this agent is highly infectious for humans by aerosol , where a single organism can cause the disease . due to q fever 's worldwide distribution and the high infectivity of c. burnetii , us military and civilian personnel deployed overseas several studies [ 13 ] showed that q fever poses a greater threat to us forces deployed in iraq than previously predicted . an investigation of febrile illness outbreak among marines in hit , iraq , highlights the fact that q fever is capable of causing localized outbreaks in exposed military personnel with attack rates up to 50% and perhaps higher . army center for health promotion and preventive medicine ( usachppm ) initiated a q fever surveillance program in early 2007 . military personnel deployed to iraq since 2007 . in addition , the largest known reported q fever outbreak involved approximately 4,000 human cases and occurred from 2007 to 2010 in the netherlands . acute q fever illness most commonly presents as a flu - like illness , pneumonia , or hepatitis . symptoms of q fever are easily confused with those due to a variety of other pathogens ( e.g. , dengue , malaria , and leptospirosis ) that may require different treatment regimens . the chronic form is infrequent ( < 5% of patients with acute infections ) , but the potentially consequent endocarditis is often fatal if left untreated [ 6 , 7 ] . although the presence of c burnetii dna can be detected occasionally in patient serum of acute phase q fever with polymerase chain reaction ( pcr ) [ 810 ] , the current diagnosis of q fever relies mainly on serological methods . these methods include the indirect immunofluorescent antibody assay ( ifa ) [ 12 , 13 ] , the complement fixation assay ( cfa ) [ 14 , 15 ] , and the enzyme - linked immunosorbent assay ( elisa ) [ 16 , 17 ] . among these tests , ifa is considered to be the reference test during an endemic situation [ 8 , 18 ] . because the screening of serum samples by ifa was laborious , elisa was used during an epidemic situation because it can be automated and is easier to perform . the antigen used in elisa is mostly whole cell preparation of phase i or phase ii c. burnetii [ 16 , 17 , 19 , 20 ] . due to the hazard and difficulty of culturing and purifying c. burnetii in a biosafety level ( bsl)-3 laboratory , the antigens are not available in most clinical laboratories . although there are commercially available ifa and elisa tests for q fever , the serological test results vary considerably among different laboratories using the same kit . this may be due to the residual egg yolk or tissue culture proteins in the whole cell antigen preparation [ 2 , 21 ] . earlier studies focused on the identification of immunogenic antigens of c. burnetii discovered protein immunogens of molecular weights from 13 to 92  kda [ 22 , 23 ] . among them , hsp60 , com1 , cbmip , p1 , and adaa have been cloned and characterized previously [ 2327 ] . more recently , several proteomic studies have identified additional immunogenic proteins by 2d - gel immunoblotting [ 28 , 29 ] and protein microarray approaches [ 30 , 31 ] . a 27  kda immunodominant antigen com1 was identified by all different methods mentioned above . in this study , we cloned and purified the com1 antigen . 33 q fever patient sera , 10 normal human sera , and 156 other febrile patient sera were used to evaluate the usage of recombinant com1 antigen for the detection of q fever specific antibodies in elisa . the results demonstrated the amplification of elisa signal may have the potential to improve the serological assay . the cloned genes were inserted into pet24a ( novagen , ca ) for the expression of com1 protein . e. coli bl21 ( de3 ) ( invitrogen , ca ) was used for expression of proteins under the control of phage t7 lac promoter . a primer pair [ com1f ( 5-cgggatccgccccctctcaattcagttttt-3 ) and com1r ( 5-aagaatgcggccgccttttctacccggtcgatttct-3 ) ] was designed by using the nucleotide sequence of the open reading frame for the com1 from strain rsa 493 ( genbank accession number nc002971.1 ) . the coding sequence for amino acids 22 to 252 of the com1 protein was amplified by pcr using genomic dna isolated from c. burnetti rsa 493 strain as the template . the pcr product was digested with bamhi and noti and ligated into the expression vector pet24a . top10 competent cells were transformed with the ligation mixture , and colonies were screened for the presence of inserts with the right size . the recombinant e. coli colony with high expression level of the com1 protein was grown overnight in overnight express medium tb ( emd biosciences , ca ) in the presence of kanamycin at 37c with shaking at 200  rpm . cell pellets from 500  ml cultures were resuspended in 20  ml of buffer a ( 20  mm tris - hcl , ph 8.0 , 1  mm edta , and 1  mm dtt ) containing 2% deoxycholic acid ( doc ) . cells extracted from sonication ( ultrasonic liquid processor model virsonic 475 , virtis company , ny ) were centrifuged at 