In the present study, the diagnostic value of IgG4-specific antibody detection by peptide-based ELISA was explored for human paragonimiasis due toP. paragonimiasis, serodiagnosis, peptide-based ELISA Rabbit polyclonal to AASS The lung flukes,Paragonimusspecies, cause paragonimiasis in humans and animals [1-3]. It is estimated that over 20 million people are infected worldwide [4] and approximately 292.8 million people are at risk of contamination [5]. WhileParagonimus westermaniis the most important human pathogen in China, Korea, and Japan [1-3,6],Paragonimus heterotremusis the main etiological agent of human paragonimiasis in southern China, Southeast Asia (including Thailand and Vietnam), and India [1-3]. Diagnosis of paragonimiasis relies on demonstration ofParagonimuseggs in BI-639667 the feces, sputum, or both [3]. However, microscopic detection ofParagonimuseggs is not very sensitive because of irregular egg production and troubles of processing sputum and fecal specimens and requirement for experienced microscopists. Furthermore, eggs are not detectable in ectopic or prepatent paragonimiasis cases. For physicians, clinical symptoms of paragonimiasis are frequently confused with those of non-parasitic respiratory diseases, such as pulmonary tuberculosis and lung malignancy. Supportive evidence of immunological tests is usually, therefore, essential for clinical diagnosis of paragonimiasis. ForP. heterotremusinfection, detection of specific antibodies in the sera of patients has been reported [7-10]. By immunoblot analysis, 31.5 kDa or 35 kDa antigens fromP. heterotremusadult worms are supposed to be the most specific antigens with the highest diagnostic value [7-9]. Detection of particular immunoglobulin subclasses often enhances the development and interpretation of serological assays. Analysis of IgG subclass antibodies can increase specificity and sensitivity of the immunological assay for diagnosis of paragonimiasis [11,12]. Development of serological diagnosis, however, is usually hampered by the limited availability of antigens as they should be prepared from live worms, which were collected from your field or prepared by experimental contamination in laboratory animals. Moreover, varying preparation methods for antigens makes inter-laboratory standardization hard. Thus, the use of a synthetic peptide as a diagnostic antigen is a good alternative and has been studied for some parasitic infections. The peptide-based ELISA for detection of IgG4-specific antibodies improved the diagnostic value for neurocysticercosis [13] and fascioliasis [14]. In the present study, the diagnostic value of IgG4-specific antibody detection by peptide-based ELISA was explored for human paragonimiasis due toP. heterotremus. A synthetic peptide of the antigenic region ofP. westermanipre-procathepsin L, corresponding to amino acids 216-227 (12 amino acids) of the enzymatic part (GenBank accession no.AAB93494), was synthesized as a carboxamide at the C-terminus and acetyl at the N-terminus: acetyl-AKIDDSIVLEKN-amide by the Mimotopes Pty Ltd (Victoria, Australia), with optimum prediction scores of epitope (>0.9) [15] and hydrophilicity (~1.3) [16]. The amino acid sequence was subjected and analyzed with software available online athttp://tools.immuneepitope.org/tools/bcell/iedb_input. Sera were obtained from serum lender of the Faculty of Medicine, Khon Kaen University or college, Khon Kaen, Thailand. Each serum was aliquoted and stored at -70 until used. The study protocol was approved by the Human Research Ethics Committee of Khon Kaen University or college (HE5000225). Informed consent was obtained from all adult participants or the parents or legal guardians. Sera were from parasitologically confirmed cases of paragonimiasis (P. heterotremus) (n=16), fascioliasis (n=9), opisthorchiasis (n=5), strongyloidiasis (n=10), capillariasis philippinensis (n=20), gnathostomiasis (n=18), angiostrongyliasis (n=6), taeniasis (n=6), cysticercosis (n=8), hookworm infections (n=4), trichinosis (n=6), bancroftian filariasis (n=1), falciparum malaria (n=1), and vivax malaria (n=1). Five sera from patients with pulmonary tuberculosis were also tested. Unfavorable control sera were obtained from 37 healthy adults whose stool examinations were unfavorable by the formalin-ether concentration method for parasite eggs at the time of the blood collection. Pooled positive and negative sera were prepared by combining equivalent volumes of confirmed paragonimiasis and healthy control sera, respectively. One serum from a paragonimiasis westermani case was provided by Prof. Yoon Kong, Department of Molecular Parasitology, Sungkyunkwan University or college School of Medicine, Korea. The method was performed as previously explained [14] with some modifications. Each well of an ELISA plate was coated with 1 g of peptide in 0.1 ml of 0.01 M phosphate buffer (PBS, pH 7.5) at 4 overnight. The wells were washed 3 times with 10 mM PBS, pH 7.5 made up BI-639667 of 0.05% Tween 20 (PBS/T) and subsequently blocked with 3% BSA in PBS/T for 1 hr at room temperature. After washing with PBS/T, the wells were incubated at 37 for 1 hr with 0.1 ml of human sera diluted 1:50 with 1% BSA in PBS/T. After another washing with PBS/T, a peroxidase-conjugated goat anti-human IgG4 subclass antibody (Zymed, South BI-639667 San Francisco, California, USA) diluted 1:500 with.