharide (5, 6, 7). After passage in culture, the bacteria changes from phase I to phase II. Phase II appears to be a manifestation of deletions of carbohydrate components from the lipopolysaccharide moiety (6).
Clinically, onset of Q fever occurs after an incubation of 18 to 21 days. Although rarely fatal, Q fever is typically abrupt, manifesting with fever, severe headache, chills, severe malaise, myalgia and chest pains. Acute and chronic infection, however, are characterized by different serological profiles (8). Early in the disease progression, Q fever resembles many infections such as influenza, salmonellosis, malaria, hepatitis, and brucellosis. For chronic Q fever, diagnosis is typically demonstrated by antibody against phase I and phase II antigens. In acute infection, diagnosis can typically only be made by direct detection of bacterial antigen or isolation of bacteria since significant serum antibodies are not available at this stage of infection.
Although it is advisable to diagnose Q fever early after infection, early after C. burnetii infection diagnosis is difficult due to the difficulty in bacterial isolation and antigen detection and because of the lack of seroconversion at these early stages. Specific complement fixation and agglutinating antibodies are useful in determining infection with agglutination test generally the more sensitive assay method (9-11). Additionally, the binding of fluorescent antibodies such as in indirect fluorescent assays (IFA) or enzyme-linked immunosorbent assays (ELISA) is also helpful in determining C. burnetii infection (12). Additional methods, such as direct tissue biopsy, culture of the organism and polymerase chain reaction (PCR) analysis are also useful but are used with much less frequency. The lack of popularity in using biopsy and culture is primarily due to the extensive equipment and expertise requirements as well as the time required to conduct the analysis. PCR offers high sensitivity and the ability to detect bacterial antigen early after infection. However, later in the course of the disease PCR's usefulness is considerably less likely to detect infection than serology (8, 13). Furthermore, PCR is often limited in use based on the availability of trained personnel and equipment.
Central to the development of improved detection and diagnostic immunoassay methods and standardization is the development of more effective antigens for use in existing antibody-based methods. Along these lines, efforts have been made to identify C. burnetii immunodominant proteins (14). Accordingly, a number of highly sera-reactive proteins have been identified, including chaperonin, translation elongation factor Tu and the 27 kDa outer membrane protein (Com-1) (14-16). Due to their high reactivity to anti-C. burnetii serum, these proteins have been suggested to be valuable antigens in detection and diagnostic assays. However, despite the utililty of immunoassays in the diagnosis of C. burnetii, currently available assays suffer from lack of sensitivity and reproducibility. Standardization of antigen will improve assay diagnostic performance and provide early and more accurate treatment regimens (13).
In order to improve standardization of diagnositic assays to C. burnetii and to increase assay sensitivity and specificity of detection, other C. burnetii immunodominant antigens need to be identified. Improved sensitivity can be achieved by defining antigens expressing a greater number of epitopes well represented in serum antibody repetoires. Alternatively or in addition, methods to ensure attachment of antigen to assay matrixes can be devised with improved orientation of the antigen on the assay matrix with concomitant reduction in steric hinderance in binding of specific anti-C. burnetii antibody in patient serum to the antigen.
SUMMARY OF THE INVENTION
Currently available antigenic moieties do not permit highly sensitive and easy to perform methods for the detection of C. burentii, the causative agent of the important infectious disease Q fever. Therefore, an object of the invention is the insertion of DNA sequences encoding immunodominant C. burnetii proteins into an expression system that when expressed yields a fusion protein composed of a biotinylated peptide and/or a T7-tag upstream of an immunodominant C. burnetii protein that can be utilized in antibody based assays for detection or diagnosis of C. burnetii. Alternatively, a construct containing a His-tag can also be constructed downstream of the C. burnetii insert. This antigen system improves assay standardization by better enabling coupling of measured and predetermined antigen onto solid matrixes for use in antibody-based assays such as ELISA and rapid flow assays. The system also ensures that antigen epitopes are free to bind available antibody.
A further aspect of the invention is the identification of new species of immunodominant C. burnetii proteins and their recombinant forms that are suitable for use as antigen in antibody-based assays.
Another object of the invention is the amplification of assay signal by rolling circle amplification or polymerase chain reaction by the incorporation into the assay secondary antibody containing specific DNA primers.
A still further object of the invention is the use of said recombinant proteins or fusion peptides as vaccine candidates.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1. (a) Map of pET-AB'C vector. In the figure T7 is the T7 promotor, RBS is the ribosomal binding site. The biotin peptide sequence encodes a 22 amino acid sequence that is selectively biotinylated, catalyzed by the down stream encoded biotin holoenzyme synthetase. FIG (b) shows a map of pET 24a containing a His-tag downstream of the bacterial insert in addition to the upstream T7-tag.
FIG. 2. Western blot of biotinylated C. burnetii recombinant proteins in pET-AB'C vector. Transferred, separated proteins were exposed to streptavidin-horse radish peroxidase to visualize the biotinylated proteins. Lane 1 shows pET-AB'C with no insert. Lane 2 shows pET-AB'C containing FtsZ. Lane 3 is the vector containing chaperonin. Lane 4 is the pET-AB'C vector containing 27 kDa outer membrane protein (Com-1).
FIG. 3. Western blot of biotyinylated C. burnetii recA and ompA-like proteins. Transferred, separated proteins were exposed to streptavidin-horse radish peroxidase to visualize the biotinylated proteins. Lane 1 shows biotinylated recA and Lane 2 shows biotinylated ompA-like protein.



