ABSTRACT
Detection of antibody specific to Leptospira by various immunological techniques has been used for leptospirosis diagnosis. However, the sensitivity of antibody detection during the first few days after infection is low. Molecular techniques are suggested to provide earlier diagnosis than antibody detection, but a rapid and easy to perform assay for Leptospira antigen detection would provide an additional useful tool for disease diagnosis. In this study, we coupled gold nanoparticles with antibody to LipL32, a protein commonly found in pathogenic Leptospira. This coupled gold reagent was used in the immunochromatographic strip for Leptospira detection. We demonstrated that the sensitivity of Leptospira detection by this strip was 10(3) ml(-1). There was no positive result detected when strips were tested with non-pathogenic Leptospira, Staphylococcus aureus, Streptococcus group B, Acinetobacter baumannii, Escherichia coli, Salmonella typhi, Klebsiella pneumoniae, Enterococcus faecalis or Enterococcus faecium. These data suggest that gold nanoparticles coupled with antibody to LipL32 could be used for Leptospira detection by a rapid test based on an immunochromatographic technique.
Subject(s)
Antibodies, Bacterial/chemistry , Chromatography, Affinity/methods , Leptospira/isolation & purification , Leptospirosis/diagnosis , Leptospirosis/microbiology , Nanoparticles/chemistry , Animals , Antibodies, Bacterial/immunology , Antibodies, Monoclonal/chemistry , Antibodies, Monoclonal/immunology , Bacterial Outer Membrane Proteins/analysis , Bacterial Outer Membrane Proteins/immunology , Chromatography, Affinity/instrumentation , Female , Gold/chemistry , Humans , Leptospira/immunology , Leptospirosis/immunology , Lipoproteins/analysis , Lipoproteins/immunology , RabbitsABSTRACT
The 30 kDa protein of B. pseudomallei is found in virulent Ara- but not avirulent Ara+ strain. The gene was cloned in Escherichia coli JM105 employing pInIII-C2 vector. The open reading frame was 870 nucleotides with a guanine plus cytosine content of 69.9%. Arginine was the most abundant amino acid in the protein, having a PI of 12.65. Nucleotide sequence of the gene was 96% identical to B. pseudomallei 1710b chromosome II sequence CP000125.1, encoding an oxidoreductase of the short chain dehydrogenase/reductase family. The 30 kDa antigen was expressed as a maltose-fusion protein with a yield of 5.25 mg/l of bacterial culture.
Subject(s)
Antibodies, Monoclonal/immunology , Antigens, Bacterial/genetics , Bacterial Proteins/genetics , Burkholderia pseudomallei/genetics , Animals , Antigens, Bacterial/analysis , Antigens, Bacterial/isolation & purification , Bacterial Proteins/analysis , Bacterial Proteins/isolation & purification , Burkholderia pseudomallei/pathogenicity , Cloning, Molecular , Escherichia coli Proteins , Gene Expression , Latex Fixation Tests , Open Reading Frames , Periplasmic Binding Proteins , Polymerase Chain Reaction , Sequence Alignment , Sequence Analysis, DNAABSTRACT
A latex agglutination test employing monoclonal antibody specific to a 30-kDa protein of Burkholderia pseudomallei was used to detect the organisms in blood culture specimens from 1,139 patients with community-acquired septicemia. The sensitivity, specificity, and positive and negative predictive values of the test were 96.75%, 99.61%, 96.75%, and 99.61%, respectively.
