doi: 10.1128/JCM.37.11.3540-3544.1999.
Serodiagnosis of human granulocytic ehrlichiosis by a recombinant HGE-44-based enzyme-linked immunosorbent assay
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- PMID:10523549
- PMCID: PMC85687
- DOI: 10.1128/JCM.37.11.3540-3544.1999
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Serodiagnosis of human granulocytic ehrlichiosis by a recombinant HGE-44-based enzyme-linked immunosorbent assay
J W IJdo et al. J Clin Microbiol.1999 Nov.
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Abstract
Current antibody testing for human granulocytic ehrlichiosis relies predominantly on indirect fluorescent-antibody assays and immunoblot analysis. Shortcomings of these techniques include high cost and variability of test results associated with the use of different strains of antigens derived from either horses or cultured HL-60 cells. We used recombinant protein HGE-44, expressed and purified as a maltose-binding protein (MBP) fusion peptide, as an antigen in a polyvalent enzyme-linked immunosorbent assay (ELISA). Fifty-five normal serum samples from healthy humans served as a reference to establish cutoff levels. Thirty-three of 38 HGE patient serum samples (87%), previously confirmed by positive whole-cell immunoblotting, reacted positively in the recombinant ELISA. In specificity analyses, serum samples from patients with Lyme disease, syphilis, rheumatoid arthritis, and human monocytic ehrlichiosis (HME) did not react with HGE-44-MBP antigen, except for one sample (specificity, 98%). We conclude that recombinant HGE-44 antigen is a suitable antigen in an ELISA for the laboratory diagnosis and epidemiological study of HGE.
Figures
HGE-44–MBP protein synthesis and immunoblot analysis. (A) Coomassie blue stain of anE. coli lysate containing HGE-44–MBP (lane 1, uninduced; lane 2, induced; lane 3, affinity-purified HGE-44–MBP). Lane M, size markers (kilodaltons). (B) Immunoblot of the gel in panel A probed with rabbit anti-MBP serum. (C) Immunoblot of the gel in panel A probed with serum from an HGE patient.
(A) HGE-44–MBP immunoblots probed with sera from patients with HGE. Lanes 1 to 3, HGE patient sera that tested negative in the ELISA; lane 4, HGE serum containing antibodies to MBP; lanes 5 to 7, HGE patient sera that tested positive in the ELISA; lanes 8 and 9, sera from healthy volunteers; lane 10, rabbit serum containing antibodies to MBP (positive control). The molecular mass of HGE-44–MBP is about 80 kDa. (B) Immunoblots containing MBP probed with sera as in panel A. Numbers at left are in kilodaltons.
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