DESCRIPTION OF THE PREFERRED EMBODIMENTS
Currently, diagnosis of C. burnetii relies principally on serology based assays such as enzyme-linked immunosorbent assay (ELISA), micro-agglutination, complement fixation and indirect-fluorescent-antibody (IFA) assays with the most commonly employed method being IFA. Although other methods are available, such as polymerase chain reaction, cell culture or immunodetection of antigen in tissue biopsy specimens, these later procedures require considerable infrastructural requirements such as specialized training for personnel, containment facilities and specialized equipment. Furthermore, most of these requirements are beyond the capacity of many health-care facilities. Additionally, many of the serology based assays, because of the lack of standardized antigen, employ a large subjective component in assay interpretation and contain variability from laboratory to laboratory.
Therefore, an aspect of this invention is a method for the detection of prior exposure to C. burnetti, which utilizes as antigen specific recombinantly-produced, C. burentii proteins that are highly sero-reactive, including ompA-like transmembrane domain protein, recA, cell division protein FtsZ, translation elongation factor Tu, chaperonin and 27 kDa outer membrane protein (Com-1). The inventive method contemplates detection of one or more of either ompA-like transmembrane domain protein, recA and cell division protein FtsZ. For greater accuracy and sensitivity, the method can be modified to also include detection of translation elongation factor Tu, chaperonin and 27 kDa outer membrane protein (Com-1).
The inventive method, therefore, by incorporating these specific proteins into an antibody-based assay format is a more accurate and potentially more sensitive assay than other antibody based methods for the detection of C. burnetti. The method, by using standardized antigens, will also enable better analysis of assay data for more definitive determination of prior C. burentii infection and diagnosis.
An additional aspect of this invention is the recombinant expression of immunodominant fusion proteins containing a C. burnetii protein down-stream of a biotin-tagged peptide sequence. The biotin-tagged fusion proteins can be used in antibody-based detection and diagnostic assays for C. burnetii. Application of this invention will result in improved sensitivity of detection of C. burnetii infection with concomitant improvement in diagnosis of Q fever over other diseases presenting with similar symptomatology, especially early after infection. Additionally, inclusion of a biotin tag improves the sensitivity of the assay by enhancing the imobilization of antigen. The biotin tag also improves standardization of the assay since matrixes containing immobilized streptavidin can be produced in high quantities at a uniformly high density with subsequent uniformly high density of streptavidin bound antigen bound via the biotin tag.
Another aspect of this invention is the identification and cloning of C. burnetii proteins that were previously described and sequenced but that were not previously recognized as immunodominant proteins. Therefore, these proteins, recombinantly produced and either used alone or with other C. burnetii recombinant proteins will confer improved standardization and concomitant assay reproducibililty and potentially sensitivity in assays for the detection and diagnostic assays for C. burnetii infection and Q fever.
