Development of specific anti-penicillium marneffel antibodies for detection of penicilliosis

Abstract

Background: Penicilliosis is emerging among the most common opportunistic infections in patients with Human Immunodeficiency Virus (HIV) in Southeast Asia. Treatment outcome depends on rapid diagnosis and initiation of antifungal therapy. The current diagnosis of penicilliosis is culture-based which takes 2-14 days for identification of Penicillium marneffei and contributes to the delay of treatment. Simple, rapid, and reliable serologic methods for detecting P. marneffei in patients’ specimen will enhance the treatment and outcome of penicilliosis. Objective: This project aims to develop sensitive and specific antiPenicillium marneffei antibody for serologic assay development. Methods: Hyperimmune sera against-Penicillium marneffei was prepared by immunizing rabbits with killed whole cell P. marneffei and Freund adjuvant. The production of antibody was tested by indirect enzyme-linked immunosorbent assay and latex agglutination assays. P. marneffei’s specificity was tested against 5 most common HIV-associated pathogens. Results: There was a four to eight fold increase of anti-Penicillium marneffei antibody production over the immunization period using ELISA assays. Agglutination assays failed to detect an antigen/antibody response. In antigen/antibody cross check experiment, rabbit hyperimmune sera was nonreactive to Salmonella spp., Cryptococcal neoformans, and E. coli, was weakly reactive to Aspergillus fumigates (2 folds), and was highly reactive (9 folds) to Candida albican. Conclusion: Immune polyclonal antibody against P. marneffei was developed in all 3 rabbits using killed whole-cell yeast form of P. marneffei. Sera from all rabbits including the control were highly reactive to Candida albican, suggesting the rabbits were either exposed to or infected with Candida albican during the experiment, limiting further optimization of the antibody produced in this experiment. Key words: Penicillium marneffei, rabbit hyperimmune sera, antiPenicillium marneffei antibody, indirect ELISA.