A novel polyclonal antibody-based sandwich ELISA for detection of Plasmodium vivax developed from two lactate dehydrogenase protein segments.
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.


CONCLUSIONS
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.
Luciana Pereira
LP
Sousa
ILMD, Instituto Leônidas and Maria Deane, 476, Teresina Street, 69057-070 Manaus, AM, Brazil. mariuba@amazonia.fiocruz.br.
Luis André Morais
LA
Mariuba
Rudson Jesus
RJ
Holanda
João Paulo
JP
Pimentel
Maria Edilene Martins
ME
Almeida
Yury Oliveira
YO
Chaves
Davi
D
Borges
Emerson
E
Lima
James Lee
JL
Crainey
Patricia Puccinelli
PP
Orlandi
Marcus Vinicius
MV
Lacerda
Paulo Afonso
PA
Nogueira
D000818:Animals; D000906:Antibodies / Q000032:analysis / Q000276:immunology; D003429:Cross Reactions; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D006801:Humans; D007770:L-Lactate Dehydrogenase / Q000097:blood* / Q000235:genetics / Q000276:immunology; D016780:Malaria, Vivax / Q000097:blood / Q000175:diagnosis* / Q000469:parasitology; D051379:Mice; D010966:Plasmodium vivax / Q000201:enzymology / Q000276:immunology / Q000302:isolation & purification*; D015800:Protozoan Proteins / Q000097:blood* / Q000235:genetics / Q000276:immunology; D011817:Rabbits; D012680:Sensitivity and Specificity
D000906:Antibodies; D015800:Protozoan Proteins; D007770:L-Lactate Dehydrogenase