Production of recombinant Bartonella henselae 17-kDa protein for antibody-capture enzyme-linked immunosorbent assay.
The Bartonella henselae 17-kDa protein was expressed in a prokaryotic expression system as a histidine-tagged fusion protein and was purified. The target gene was cloned into a recombinant expression construct, pTri-17kd. The expressed protein was purified to near homogeneity by a nickel-agarose column chromatography. Protein recovery was estimated to be 2.9 mg from 100 mL of bacterial culture. The purified 17-kDa protein was recognized by serum from patients infected with B. henselae and Bartonella quintana, suggesting antigenic integrity. The sensitivity and specificity of the IgG enzyme-linked immunosorbent assay (ELISA) relative to immunofluorescent antibody assay testing were 71.1% and 93.0%, respectively. According to the receiver operating characteristic curve analysis, the area under the curve was 0.823. These results indicate that the expressed 17-kDa protein is a suitable source of antigen for development of an antibody-capture ELISA for the detection of antibodies to B. henselae.
Department of Research and Development, Medical Diagnostic Laboratories, L.L.C., Hamilton, NJ 08690, USA.
Chien Chang
CC
Loa
Eli
E
Mordechai
Richard C
RC
Tilton
Martin E
ME
Adelson
D000595:Amino Acid Sequence; D000818:Animals; D000907:Antibodies, Bacterial / Q000097:blood*; D000942:Antigens, Bacterial / Q000276:immunology*; D019540:Area Under Curve; D001426:Bacterial Proteins / Q000235:genetics / Q000276:immunology*; D018416:Bartonella henselae / Q000235:genetics / Q000276:immunology*; D018413:Bartonella quintana / Q000235:genetics / Q000276:immunology; D015153:Blotting, Western; D002415:Cats; D002847:Chromatography, Agarose; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics; D019084:Fluorescent Antibody Technique, Indirect; D005784:Gene Amplification; D005786:Gene Expression Regulation / Q000276:immunology*; D005822:Genetic Vectors; D006801:Humans; D007074:Immunoglobulin G / Q000032:analysis; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology*
D000907:Antibodies, Bacterial; D000942:Antigens, Bacterial; D001426:Bacterial Proteins; D007074:Immunoglobulin G; D011994:Recombinant Proteins