C. burnetii immunodominant proteins were identified and selected by intensity of reactivity to C. burnetii infected patient sera in western blots from 2-dimensional gel electrophoresed C. burnetii proteins. This was accomplished by lysing C. burentii bacteria (Henzeerling strain) and their protein components separated by 2-dimensional (2-D) gel electrophoresis. The 2-D gel separated proteins were then transferred to a polyvinylidene fluoride (PVDF) matrix by western blotting and the immunodominate spots identified by exposing the PVDF to sera from C. burnetii infected patients. The most prominent antigen spots were excised (cut out) from the 2-D gel and subjected to N-terminal amino acid sequence analysis and mass spectrometry (MS) to definitively identify the proteins. Prior to protein identification, the excised 2-D spots were reduced with DTT, alkylated with isoacetamide and subjected to trypsin enzymatic digestion. In order to determine the internal amino acid sequences, the digests were then subjected to further analysis by liquid chromatography (LC) and MS for definitive identification of the digest peptide constituents and ultimately the identity of the protein (FIG. 3).
Western blot studies, as described above, demonstrated a number of proteins that exhibited strong reactivity to anti-C. burnetii serum and therefore were likely candidates for diagnostic assays for Q-fever. Several of these proteins: translation elongation factor Tu, chaperonin and 27 kDa outer membrane protein (Com-1) were previously described to be reactive to C. burnetii serum (12-14). However, three other proteins were identified as also being highly reactive to anti-C. burnetii serum, ompA-like transmembrane domain protein, recA and cell division protein FtsZ. These observations strongly suggest that these other proteins, alone or used together, are valuable as antigens in diagnostic assays.
The following examples illustrate utilization of the C. burnetii recombinant peptides in antibody-based detection of C. burnetii. 
EXAMPLE 1 
Use of C. burnetii Peptides as Diagnostic Assay Reagent in ELISA
Currently available diagnostic assays or Q fever primarily rely on serum-based assays for the detection of antibodies to C. burnetii with IFA the primary assay method. However, this assay, is often not sensitive and can lead to inaccurate results due to the somewhat subjective nature of the assay procedure. Therefore, other, more objective means of detection of C. burnetii infection and diagnosis of Q fever are needed. One such method is enzyme-linked immunosorbent assay (ELISA) or other antibody-based assay. In order to sensitively and reliably diagnosis by this method, suitable, standardized antigen must be available. An aspect of this invention is the use of the recombinant protein antigens in antibody-based assays such as ELISA, lateral flow or immunochromatigraphic assays.
A number of proteins have been identified in western blot studies as being highly immunoreactive to anti-C. burnetii antibody from C. burnetii infected patient serum. For example, the proteins translocation elongation factor Tu, 27 kDa outer membrane protein (Com-1) and chaperonin have been previously determined to be reactive to anti-C. burnetii serum (12-14). However, based on western blotting of 2D gels, using anti-C. burnetii serum, as described above, other proteins have been identified that have equal or greater immunoreactivity. These include cell division protein FtsZ, recA and ompA-like transmembrane domain protein.
Construction of recombinant C. burnetii proteins can be carried out by first producing a cDNA copy of the gene sequenced by polymerase chain reaction. The sequence of the amplified DNA of C. burnetii ompA-like transmembrane domain protein, rec A and cell division protein FtsZ are shown in SEQ ID No. 13, 14 and 15, respectively and their encoded amino acid sequences as SEQ ID No. 16, 17 and 18, respectively. The DNA sequences C. burnetii translation elongation factor Tu, chaperonin and 27 kDa outer membrane protein (Com-1) are represented by SEQ ID No. 19, 20, and 21, respectively and their amino acid sequences shown as SEQ ID No. 22, 23, and 24, respectively. The SEQ ID numbers of the proteins is also summarized in Table 1.