10,000  g for 30  min in a thermo centrifuge ( model iec multirf ) . the pellets were resuspended in hisbind buffer ( 20  mm tris , ph 8.0 , 0.5  m nacl , 10  mm imidazole ) containing 8  m urea by vortexing , placed on a shaker at room temperature for an additional 10  min , and centrifuged for 30  min at 10,000  g . the supernatant from one liter of culture was applied onto a 1  ml nickel - column ( ni - nta ) equilibrated with the same buffer containing 8  m urea . the recombinant protein was eluted from the column in a step gradient of 25 , 50 , 100 , 200 , 400 , and 600  mm imidazole in hisbind buffer containing 8  m urea . refolding of rcom1 protein in 8 m urea in hisbind buffer was achieved by sequential dialysis as described previously . different amounts ( 0.15 , 0.3 , 0.45 , and 0.6  g per well ) of rcom1 were used to coat the elisa plate to determine the optimum amount for coating . the optimal amount of rcom1 was determined to be 0.3  g per well as coating plate with 0.45 and 0.6 did not increase the signal . the optimization was also performed for whole cell antigens and determined to be 0.15  g per well . after incubation with diluted patient sera , the biotinylated anti - human igm ( thermo scientific , il ) or anti - human igg ( santa cruz biotechnology , ca ) at 1  :  5000 dilution in phosphate buffered saline ( pbs ) with 5% bovine serum albumin ( bsa ) was added . after 1  h of incubation at room temperature , the plates were washed as previously described then incubated with streptavidin - peroxidase polymer ( sigma , ms ) at 1  :  8000 dilution in pbs with 5% bsa for another hour . at the end of incubation , the plates were washed again before the addition of the abts substrate . optical densities at 405  nm ( od405 ) were measured after 30 minutes as previously described . 33 archived q fever ifa positive patient sera were received from naval medical research unit number 3 , cairo , egypt . 10 archived normal human sera and 156 archived sera from patients with other febrile illness identified by their respective ifa ( 27 with scrub typhus , 45 with murine typhus , 56 with spotted fever - type rickettsioses , and 28 with oroya fever ) were used as control ( tables 1 and 4 ) . the study protocol was approved by the naval medical research center institutional review board ( case number pjt23 ) in compliance with all applicable federal regulations governing the protection of human subjects . the gene coding for amino acids 22 to 252 of com1 protein was cloned into vector pet24a and was expressed as a histidine - tag fusion proteins . the rcom1 was highly purified after a single step as a 27  kda fusion protein ( figure 1 ) . the refolded purified rcom1 was used as antigen to detect the presence of q fever specific igg and igm in an elisa . a panel of six positive serum samples ( p1 to p6 ) with ifa titers and two normal serum samples ( c1 and c2 ) was used . the results of elisa showed only one sample ( p4 ) had significant higher levels of specific igg against rcom1 than the controls ( table 2 ) and none of them had detectable levels of specific igm against rcom1 ( data not shown ) . it appears that by using rcom1 as the antigen , we can only detect the presence of specific igg in samples with high ifa titer ( greater than 1024 ) . to improve the sensitivity of the elisa , biotinylated anti - of the six positive samples , six and four had greater signal levels of specific igg and igm against rcom1 than the controls , respectively . however , the igm ifa titers and the amplified igm elisa signal values do not correlate very well . 33 q fever serum samples confirmed by ifa were used to perform standard and/or amplified elisa to detect specific antibody against rcom1 and standard elisa to detect specific antibody against c. burnetii phase i and phase ii whole cell antigen ( figure 2 and table 3 ) . of those samples , 26 ( 79% ) samples had specific igg against phase i whole cell antigen , 33 ( 100% ) samples had specific igg against phase ii whole cell antigen , and 29 ( 88% ) had specific igg against rcom1 . the signals from igg against rcom1 and phase ii whole cell antigens in these samples were higher than those against phase i whole cell antigen ( figure 2(a ) ) . for igg measurement , cohen 's kappa values were 0.78 for rcom1 to phase ii whole cell antigen and 0.67 for rcom1 to phase i whole cell antigen . in igm elisa , 26 ( 79% ) samples had a specific antibody against phase i whole cell antigen , 31 ( 94% ) samples had a specific antibody against phase ii whole cell antigen , and 21 ( 64% ) samples had a specific antibody against rcom1 . with amplification , 29 samples had detectable specific antibody activity against rcom1 ( figure 2(b ) ) . the kappa values were 0.65 for rcom1 ( amplified ) to phase ii whole cell antigen and 0.55 for rcom1 ( amplified ) to phase i whole cell antigen in igm assay . less than 5% ( 5 out of 156 ) of other febrile illness samples had detectable igg