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Clin. Vaccine Immunol. doi:10.1128/CVI.00083-07
Copyright (c) 2007, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Production and Evaluation of Reagents to Detect Histoplasma capsulatum Antigenuria by Enzyme Immunoassay

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

^* To whom correspondence should be addressed. Email: mlindsley{at}cdc.gov.

	Abstract

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: 1) detection of a largely uncharacterized antigen and 2) 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: 1) purified H. capsulatum C-Ag (C-Ag) and 2) antigenuria. Reported as enzyme immunoassay (EI) units (A_450nm with antigen ÷ A_450nm without antigen), results demonstrated that intravenous immunization of rabbits with whole, killed yeast-phase cells ("Yeast iv") produced antibodies giving the highest EI values in the C-Ag EIA (mean EI units±SD: 14.9±0.6 vs. 6.4±0.4 for rabbits immunized with C-Ag vs. 2.4±0.3 for all other regimens combined). "Yeast iv" antibodies were highly sensitive for the detection of antigenuria in patients with histoplasmosis (12/12 patients compared to 10/12, 6/12, 3/12 and 3/12, respectively, for antibodies from rabbits immunized with: a) C-Ag; b) whole, killed yeast-phase cells administered subcutaneously and intramuscularly; c) yeast-phase culture filtrates; or d) HPA-positive urine). Rabbits immunized using the "Yeast iv" regimen also gave the highest peak antibody titers compared to rabbits immunized by any other regimen (P<0.03) and 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 iv" regimen produced the highest titered and most sensitive antibodies to detect C-Ag and antigenuria in histoplasmosis patients.

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]