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https://www.arca.fiocruz.br/handle/icict/20925
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ArtigoDireito Autoral
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2015-03-19
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A NOVEL POLYCLONAL ANTIBODY-BASED SANDWICH ELISA FOR DETECTION OF PLASMODIUM VIVAX DEVELOPED FROM TWO LACTATE DEHYDROGENASE PROTEIN SEGMENTS.
Autor(es)
Afiliação
Instituto Leônidas e Maria Deane
Universidade Federal do Amazonas
Fundação de Medicina Tropical Heitor Vieira Dourado
Universidade Federal do Amazonas
Fundação de Medicina Tropical Heitor Vieira Dourado
Resumo em Inglês
Abstract
Background: Immunoassays for Plasmodium detection are, presently, most frequently based on monoclonal
antibodies (MAbs); Polyclonal antibodies (PAbs), which are cheaper to develop and manufacture, are much less
frequently used. In the present study we describe a sandwich ELISA assay which is capable of detecting P. vivax Lactate
Dehydrogenase (LDH) in clinical blood samples, without cross reacting with those infected with P. falciparum.
Methods: Two recombinant proteins were produced from different regions of the P. vivax LDH gene. Two sandwich
ELISA assay were then designed: One which uses mouse anti-LDH 1-43aa PAbs as primary antibodies (“Test 1”) and
another which uses anti-LDH 35-305aa PAbs (“Test 2”) as the primary antibodies. Rabbit anti-LDH 1-43aa PAbs were
used as capture antibodies in both ELISA assays. Blood samples taken from P. vivax and P. falciparum infected patients
(confirmed by light microscopy) were analysed using both tests.
Results: “Test 2” performed better at detecting microscopy-positive blood samples when compared to “Test 1”,
identifying 131 of 154 positive samples (85%); 85 positives (55%) were identified using “test 1”. “Test 1” produced
one false positive sample (from the 20 malaria-free control) blood samples; “test 2” produced none. Kappa coefficient
analysis of the results produced a value of 0.267 when microscope-positive blood smears were compared with “test 1”,
but 0.734 when microscope-positive blood smears were compared with the results from “test 2”. Positive predictive
value (PPV) and negative predictive value (NPV) were observed to be 98% and 22% respectively, for “Test 1”, and 99%
and 45%, for “test 2”. No cross reactivity was detected with P. falciparum positive blood samples (n = 15) with either test
assay.
Conclusion: Both tests detected P. vivax infected blood and showed no evidence of cross-reacting with P. falciparum.
Further studies will need to be conducted to establish the full potential of this technique for malaria diagnostics. As
well as representing a promising new cost-effective novel technique for P. vivax diagnosis and research, the method
for developing this assay also highlights the potential for PAb-based strategies for diagnostics in general.
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