An arrow indicates band at approximate of 748 base pairs

An arrow indicates band at approximate of 748 base pairs. features, history of eating parasite-contaminated foods, blood eosinophilia, and serological outcomes, i.e., enzyme-linked immunosorbent assays13C16 or immunoblotting using AL3 extract, including an antigenic peptide with an approximate molecular mass of 24 kDa17C19 and below 27C29 kDa.20 Two-dimensional gel electrophoresis (2-DE) and immunoblotting revealed that AL3 antigenic spots with an approximate molecular mass of 23C25 kDa and pof 8.3C8.5 revealed a high potential for the serodiagnosis of human gnathostomiasis spinigerum.21 The amino acid sequence of these antigenic spots was determined by liquid chromatography tandem mass spectrometry (LC/MS-MS) and the LC/MS-MS spectra22 and one of the peptide sequences showed high similarity with a matrix metalloproteinase (MMP)-like protein of database (GenBank accession no. “type”:”entrez-protein”,”attrs”:”text”:”AAF82802″,”term_id”:”9082310″,”term_text”:”AAF82802″AAF82802).23 Cloning and expression of genes such as MMP-like protein,23 cathepsin L-like cysteine protease,24 and cyclophilin protein25 have been reported. However, the diagnostic values of those recombinant proteins for human gnathostomiasis have not been validated. In this study, we produced a recombinant MMP-like protein of and evaluated its sensitivity and specificity in immunodiagnosis for human gnathostomiasis. We selected MMP-like protein because its molecular mass and pcorresponded well with the 2-DE immunoreactive spots detected by the confirmed human gnathostomiasis sera.22 The goal of this study is to setup a stable mass-production system for the standardized immunodiagnostic kit with recombinant MMP-like protein antigen. Materials and Methods Human sera. All serum samples were supplied by the serum bank of the Faculty of Medicine, Khon Kaen University. Serum samples consisted of three groups: 1) Negative control group, which included samples from healthy adult volunteers who were free from any intestinal parasitic infection and checked by stool examination by the formalin ethyl acetate concentration technique26 at the time of blood collection. A pooled sera from all those healthy individuals was also used as negative Tetrodotoxin control for each assay. 2) Gnathostomiasis group, which included samples from parasitologically confirmed gnathostomiasis patients and from patients showing clinical symptoms Tetrodotoxin of suspected cutaneous and visceral gnathostomiasis,2,27,28 with a history of eating food possibly contaminated with larvae and were positive 24 kDa antigen by immunoblotting.29 3) The third group (= 83) consisted of serum samples Tetrodotoxin from patients with other parasitic infections than gnathostomiasis. Their infections were confirmed by parasitological methods except that cysticercosis cases were diagnosed by a computerized tomography scan and found positive by the immunological method Table 1. Informed consent was Tetrodotoxin obtained from all human adult participants and from parents or legal guardians of minors. The study protocol was approved by the Khon Kaen University Ethics Committee for Human Research (“type”:”entrez-nucleotide”,”attrs”:”text”:”HE541293″,”term_id”:”288736136″,”term_text”:”HE541293″HE541293). Table 1 Type of human sera and immunoblotting results against the purified fusion-tagged recombinant MMP-like protein* AL3 were collected from mice inoculated orally with early third-stage larvae recovered from copepod.30 The worms (= 40) were then placed into RNAlater (Promega, Madison, WI). The total Tetrodotoxin RNA was extracted using Trizol reagent (Invitrogen, Carlsbad, CA) and was finally dissolved in diethylpyrocarbonate-treated deionized water and stored at ?70C unit use. Based on the DNA sequence of a MMP-like protein from the published data23 (GenBank accession no. “type”:”entrez-nucleotide”,”attrs”:”text”:”AF277294″,”term_id”:”9082309″,”term_text”:”AF277294″AF277294), we designed a primer pair to obtain the complete open reading frame of the MMP-like sequence. The primers used were as follows: GS-F1 5-CAGTAAAGATGAAACTACAGAGTGTG-3 and Rabbit Polyclonal to HSL (phospho-Ser855/554) GS-R1 5-GACGTTTACGGCATTGGAG-3 (The start and stop codons are indicated in bold). A reverse transcription-polymerase chain reaction (RT-PCR) was performed using the RobusT II RT-PCR kit (Finnzymes, Espoo, Finland) according to the manufacturer’s protocol. The PCR parameters were as follows: cDNA synthesis at 40C for 60 minutes and at 94C for 2 minutes;.