TABLE 1







Sequence



description



















SEQ ID No. 1
cell division protein




FtsZ forward primer



SEQ ID No. 2
cell division protein




FtsZ reverse primer



SEQ ID No. 3
ompA-like




transmembrane




domain protein




forward primer



SEQ ID No. 4
ompA-like




transmembrane




domain protein




reverse primer



SEQ ID No. 5
rec A forward




primer



SEQ ID No. 6
rec A reverse primer



SEQ ID No. 7
translation




elongation factor Tu




forward primer



SEQ ID No. 8
translation




elongation factor Tu




reverse primer



SEQ ID No. 9
chaperonin, 60 kDa




forward primer



SEQ ID No. 10
chaperonin, 60 kDa




reverse primer



SEQ ID No. 11
27 kDa outer




membrane protein




(Com-1) forward




primer



SEQ ID No. 12
27 kDa outer




membrane protein




(Com-1)




reverse primer



SEQ ID No. 13
ompA-like




transmembrane




domain protein




DNA seq.



SEQ ID No. 14
rec A DNA seq.



SEQ ID No. 15
cell division protein




FtsZ DNA seq.



SEQ ID No. 16
ompA-like




transmembrane




domain protein




amino acid seq.



SEQ ID No. 17
rec A amino acid




seq.



SEQ ID No. 18
cell division protein




FtsZ amino acid




seq.



SEQ ID No. 19
translation elong.




factor Tu DNA seq.



SEQ ID No. 20
chaperonin, 60 kDa




DNA seq.



SEQ ID No. 21
27 kDa outer




membrane protein




(Com-1) DNA seq.



SEQ ID No. 22
translation elong.




factor Tu amino




acid seq.



SEQ ID No. 23
chaperonin, 60 kDa




amino acid seq.



SEQ ID No. 24
27 kDa outer




membrane protein




(Com-1) amino acid




seq.










Expression of the C. burnetii protein products can be accomplished by inserting the encoding DNA into a suitable expression system, such as pET 24a. The C. burnetii protein can be utilized as an antigen either as an unpurified E. coli lysate or purified by any number of methods and subsequently used as antigen in detection or diagnostic assays.
Assays using the recombinantly produced proteins include antibody-based assays such as enzyme-linked immunosorbent assays. As previously mentioned, antigen for the assay can be in the form of unpurified E. coli lysate. However, for increased assay sensitivity and reduced background, purified recombinant C. burnetii proteins can be used. The preferred method for conducting the assay comprises the following steps:

    
    
        1. Microtiter plates with 96 wells are coated with 0.3 μg/well of any or all of the recombinant proteins represented by SEQ ID No. 16-18 and/or 22-24 and stored in 4° C. for 2 days.
        2. Plates are washed 3X with wash buffer (0.1% TWEEN-20, polyoxyethylenesorbitan monolaurate, in PBS).
        3. Plates are blocked with 200 μl/well of blocking buffer (5% skim milk in wash buffer)×45 minutes and then rinsed three times.
        4. Sera is diluted in blocking buffer and 100 μl/well is added and incubated ×1 to 2 hours.
        5. Plates are washed three times with wash buffer.
        6. Plates are then incubated with 100 μg/well of enzyme-labeled (e.g. peroxidase) anti-human immunoglobulin for 1 hour.
        7. The plates are washed three times with wash buffer.
        8. Substrate is added to the wells and read after 15 to 30 minutes.
    
