Padronização de um ELISA do tipo sandwich para captura do NS1 do vírus Zika
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2023-01-25
Autores
Lino, Nikolas Alexander Borsato
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Universidade Estadual Paulista (Unesp)
Resumo
O vírus Zika (ZIKV) foi associado com o incomum aumento de casos de microcefalia no Brasil entre os anos de 2015 e 2016. Em decorrência desta epidemia causada por arbovírus nas Américas, a Organização Mundial de Saúde declarou emergência de saúde pública de interesse internacional. Desde então, o ZIKV tem sido o foco de pesquisas voltadas a mitigar os seus efeitos na saúde humana, incluindo estudos voltados à vigilância epidemiológica molecular, antivirais e formas de prevenção. Testes laboratoriais voltados à identificação acurada do ZIKV são de suma importância, porque, durante a doença, o quadro clínico é inespecífico. Diferentemente dos ensaios de captura de IgM e IgG anti-ZIKV, a NS1 representa um biomarcador de maior precocidade, porque é secretado pelas células infectadas e independe do período de janela imunológica. Outra vantagem da NS1 está relacionada à menor possibilidade da ocorrência de reações cruzadas, as quais são frequentes na sorologia por anticorpos entre os flavivírus por estes compartilharem vários epítopos. Portanto, a relevância do presente estudo situa-se nessa problemática por visar padronizar um ensaio de ELISA sandwich para captura da proteína não estrutural 1 (NS1) do ZIKV, ou seja, a proteína que será testada representa um IgG anti-NS1 (anticorpo teste). De resultados principais, foi observado que a caseína na concentração a 0.5% foi o melhor bloqueio, dentre a testagem com leite em pó desnatado e albumina sérica bovina, pois gerou menor background nos experimentos. O anticorpo teste revelou positividade para o NS1-ZIKV recombinante nas concentrações de 2.14 µg/mL a 0.03 µg/mL. Por outro lado, satisfatoriamente o anticorpo teste não reagiu de forma cruzada com o NS1 recombinante do vírus dengue (DENV) nas concentrações de 5.32 µg/mL a 0.16 µg/mL. Para o teste de amostras séricas NS1-ZIKV positivas por padrão ouro (PRNT 50), houve sensibilidade de 55.5% (15/27) e especificidade de 88% (30/34). Em síntese, neste estudo piloto os dados gerados mostram que o anticorpo teste não reage de forma cruzada com o NS1-DENV e pode ser um alvo interessante para detecção do NS1-ZIKV, porém testes adicionais são necessários com o intuito de otimizar a sua sensibilidade.
Zika virus (ZIKV) was associated with an unusual increase in microcephaly cases in Brazil between 2015 and 2016. As a result of this epidemic caused by arbovirus in the Americas, the World Health Organization declared a public health emergency of international concern. Since then, ZIKV has been the focus of research aimed at mitigating its effects on human public health, including studies aimed at molecular epidemiological surveillance, antivirals and forms of prevention. Laboratory tests aimed at the accuracy of ZIKV are of paramount importance, because, during the disease, the clinical picture is nonspecific. Unlike anti-ZIKV IgM and IgG capture assays, NS1 represents an early biomarker, because it is secreted by infected cells and it is independent of the window period. Another advantage of NS1 is related to the lower possibility of the occurrence of cross-reactions, which are frequent in serology for antibodies between flaviviruses because it shares several epitopes. Therefore, the relevance of the present study lies in this problem as it aims to standardize a sandwich ELISA assay for the capture of ZIKV non-structural protein 1 (NS1), that is, the protein that will be tested represents an anti-NS1 IgG (test antibody). From the main results, it was observed that casein at a concentration of 0.5% was the best blocker, among the tests with skimmed milk powder and bovine serum albumin, as it generated a smaller background in the experiments. The test antibody revealed positivity for recombinant NS1-ZIKV at concentrations from 2.14 µg/mL to 0.03 µg/mL. On the other hand, the test antibody did not satisfactorily cross react with dengue virus recombinant NS1 (DENV) at concentrations from 5.32 µg/mL to 0.16 µg/mL. For testing NS1-ZIKV positive serum samples by gold standard (PRNT 50), there was a sensitivity of 55.5% (15/27) and a specificity of 88% (30/34). In summary, in this pilot study, the data generated show that the test antibody does not cross-react with NS1-DENV and may be an interesting target for detection of NS1-ZIKV, but additional tests are needed in order to optimize its sensitivity.
Zika virus (ZIKV) was associated with an unusual increase in microcephaly cases in Brazil between 2015 and 2016. As a result of this epidemic caused by arbovirus in the Americas, the World Health Organization declared a public health emergency of international concern. Since then, ZIKV has been the focus of research aimed at mitigating its effects on human public health, including studies aimed at molecular epidemiological surveillance, antivirals and forms of prevention. Laboratory tests aimed at the accuracy of ZIKV are of paramount importance, because, during the disease, the clinical picture is nonspecific. Unlike anti-ZIKV IgM and IgG capture assays, NS1 represents an early biomarker, because it is secreted by infected cells and it is independent of the window period. Another advantage of NS1 is related to the lower possibility of the occurrence of cross-reactions, which are frequent in serology for antibodies between flaviviruses because it shares several epitopes. Therefore, the relevance of the present study lies in this problem as it aims to standardize a sandwich ELISA assay for the capture of ZIKV non-structural protein 1 (NS1), that is, the protein that will be tested represents an anti-NS1 IgG (test antibody). From the main results, it was observed that casein at a concentration of 0.5% was the best blocker, among the tests with skimmed milk powder and bovine serum albumin, as it generated a smaller background in the experiments. The test antibody revealed positivity for recombinant NS1-ZIKV at concentrations from 2.14 µg/mL to 0.03 µg/mL. On the other hand, the test antibody did not satisfactorily cross react with dengue virus recombinant NS1 (DENV) at concentrations from 5.32 µg/mL to 0.16 µg/mL. For testing NS1-ZIKV positive serum samples by gold standard (PRNT 50), there was a sensitivity of 55.5% (15/27) and a specificity of 88% (30/34). In summary, in this pilot study, the data generated show that the test antibody does not cross-react with NS1-DENV and may be an interesting target for detection of NS1-ZIKV, but additional tests are needed in order to optimize its sensitivity.
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Palavras-chave
Vírus Zika, ELISA sandwich, Diagnóstico NS1, Zika Vírus, Diagnostic