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Open Access Methodology

Performance of coumarin-derived dendrimer-based fluorescence-linked immunosorbent assay (FLISA) to detect malaria antigen

Seon-Ju Yeo1, Dinh Thi Huong1, Jin-Hee Han2, Jung-Yeon Kim3, Won-Ja Lee3, Ho-Joon Shin4, Eun-Taek Han2* and Hyun Park1*

Author Affiliations

1 Zoonosis Research Center, Department of Infection Biology, School of Medicine, Wonkwang University, Iksan, Jeonbuk 570-749, Republic of Korea

2 Department of Medical Environmental Biology and Tropical Medicine, School of Medicine, Kangwon National University, Chuncheon, Gangwon-do, Republic of Korea

3 Division of Malaria and Parasitic Disease, Korea National Institute of Health, Osong 363-951, Republic of Korea

4 Department of Microbiology, Ajou University School of medicine, Suwon 443-721, Republic of Korea

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Malaria Journal 2014, 13:266  doi:10.1186/1475-2875-13-266

Published: 10 July 2014

Abstract

Background

Due to limitation of conventional malaria diagnostics, including microscopy, polymerase chain reaction (PCR), and enzyme-linked immunosorbent assay (ELISA), alternative accurate diagnostics have been demanded for improvement of sensitivity and specificity.

Methods

Serially diluted Plasmodium LDH antigens, Plasmodium falciparum-infected human red blood cells (RBC) derived from in vitro culture or patient’s samples were used for evaluation of the performance of fluorescence-linked immunosorbent assay (FLISA). Microscopic examination was used to determine parasite density and the performance of FLISA was compared to ELISA. Finally, sensitivity and specificity of FLISA was determined by human specimens infected with P. falciparum, Plasmodium vivax, Toxoplasma gondii, and amoebae.

Results

As a result of FLISA, the fluorescent intensity was highly correlated with antigen amount and FLISA was more sensitive than ELISA. FLISA detected at least 0.01 ng/ml of pLDH antigen, which showed 1,000-fold higher sensitivity than ELISA. In vitro-cultured P. falciparum was detected up to 20 parasite number/μL in FLISA but 5120 parasite number/μLin sandwich ELISA. In vitro P. falciparum-infected RBC number was highly correlated with fluorescent intensity (R2 = 0.979), showing that FLISA was reliable for detection of P. falciparum and available for quantification of parasite numbers. Furthermore, eighteen patient samples infected with P. falciparum (n = 9) and P. vivax (n = 9) showed 100% of sensitivity (18/18). FLISA showed 96.3% of specificity (26/27) because one sample of patient blood infected with T. gondii gave a false positive reactivity among healthy donors (n = 9), T. gondii-infected patients (n = 9), and amoeba-infected patients (n = 9).

Conclusion

FLISA has a keen and high performance to detect malaria antigen, suggesting a potential assay as malaria immunodiagnostic.

Keywords:
Coumarin-derived dendrimer; Fluorescence-linked immunosorbent assay (FLISA); Lactate dehydrogenase (LDH); ELISA; Plasmodium falciparum; Plasmodium vivax