This reinforces the potential of the SPR-HAVP1 strategy as a new tool in serological monitoring for HA. The relatively wide linearity range and high sensitivity observed from this study will be valuable for early diagnosis of acute hepatitis A, even during the period of infection, when the levels of anti-HAV IgM antibodies are low, thereby reducing the immunological window period of this infection. Current gold standard immunoassays used for hepatitis A diagnosis employ a long incubation period for antibodyCantigen association, indirect format [10,11], and can be relatively time-consuming when handling large sample sizes, as in an epidemic [15]. assays. SPR-HAVP1 assays showed good performance in the detection of IgM polyclonal antibody anti-HAV. These assays were performed using a COOH5 sensor chip functionalized with VP1 protein. The sensorgram record showed a significant difference between positive and negative serum samples, which was confirmed by analysis of variation of initial and final dissociation values through time (RUd/t). The data gathered here are unequivocal evidence that this SPR-HAVP1 strategy can be applied to detect IgM antibodies in human serum positive to the HAV. This is a new tool to be explored to diagnose human HAV infections. Keywords: hepatitis A computer virus, major capsid protein VP1, IgM, surface plasmon resonance 1. Introduction Hepatitis A is an acute liver disease caused by the hepatitis A computer virus (HAV). HAV is usually classified within the family, in the genus AC260584 = 0.0006) between positive and negative serum samples was more evident after performing analysis of the variation of initial and final dissociation values in the time of 173 s (RUd/t). Open in a separate window Physique 3 Binding avidity evaluation of anti-HAV for serum samples. (A) Human serum (1:1000) positive [1378 (light green) and 1398 (light orange)] and unfavorable [104 (light blue), 106 (red ), 107 (dark blue), 110 (green Rabbit Polyclonal to GPRC6A musk), and 111(pink)] from hepatitis A computer virus. (B) The difference between positive () and unfavorable () serum was analyzed from the variation of the initial and final dissociation values in 177 s (RUd/t). The results are shown as resonance models (RU) and are representative of the average response between 1 and 800 s. These results are representative of three impartial assays. * = 0.0006. The initial dissociation phase (RUid = 209.27) and the final dissociation (RUfd = 106.21) of serum 1398 showed higher values than serum 1378 (RUid = 85.66 and RUfd = 19.35). Unfavorable sera had more homogeneous AC260584 RUid values from 55.14 to 82.25 and RUfd ranging from 1.3 to 13.17, except for serum 111, which presented RUid = 55.16 and did not present positive RUfd. The sensorgram generated from serum samples allowed us to evaluate the avidity of each serum sample based on the RU variation of dissociation divided by time. In this way, it was possible to determine a cutoff value for discrimination between positive (0.25) and negative (0.15) serum samples (Determine 3B). Additionally, the CV generated by the repeated injections of serum samples (triplicate) onto the chip sensor functionalized with VP1 was found to be from 1.15% to 6.86%, indicating high reproducibility of the assay. 4. Discussion The specific diagnosis of acute hepatitis A depends on the detection of serum IgM antibody to HAV [14]. Currently, this diagnosis is mainly based on ELISA and chemiluminescence immunoassays. Although these assays show good sensitivities and can be automated, they are not high-throughput assays and do not allow large-scale testing [8]. Most of these immunodiagnostic assessments for anti-HAV detection rely on the use of inactivated HAV particles as a tool for antibody detection [21]. However, HAV grows slowly and produces low titers in most cell culture systems [22,23], a feature that hampers its mass production for diagnostic assessments. Troubles in producing HAV by cell culture may be circumvented by the use of well-defined antigens. Alternatively, the use of recombinant VP1 proteins may overcome this issue to obtain large amounts of antigen in a faster and cheaper approach, for application in diagnostic assessments for HA. Thus, this work explores, for the first time, the association of the recombinant VP1 with SPR technology as a new tool (SPR-HAVP1) for HA diagnosis. AC260584 The immunodominant neutralization site of HAV mainly involves residues of VP1 and VP3 and a potentially impartial site involving residue 221 of VP1 [24]. Due to the acknowledged role of AC260584 VP1 in the humoral immune response during contamination, this protein has been the main target.