Curr Protein Pept Sci 4:299C308. the results of the immunoscreening. PNZ5 The coding regions of five of the characterized antigens (enolase, tryparedoxin peroxidase, eukaryotic initiation factor 5a, -tubulin, and one of the hypothetical proteins) were cloned in a prokaryotic expression vector, and the corresponding recombinant proteins were purified PNZ5 and evaluated for the serodiagnosis of TL. The antigens presented sensitivity and specificity values ranging from 95.4 to 100% and 82.5 to 100%, respectively. As a comparative antigen, a preparation of extract showed sensitivity and specificity values of 65.1 and 57.5%, respectively. The present study has enabled the identification of proteins able to be employed for the serodiagnosis of TL. INTRODUCTION Leishmaniasis consists of a spectrum of diseases caused by protozoan parasites of the genus (((is the most important etiological agent of the disease (6, 7). At present, there is no gold standard test for TL diagnosis, and a combination of diagnostic methods is frequently needed to obtain more precise results (8). Parasitological diagnosis is usually definitive and consists PNZ5 of the microscopic examination of Giemsa-stained biopsy specimen smears, of histopathological examination or immunohistochemical approaches in lesion fragment triturates, and by parasite cultures, as well as PCR. However, these assessments’ sensitivity varies according to when the lesions occurred, and a Pax1 low number of parasites can be present in the late lesions, hampering the sensitivity of the assessments (8,C13). Additionally, these assessments require invasive procedures of sample collection, which limit their use. The Montenegro skin test (MST) has PNZ5 been employed for the immunological diagnosis of TL, although it does not make it possible to distinguish if the patients present either the acute or chronic disease or if they have already been cured or are noninfected subjects living in areas where the disease is usually endemic (14). In this context, assessments based on antileishmanial serology could be used to contribute to the laboratory diagnosis of the disease (15). However, the antigens usually employed can indicate false-positive results in serum samples from noninfected subjects living in areas of endemicity where leishmaniasis is commonly found or in those infected by other trypanosomatids, such as antigens (21,C23). Although the role of antibodies in TL is not completely comprehended, evidence has shown that this humoral response determined by specific IgG reactivity to antigens could well be used as an indicator of the development of the disease, since some authors have positively correlated the clinical cure of disease with a decrease in the antileishmanial antibody titers, as well as to assist the clinical follow-up during and after healing by the treatment of the patients (23,C27). In this context, the search for more refined antigens of spp. is necessary to identify molecules able to improve the sensitivity and specificity values for the serodiagnosis of TL. In addition, the identification of proteins able to predict the success of a given treatment will help the follow-up of patients after treatment. Only a few reports that applied proteomic methods for the identification of using sera from TL patients. The objective was the identification of the most antigenic proteins to be applied for the improvement of serodiagnosis of TL. With the purpose of refining the selection of the candidate antigens, sera from noninfected subjects living in regions of TL endemicity, as well as sera from Chagas disease (CD) patients, PNZ5 were also used in the immunoblots to exclude the cross-reactive proteins. MATERIALS AND METHODS Ethics statement. This study was conducted according to the Declaration of Helsinki principles, and it was approved by the Ethics Committee from Federal University of Minas Gerais (protocol no. CAAEC323431 14.9.0000.5149), Belo Horizonte, Minas Gerais, Brazil. All patients received an individual copy of the study policy, which was reviewed by an independent person, and all participants gave their consent form in Portuguese before the collection of their blood sample. Parasite. (strain MHOM/BR/1975/M2904 was used. Parasites were used until the 5th culture passage. The promastigotes were produced at 24C in Schneider’s medium (Sigma, St. Louis, MO), which was supplemented with 10% inactivated fetal bovine serum (FBS) (Sigma), 20 mM l-glutamine, 100 U/ml penicillin, and 100 g/ml streptomycin (pH 7.4) after reaching the stationary phase of the culture. The soluble antigenic extract (SLA) was prepared as described previously (30). For the preparation of the amastigote-like forms of experiments to infect macrophages as.