title: Gold nanoparticle-based fluorescence immunoassay for malaria antigen detection.
abstract: The development of rapid detection assays for malaria diagnostics is an area of intensive research, as the traditional microscopic analysis of blood smears is cumbersome and requires skilled personnel. Here, we describe a simple and sensitive immunoassay that successfully detects malaria antigens in infected blood cultures. This homogeneous assay is based on the fluorescence quenching of cyanine 3B (Cy3B)-labeled recombinant Plasmodium falciparum heat shock protein 70 (PfHsp70) upon binding to gold nanoparticles (AuNPs) functionalized with an anti-Hsp70 monoclonal antibody. Upon competition with the free antigen, the Cy3B-labeled recombinant PfHsp70 is released to solution resulting in an increase of fluorescence intensity. Two types of AuNP-antibody conjugates were used as probes, one obtained by electrostatic adsorption of the antibody on AuNPs surface and the other by covalent bonding using protein cross-linking agents. In comparison with cross-linked antibodies, electrostatic adsorption of the antibodies to the AuNPs surfaces generated conjugates with increased activity and linearity of response, within a range of antigen concentration from 8.2 to 23.8 μg.mL(-1). The estimated LOD for the assay is 2.4 μg.mL(-1) and the LOQ is 7.3 μg.mL(-1). The fluorescence immunoassay was successfully applied to the detection of antigen in malaria-infected human blood cultures at a 3% parasitemia level, and is assumed to detect parasite densities as low as 1,000 parasites.μL(-1).
authors.affiliation: REQUIMTE, Departamento de Química, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, 2829-516 Caparica, Portugal.
authors.forename: Bassem S S
authors.initials: BS
authors.lastname: Guirgis
authors.forename: Cláudia
authors.initials: C
authors.lastname: Sá e Cunha
authors.forename: Inês
authors.initials: I
authors.lastname: Gomes
authors.forename: Miguel
authors.initials: M
authors.lastname: Cavadas
authors.forename: Isabel
authors.initials: I
authors.lastname: Silva
authors.forename: Gonçalo
authors.initials: G
authors.lastname: Doria
authors.forename: Gregory L
authors.initials: GL
authors.lastname: Blatch
authors.forename: Pedro V
authors.initials: PV
authors.lastname: Baptista
authors.forename: Eulália
authors.initials: E
authors.lastname: Pereira
authors.forename: Hassan M E
authors.initials: HM
authors.lastname: Azzazy
authors.forename: Maria M
authors.initials: MM
authors.lastname: Mota
authors.forename: Miguel
authors.initials: M
authors.lastname: Prudêncio
authors.forename: Ricardo
authors.initials: R
authors.lastname: Franco
mesh_terms: D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000276:immunology; D000953:Antigens, Protozoan / Q000032:analysis* / Q000276:immunology; D002232:Carbocyanines / Q000737:chemistry; D005455:Fluorescent Antibody Technique / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry; D006046:Gold / Q000737:chemistry*; D018840:HSP70 Heat-Shock Proteins / Q000032:analysis / Q000276:immunology; D006801:Humans; D008288:Malaria / Q000097:blood / Q000175:diagnosis* / Q000276:immunology; D053758:Nanoparticles / Q000737:chemistry*; D010963:Plasmodium falciparum / Q000276:immunology / Q000302:isolation & purification*; D015800:Protozoan Proteins / Q000032:analysis / Q000276:immunology; D011994:Recombinant Proteins / Q000032:analysis / Q000276:immunology; D012680:Sensitivity and Specificity
chemical_list: D000911:Antibodies, Monoclonal; D000953:Antigens, Protozoan; D002232:Carbocyanines; D005456:Fluorescent Dyes; D018840:HSP70 Heat-Shock Proteins; D015800:Protozoan Proteins; D011994:Recombinant Proteins; C089187:cyanine dye 3; D006046:Gold