    


A standard curve can be constructed by conducting the above ELISA procedures with the recombinant proteins but utilizing a range of concentrations of specific antibody to C. burnetii. The extent of measured binding of patient serum antibody is compared to a graphic representation of the binding of the C. burnetii-specific antibody concentrations.
Sensitivity of antibody-based assays, such as ELISA, can be enhanced by substituting the enzyme-substrate step with a molecular detection method. An example of a molecular method employed is the amplification of circular DNA by rolling circle amplification (RCA). In RCA, secondary antibody is conjugated with a single stranded DNA primer comprising the following steps:

    
    
        a. 1 mg of sulfo-GMBS powder was added to 4 mg of antibody F(ab′)2 in 1 ml, in the dark, for 30 minutes at 37° C., followed by 30 minutes at room temperature;
        b. 2 ml of phosphate buffered saline (PBS) was added to the reaction mixture from a;
        c. the reaction mixture in b, above, was applied unto a pre-equilibrated Presto Desalting Column® (Pierce Biotechnology, Inc, Rockford, Ill.);
        d. the applied material was eluted with PBS and the eluted fractions monitored by absorbance at 280 nm;
        e. pooled fractions containing maleimide-acitivated antibody was concentrated and stored at 4° C. in the dark until used;
        f. activated DNA was prepared by res-suspending 0.44 mg of thio-DNA (C6 S-S®) (MWG-Biotech Inc, High Point, N.C.) in 150 μl TE buffer with 15 μl of 1 M DTT and incubated at room temperature for 30 minutes;
        g. the DTT was removed from the mixture of step g by applying the mixture to a G-50 micro column and spinning the column at 735×g for 2 minutes;
        h. the activated antibody and activated thio-DNA was then mixed and the mixture incubated in the dark at room temperature for 1 hour then overnight at 4° C.;
        i. product from step h was analyzed by gel electrophoresis.
    
    


RCA reactions were undertaken the method comprising the following steps:

    
    
        a. mix together on ice 5 nM of primer-conjugated antibody, 10 nM circular DNA, 200 ng of E. coli, single-stranded DNA binding protein (SSB), 13 units of T7 Sequenase and 0.4 mM each of dATP, dCTP, dGTP, 0.3 mM dTTP and 0.1 mM FITC-dUTP in 25 μl of reaction buffer at pH 7.9 containing 20 mM Tris-acetate, 10 mM magnesium acetate, 50 mM potassium acetate and 1 mM DTT;
        b. incubate the mixture of step at 37° C. for up to 30 minutes;
        c. RCA products are then analyzed by measuring fluorescence incorporation of DNA product.
    
    