or igm against rcom1 suggesting that rcom1 is q fever specific ( table 4 ) . these data indicate that the amplified elisa with the rcom1 is specific and sensitive for the detection of antibodies against c. burnetii . the purified and refolded recombinant com1 antigen without its signal peptide can be recognized by both human igg and igm to c. burnetii in elisa . the recombinant protein antigen rcom1 offers a considerable advantage over the whole cell antigen of c. burnetii . it can be easily purified in large quantity and its quality can be more consistent from batch to batch . the amplified elisa reported in this study involves the binding of biotinylated anti - human antibody ( igg or igm ) to the captured primary antibody followed by the addition of streptavidin labeled peroxidase polymers . the amplified method takes the advantage of the high affinity between streptavidin and biotin to form a stable complex and multiple peroxidases on each polymer chain to increase the peroxidase enzyme signal as compared to the standard elisa . the additional step in the amplified elisa does require longer processing time to perform the assay . because skim milk contains endogenous biotin , it is incompatible with the amplification using streptavidin . bsa was used in the buffer for dilution of biotinylated anti - human antibodies and streptavidin - peroxidase polymer . this amplified method can also be used to determine igg and igm titers ( data not shown ) . by using the ifa as the reference assay , the rcom1 antigen can only detect the q fever specific antibody in elisa without amplification when the titer is greater than 1024 , as indicated in table 2 . the amplification steps in elisa yield a greater gap between the signal of the q fever patient sera and the normal human sera ( table 2 ) or other febrile diseases patient sera ( data not shown ) . with the amplification , we were able to detect q fever specific antibody in some samples with very low ifa titers . in our study , close to 90% ( 29/33 ) q fever patient sera had detectable igg and igm against rcom1 . we were able to detect the com1 specific igg antibody in the majority ( 88% ) of the q fever patient sera without amplification , suggesting that those sera may have high ifa titers . both igg and igm results of using rcom1 were in substantial agreement with that of phase ii whole cell antigen as indicated by the kappa values , 0.78 and 0.65 , respectively . there are fewer elisa positive samples against phase i whole cell antigen than that of the phase ii whole cell antigen . as demonstrated by previous studies , the phase i and phase ii antigens interact differently with antisera collected in the early time of infection [ 3437 ] . phase ii antigens can be recognized by both early ( < 20 days after infection ) and late ( > 20 days after infection ) infection - derived sera , while phase i antigens were recognized predominantly by antisera collected in the late time of infection . since we do not have record of the sample collection date after onset of symptoms , we are unable to make the same conclusion . in our elisa , all 33 q fever sera were igg positive but only 31 sera were igm positive using whole bacteria as the antigens . it could be that those samples have very low igm titers , as we observed in table 2 . the com1 protein is a 27  kda outer membrane - associated immunoreactive protein found in both acute and chronic disease strains of the pathogenic bacteria c. burnetii . it contains a cxxc motif that is homologous to the catalytic site of protein disulfide oxidoreductases . zhang et al .   reported the detection of igg in human patient sera using a partially purified rcom1 . in this study , we were able to produce rcom1 with greater than 95% purity to eliminate nonspecific signal due to impurity and detect both igg and igm with signal amplification in elisa . more well - characterized positive and negative samples from different parts of the world are needed to further evaluate the performance of rcom1 .
currently , the accepted method for q fever serodiagnosis is indirect immunofluorescent antibody assay ( ifa ) using the whole cell antigen . in this study , we prepared the recombinant antigen of the 27-kda outer membrane protein ( com1 ) which has been shown to be recognized by q fever patient sera . 
 the performance of recombinant com1 was evaluated in elisa by ifa confirmed serum samples . due to the low titers of igg and igm in q fever patients , 
 the standard elisa signals were further amplified by using biotinylated anti - human igg or igm plus streptavidin - hrp polymer . 
 the modified elisa can detect 88% ( 29 out of 33 ) of q fever patient sera collected from marines deployed to iraq . 
 less than 5% ( 5 out of 156 ) of the sera from patients with other febrile diseases reacted with the com1 . 
 these results suggest that the modified elisa using com1 may have the potential to improve the detection of q fever specific antibodies .