ELISA based on a recombinant Paragonimus heterotremus protein for serodiagnosis of human paragonimiasis in Thailand

Abstract Background 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. This study aimed to use a P. heterotremus cDNA library to create a recombinant version of this antigen for use in immunodiagnosis of paragonimiasis. Methods To 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. Results The 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). Conclusions Given 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

Additional file 1: of ELISA based on a recombinant Paragonimus heterotremus protein for serodiagnosis of human paragonimiasis in Thailand

Figure S1. Sizes of the inserted cDNA in randomly selected clones from the P. heterotremus cDNA library. PCR products resulting from amplification with the pJET forward primer and pJET reverse primer were separated on a 1.2% agarose gel. The sizes of inserted cDNA fragments from random clones (Lanes 1–14) were determined by comparison with the Kapa universal ladder (Lane M). (TIF 1102 kb

Additional file 2: of ELISA based on a recombinant Paragonimus heterotremus protein for serodiagnosis of human paragonimiasis in Thailand

Figure S2. The sizes of CE3 PCR products. PCR was performed on CE3 and the resulting products were run on a 1.2% agarose gel. Lane M: marker; Lane 1: CE3 clone. (TIF 780 kb