Evaluation of a Recombinant 27‐kDa Outer Membrane Protein of Coxiella burnetii as an Immunodiagnostic Reagent

Abstract

The 27-kDa outer membrane protein from eight strains of Coxiella burnetii was expressed in the pET-21c protein expression system. Two fusion proteins with molecular masses of 30 and 32 kDa were evident in all eight of the recombinants by SDS-PAGE and immunoblotting. A protein having an approximate size of 30 kDa was purified from the Escherichia coli lysates by one-step affinity purification. The utility of the purified recombinant protein in ELISA was also evaluated by testing its reactivity with human sera and comparing this reactivity with that of Nine Mile phase II antigen. All of the 40 IF-positive serum samples were ELISA-positive for both the Nine Mile phase II and recombinant antigens, and negative serum controls were negative for both antigens. These results suggest that ELISA with the 27-kDa recombinant antigen is a sensitive and specific method for detecting anti-C. burnetii antibodies in human sera.

Role of B Cells in Host Defense against Primary Coxiella burnetii Infection

“…kit (eBioscience) according to manufacturer specifications. ELISAs for detection of C. burnetii-specific antibodies were performed as described previously (32). Briefly, plates were coated with 100 l of inactivated NMI antigen at 0.5 mg/ml in 0.05 M carbonate/bicarbonate coating buffer (pH 9.6) for 48 h at 4°C.…”

Identification and Characterization of an Immunodominant 28-Kilodalton Coxiella burnetii Outer Membrane Protein Specific to Isolates Associated with Acute Disease

“…Guinea pig serum was collected at 4 weeks postaerosol infection with 10 6 organisms of the Nine Mile phase I, Ohio, Q217, or Q229 strain and stored at Ϫ80°C until use. C. burnetii Nine Mile whole-cell lysate and purified rCom1, which is a protein common to all isolates tested (29,30), were used as a control to confirm the presence of the antibodies to C. burnetii antigens in infection-derived sera. SDS-PAGE and immunoblotting were performed as described previously (31).…”

“…E. coli containing the recombinant plasmid was cultured in LB supplemented with 4 mM IPTG (isopropyl-␤-D-thiogalactopyranoside) at 37°C overnight, and then cells were pelleted by centrifugation. The cell pellet was analyzed by SDS-PAGE and immunoblotting with rabbit anti-Nine Mile serum as described previously (30).…”

Mechanisms of Vaccine-Induced Protective Immunity against Coxiella burnetii Infection in BALB/c Mice

“…Mouse sera from PI-V-or PII-V-vaccinated mice at prechallenge and 14 days postchallenge were tested for total IgG, IgG1, and IgG2a to PI or PII Ag by ELISA as modified from the method described previously (30). Briefly, 50 l of inactivated PI or PII Ag at 500 ng/ml in 0.05 M carbonate/ bicarbonate coating buffer (pH 9.6) were added to each well of a 96-well microtiter plate and coated at 4°C for 48 h. The plates were blocked with 1% BSA in PBST buffer (0.05% Tween 20 in PBS) and then incubated with 100 l of serially diluted mouse sera at room temperature for 2 h. After washing four times with PBST buffer, the plates were incubated with 100 l of HRP-conjugated goat anti-mouse IgG, IgG1, or IgG2a (1/5000 dilution) at room temperature for 2 h. The Sigma Fast O-Phenylenediamine Dihydrochloride Tablet Sets (Sigma-Aldrich) were used as substrates, and OD was measured at 490 nm by the Spectra Max M2 system using the SoftMax program (Molecular Devices).…”