As alternative to RCA, PCR can be utilized using a primer complimentary to the antibody-conjugated DNA, made as described for RCA. Amplification is conducted by utilizing a DNA primer complementary to a template sequence contained on the conjugated DNA.
EXAMPLE 2 
Expression of Tagged Recombinant C. burnetii Fusion Proteins
Antigen reactivity to patient serum can be enhanced by either using antigen with increased number of B-cell epitopes, improving efficiency of antigen attachment to the solid matrix prior to serum exposure or by reducing the potential for steric hinderance by improving antigen orientation on the solid matrix.
Previously ELISA methods require direct adhering of assay antigen onto the ELISA plates. Alternatively, antigen can be captured by specific antibody bound to the plates. However, either technique has distinct disadvantages. By adhering antigen directly to plates, potentially reactive epitopes are masked from binding to antibody in test sera. Furthermore, adherence of antigen cannot be completely controlled since the non-specific binding characteristics vary from one lot of plates to another or plate to plate within a lot. Furthermore, non-specific adherence to the plate can vary depending on the conditions used to bind the protein. This variability can be circumvented to a considerable degree by capturing antigen by specific antibody previously adhered to the plates. This suffers, however, by consuming epitopes on the antigen that would normally be available for binding to specific antibody in the test sera. However, another approach to addressing the problem of reproducibly adhering antigen to ELISA plates but still maximally retaining antigen epitopes is to adhere antigen via a biotyin-streptavidin bridge.
The vector pET-AB'C vector (17) is used to construct a fusion protein comprising C. burnetii antigen containing a biotinylated leader sequence as illustrated in FIG. 1(a). Referring to FIG. 1(a), biotinylation is catalyzed by biotin holoenzyme sysnthetase down-stream of the insertion site. The biotinylated fusion antigen is then bound to ELISA plates via strepavidin previously coated onto the plates. This method offers an improvement in assay design by permitting standardization antigen by controlling the amount of strepavidin bound to the plate. Additionally, the method improves the orientation of C. burnetii antigen bound to the plate since the antigen will be bound via a biotin attached to streptavidin rather than attached in any number of orientations to the plate itself.
Construction of the pET-AB'C C. burnetii expression vector is carried out by first amplifying DNA encoding the C. burnetii proteins translocation elongation factor Tu; ompA-like transmembrane domain protein and; recA, cell division protein FtsZ, chaperonin or 27 kDa outer membrane protein (Com-1) as described in Example 1. The forward and reverse primers used for amplification are SEQ ID No. 1 -12 as summarized in Table 1. Amplification with these primers will yield DNA, encoding C. burnetii immunodominant proteins, containing Not I enzyme restriction sites. The insertion site on the pET-AB'C vector is flanked by Not I sites, as well. This permits efficient, site-directed insertion of the sequences into the vector. Any of the C. burnetii immunodominant proteins can be inserted and expressed using this vector. FIG. 2 shows the expression of FtsZ, chaperonin and 27 kDa outer membrane protein (Com-1) using the pET-AB'C expression system.
Once constructed, the recombinant, biotinylated C. burnetii antigen can then be bound to ELISA plates that have been pre-coated with streptavidin. The ELISA procedure is then conducted as described in Example 1 from step 2. As previously mentioned, this procedure permits the construction of standardized antigen, in recombinant form that can be reproducibly coupled to ELISA plates. Standardization of antigen coating is also enhanced by prior quality assurance and control of the streptavidin coated ELISA plates to ensure measured amounts of stretavidin are present per well. Furthermore, the sensitivity and reproducibility of assay results are enhanced since the antigen epitopes are conserved for exposure to antibody in patient serum and not bound directly to the plate.
Recombinant C. burnetii antigens were also designed such that C. burnetii proteins were fused to a His-tag at the C-terminal end and a T7-tag at the opposite end (N-terminal) (FIG. 1(b)). As an example, Com-1 or OmpA-like genes were cloned into the pET24a expression vector using the primers SEQ ID No. 25 and 26, for Com-1 forward and reverse primers, respectively and SEQ ID No. 27 and 28 for OmpA forward and reverse primers respectively. The ensuing fusion protein contained an N-terminal T7 tag sequence plus a C-terminal His-tag sequence. Bacteria expressing Com-1 or OmpA-like genes were then grown in Overnight Express Instant TB medium (Novagen (EMD Biosciences, San Diego, Calif.)) with Kanamycin (50 mg/l) at 37° C. for overnight.
Approximately 3 g of bacterial cells (from a 250 ml culture) were then re-suspended in 15 ml lysis solution (BugBuster Master Mix, Novagen (EMD Biosciences, San Diego, Calif.)). The cell suspension was incubated on a rotating mixer at a slow setting for 20 min at room temperature. After centrifugation at 14,000 g for 15 min at 4° C., both Com-1 and OmpA-like fusion proteins were precipitated in the pellets.
The Com-1 pellet was re-suspended in 10 ml of HisBind buffer A (20 mM TrisHCl, pH 8.0, 0.5 M NaCl, and 5 mM imidazole, 8 M urea). Protein sample was applied onto 1 ml Ni-NTA column equilibrated with the same solution except with 10 mM imidazole. After an extensive wash, Com-1 fusion protein was eluted with increased imidazole concentrations (50 mM, 100 mM, and 200 mM). The most pure of the Com-1 fusion fractions were pooled and dialyzed to remove urea against 1× PBS containing 6M, 4M, 2M, 1M, and no urea at 4° C. Refolded Com-1 fusion protein was recovered and stored at −20° C. The refolded pure Com-1 protein was used to raise hyper-immune rabbit sera. The titers were greater than 10e6.
Unlike Com-1 fusion protein, OmpA-like fusion protein was not soluble in 8 M urea. Therefore, the OmpA-like pellet was re-suspended in 20 mM TrisHCl, pH 8.0, 0.5 M NaCl, and 5 mM imidazole 6 M Guanidine Hydrochloride (HisBind buffer B, Novagen). A sample was applied onto 1 ml Ni-NTA column equilibrated with the same buffer. The column was first washed with HisBind buffer B, then washed with buffer A, followed by buffer A except with 10 mM imidazole. After extensive washing, the OmpA-like fusion protein was eluted. The fractions containing Com-1 and OmpA-like fusion proteins that were the most pure were then pooled and dialyzed to remove urea against 1× PBS containing 6M, 4M, 2M, 1M, and no urea at 4° C. OmpA-like fusion protein forms precipitate as urea concentration decreased to 4 M.
REFERENCES


