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Production and Evaluation of Reagents for Detection of Histoplasma capsulatum Antigenuria by Enzyme Immunoassay

Mark D. Lindsley,^1* Heather L. Holland,^1, Sandra L. Bragg,¹ Steven F. Hurst,¹ Kathleen A. Wannemuehler,² and Christine J. Morrison^1,

Mycotic Diseases Branch,¹ Office of the Director, Division of Food-borne, Bacterial, and Mycotic Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia²

Received 12 February 2007/ Returned for modification 19 March 2007/ Accepted 29 March 2007

The detection of urinary Histoplasma capsulatum polysaccharide antigen (HPA) by enzyme immunoassay (EIA) has proven useful for the presumptive diagnosis of histoplasmosis in AIDS patients. Assay limitations include (i) detection of a largely uncharacterized antigen and (ii) difficulty in reproducibly generating antibodies for use in the EIA. To improve antibody production for use in this test and to better understand the antigen being detected, we compared rabbit antibodies elicited using various immunization schedules, routes, and H. capsulatum-derived antigens. Antibodies were evaluated by EIA for their ability to detect purified H. capsulatum C antigen (C-Ag) and antigenuria. Reported as enzyme immunoassay (EI) units (the A₄₅₀ with antigen divided by the A₄₅₀ without antigen), results demonstrated that intravenous immunization of rabbits with whole, killed yeast-phase cells (yeast-i.v. regimen) produced antibodies giving the highest EI values in the C-Ag EIA (mean EI units ± standard deviation, 14.9 ± 0.6 versus 6.4 ± 0.4 for rabbits immunized with C-Ag versus 2.4 ± 0.3 for all other regimens combined). Yeast-i.v. antibodies were highly sensitive for the detection of antigenuria in patients with histoplasmosis, as shown by the following results: 12/12 patients compared to 10/12, 6/12, 3/12, and 3/12, respectively, for antibodies from rabbits immunized with (i) C-Ag; (ii) whole, killed yeast-phase cells administered subcutaneously and intramuscularly; (iii) yeast-phase culture filtrates; and (iv) HPA-positive urine. Rabbits immunized using the yeast-i.v. regimen also gave higher peak antibody titers than rabbits immunized by any other regimen (P < 0.03), and their antibodies were most comparable in reactivity to antibodies produced for use in the standard HPA-EIA test (P < 0.001). Therefore, rabbits immunized using the yeast-i.v. regimen produced the most sensitive antibodies with the highest titers for detection of C-Ag and antigenuria in histoplasmosis patients.

Published ahead of print on 11 April 2007.

Present address: Strategic Science and Program Unit, Coordinating Center for Infectious Diseases, Mailstop A-02, Centers for Disease Control and Prevention, 1600 Clifton Road, NE, Atlanta, GA 30333.

Present address: Office of Public Health Research, Office of the Director, Mailstop D-72, Centers for Disease Control and Prevention, 1600 Clifton Road, NE, Atlanta, GA 30333.

This article has been cited by other articles:

• Wheat, L. J., Lindsley, M. D., Holland, H. L., Bragg, S. L., Hurst, S. F., Wannemuehler, K. A., Morrison, C. J. (2007). Evaluation of Reagents for Detection of Histoplasma capsulatum Antigenuria. CVI 14: 1387-1388 [Full Text]