Synthetic peptides for efficient discrimination of anti-enterovirus antibodies at the serotype level.

Enteroviruses are important human pathogens, causing a broad spectrum of diseases from minor common colds to fatal myocarditis. However, certain disease syndromes are caused by one or few serotypes. Serotype identification is difficult due to the laborious neutralization tests that lack of sensitivity, while in commercial ELISAs homotypic antibodies' activities are largely masked by the recognition of genera-specific epitopes by heterotypic antibodies. In the present study homotypic assays were developed with the ability to discriminate different enterovirus serotypes. Seventy-three children sera, positive for IgM antibodies against enterovirus genus and 49 healthy children were examined for the presence of antibodies against 14 synthetic peptides derived from a non-conserved region of the VP1 protein of coxsackieviruses B2, B3, B4, B5, A9, A16, A24, echoviruses 6, 7, 9, 11, 30, enterovirus 71 and parechovirus 1. 50% of the anti-enterovirus IgM positive sera (>150 BU) reacted with the peptides with the majority of them to preferentially recognize one of them, supporting the homotypic nature of our assay. Inhibition studies yielded homologous inhibition rates 67-95% suggesting that specific peptide recognition actually occurred. The diagnostic value of our assay was tested in blood samples drawn over a 1.5-year period from a 5-year old patient. The anti-enterovirus reactivity was clearly attributed to echovirus serotype 11. The IgM/IgG antibody ratio was reversed 4 months later and subsequently IgM antibodies dropped below the cutoff point. In this paper we demonstrate that our assay can be used to discriminate between antibodies targeting different enterovirus serotypes.

Estimation of seroprevalence of the pandemic H1N1 2009 influenza virus using a novel virus-free ELISA assay for the detection of specific antibodies.

The pandemic H1N1 2009 influenza A emerged in April 2009 and spread rapidly all over the world. In Greece, the first case of the pandemic H1N1 was reported on May 18, 2009, while a considerable increase in the number of cases was noticed at the beginning of July 2009. The need for surveillance of the immune status of the Greek population led us to develop a virus-free ELISA that specifically recognizes pandemic H1N1 2009 influenza virus antibodies in human sera. The method is based on the use of synthetic peptides (H1-pep and N1-pep) that are derived from the hemagglutinin and neuraminidase of the 2009 pandemic strain, respectively, and differentiate the swine-origin influenza A/California/14/2009 (H1N1) from the seasonal influenza A viruses. Serum samples were obtained from 271 healthy blood donors during May, November, and December 2009. Among sera collected during May, November, and December, IgG antibodies against the peptide H1-pep were detected in 7.4, 13.8, and 19.3% of the donors, respectively, while IgG antibodies against the peptide N1-pep were detected in 5.3, 9.6, and 16.9% of the donors, respectively. The application of the immunoassay indicated a time-dependent increase of the prevalence of anti-H1-pep and anti-N1-pep IgG antibodies during the pandemic H1N1 outbreak in Greece. The method could be also indicative for the discrimination of immune persons from those susceptible to infection with the pandemic H1N1 strain, as well as for the establishment of effective vaccination programs.

MAP kinases mediate phagocytosis and melanization via prophenoloxidase activation in medfly hemocytes.

E. coli phagocytosis by medfly hemocytes, in contrast to mammalian macrophages, associates with E. coli-challenged hemocyte secretion by mitogen activating protein (MAP) kinases. In the present work, we examined whether this system links with the proteolytic activation of prophenoloxidase (proPO). ProPO and prophenoloxidase-activating proteinases (PAPs) were initially identified within freshly isolated medfly hemocytes. Moreover, flow cytometry and immunocytochemical analysis revealed the constitutive expression of proPO and its stable association with hemocyte surface. The expression level of hemocyte surface proPO is not affected by E. coli infection. In addition, flow cytometry analysis in freshly isolated hemocytes showed that E. coli phagocytosis is markedly blocked by antibodies against proPO or PAPs, as well as by several serine protease inhibitors, strongly supporting the involvement of proPO cascade in the phagocytosis process. Similarly, it was shown that melanization process depends on proPO activation. MAP kinases appeared to control both phagocytosis and melanization, since they regulate PAPs secretion, a prerequisite for the conversion of proPO to active PO. From this and previous studies, hemocytes appear to be central to immune response in medfly.