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Having described the invention, one of skill in the art will appreciate in the appended claims that many modifications and variations of the present invention are possible in light of the above teachings. It is therefore, to be understood that, within the scope of the appended claims, the invention may be practices otherwise than as specifically described.

## Claims

1. A method for the detection of antibodies that bind to Coxiella burnetii comprising the steps:
a) expressing recombinant Coxiella burnetii antigens, wherein said recombinant Coxiella burnetii antigens consist of an ompA-like transmembrane domain protein comprising the sequence of SEQ ID NO:16 and a 27 kDa outer membrane protein Corn-1 antigen comprising the sequence of SEQ ID NO:24;
b) contacting said recombinant Coxiella burnetii antigens with a patient serum sample; and
c) detecting binding of said recombinant Coxiella burnetii antigens to antibodies in said patient serum sample.

2. The method of claim 1 wherein said recombinant Coxiella burnetii ompA-like transmembrane protein and 27 kDa outermembrane protein Coin-1 antigen are encoded by polynucleotides with the nucleotide sequences set forth in SEQ ID NO:13 and SEQ ID NO:21, respectively.

3. The method of claim 1, wherein said recombinant Coxiella burnetii antigens are immobilized on a matrix.

4. The method of claim 3, wherein said recombinant Coxiella burnetii antigens are immobilized on said matrix by a method comprising adding said recombinant Coxiella burnetii antigens to immobilized streptavidin, wherein said recombinant Coxiella burnetii antigens are components of recombinant fusion polypeptides containing a biotinylated leader sequence fused, either directly or via another peptide sequence, to the N-terminal end of each recombinant Coxiella burnetii antigen that is operatively linked to a biotin holoenzyme synthetase; and wherein said biotinylated leader-Coxiella burnetii antigen recombinant fusion polypeptide is immobilized to streptavidin via biotin.

5. The method of claim 3, wherein said recombinant Coxiella burnetii antigens are immobilized on said matrix by a method comprising adding said recombinant Coxiella burnetii antigens to either immobilized anti-T7 or immobilized anti-His antibody, wherein each of said recombinant Coxiella burnetii antigens is a component of a recombinant fusion polypeptide containing a T7 tag fused to the N-terminal end of the recombinant Coxiella burnetii antigen that is fused to a His tag at the C-terminal end of the recombinant Coxiella burnetii antigen.

6. The method of claim 4, wherein said recombinant Coxiella burnetii ompA-like transmembrane protein and 27 kDa outermembrane protein Coin-1 antigen are encoded by the nucleotide sequences set forth in SEQ ID NO:13 and SEQ ID NO:21, respectively.

7. The method of claim 1, wherein sensitivity of detection is enhanced by detecting said binding of said patient antibody by the following additional steps:
d. exposing said antigen bound patient antibody to a DNA template-conjugated secondary antibody;
e. amplifying said DNA template by polymerase chain reaction.

8. The method of claim 1, wherein sensitivity of detection is enhanced by detecting said binding of said patient antibody by the following additional steps:
d. adding DNA primer-conjugated secondary antibody;
e. amplifying said DNA primer by rolling circle amplification.