AN INDIRECT IMMUNOFLUORESCENCE ASSAY USING A CELL CULTURE-DERIVED ANTIGEN FOR DETECTION OF ANTIBODIES TO THE AGENT OF HUMAN GRANULOCYTIC EHRLICHIOSIS

An indirect immunofluorescence assay for the detection of human antibo dies to the agent of human granulocytic ehrlichiosis (HGE) was develop ed and standardized. Antigen was prepared from a human promyelocytic l eukemia cell line (HL-60) infected with a tick-derived isolate of the HGE agent (USG3). Suitable antigen presentation and preservation of ce llular morphology were obtained when infected cells were applied and c ultured on the slide, excess medium was removed, and cells were fixed with acetone. Use of a buffer containing bovine serum albumin and goat serum reduced background fluorescence, and use of an immunoglobulin G (gamma-specific) conjugate reduced nonspecific binding. The assay rea dily detected specific antibody from HGE patients and did not detect a ntibody from healthy individuals. No significant reactivity was noted in sera from patients with high titers of antibodies to other ricketts ial species. We were able to identify antibodies reactive to USG3 anti gen in samples from areas where HGE is endemic that had tested negativ e to other rickettsial agents. Animal sera reactive against Ehrlichia equi or Ehrlichia phagocytophila bound to the HGE antigen, indicating that the assay may be useful for veterinary use. Comparability between two different laboratories was assessed by using coded human sera exc hanged between laboratories. Results from the two laboratories were si milar, indicating that the assay can be easily integrated into use for routine testing for HGE. The assay was then compared to an assay usin g horse neutrophils infected with ehrlichiae. The two assays gave comp arable results, indicating that the cell culture-derived antigen can b e used for testing samples that have been previously tested with E. eq ui as an antigen. The new assay offers several advantages over other i mmunofluorescence methods that use animal-derived antigen and is suita ble for use in testing for human antibodies to the HGE agent.