Abstract
BackgroundParagonimus heterotremus is the main causative agent of paragonimiasis in Thailand. In Western blot diagnostic assays for paragonimiasis, the 35 kDa band present in crude P. heterotremus somatic extracts represents one of the known diagnostic bands. This study aimed to use a P. heterotremus cDNA library to create a recombinant version of this antigen for use in immunodiagnosis of paragonimiasis.MethodsTo accomplish this aim a cDNA expression library was constructed from adult worm mRNA and immuno-screened using antibodies from mice that had been immunized with the 35 kDa antigen. Screening resulted in the identification of an immunoreactive protein encoded by clone CE3, which contained an inserted sequence composed of 1292 base pairs. This clone was selected for use in the construction of a recombinant P. heterotremus protein because of its similarity to proactivator polypeptide. For recombinant protein expression, the CE3 gene sequence was inserted into the plasmid vector pRset and the resulting product had the expected molecular weight of 35 kDa. An IgG-ELISA based on the CE3 recombinant protein was evaluated by using sera from healthy individuals, from patients with paragonimiasis and other parasitic infections. This ELISA was performed by using human sera diluted at 1:2000, an optimized antigen concentration of 1 μg/ml, and anti-human IgG diluted at 1:4000.ResultsThe cut-off optical density value was set as the mean + 2 standard deviations (0.54), which resulted in the test having a sensitivity of 88.89% and a specificity of 95.51%. The recombinant antigen could react with antibodies from P. heterotremus, P. pseudoheterotremus and P. westermani infections. Cross-reactivity occurred with a few cases of Blastocystis hominis infection (2/3), Bancroftian filariasis (1/10), opisthorchiasis (3/10), strongyloidiasis (4/10) and neurocysticercosis (1/11).ConclusionsGiven the high test sensitivity and specificity, reflected in the low level of heterologous infection cross-reactivity (11/215 serum samples), observed in the IgG-ELISA, this 35 kDa antigen may be useful for the detection of paragonimiasis.
Highlights
Paragonimus heterotremus is the main causative agent of paragonimiasis in Thailand
It should be noted by including serum samples from patients infected with the three human Paragonimus spp: P. heterotremus, P. pseudoheterotremus and P. westermani, we found that the Enzyme-linked immunosorbent assay (ELISA) was not able to discriminate between species-specific infections
In the present study, a novel recombinant protein antigen was produced from the complementary DNA (cDNA) of P. heterotremus worms
Summary
Paragonimus heterotremus is the main causative agent of paragonimiasis in Thailand. In Western blot diagnostic assays for paragonimiasis, the 35 kDa band present in crude P. heterotremus somatic extracts represents one of the known diagnostic bands. Immunoblotting tests using P. heterotremus crude extracts of adult worms show high sensitivity and specificity to paragonimiasis heterotremus with diagnostic bands corresponding to 32.5, 33, and 35 kDa native antigens [8] and can differentiate between P. heterotremus and P. westermani Korean strain [9]. The aim of the present study was to apply a molecular approach to generate P. heterotremus DNA recombinant antigens for use in immunodiagnosis of human paragonimiasis. To this end, we constructed a complementary DNA (cDNA) library from adult P. heterotremus worms and used a molecular cloning approach to identify and express recombinant proteins that exhibited selective immunoreactivity with antibodies recognizing the 32.5, 33 and 35 kDa bands comprising P. heterotremus native antigens. Results of testing against a panel of human sera indicate that the recombinant protein generated by this study may be useful in the serodiagnosis of human paragonimiasis
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