Diagnosis of human toxocariasis currently relies on serologic tests that use Toxocara excretory-secretory (TES) antigen to detect immunoglobulin G (IgG) antibodies to the larvae. In general, however, these assays do not have adequate specificity for use in countries in which other soil-transmitted helminths are endemic. The use of recombinant antigens in these assays, however, is promising for improving the specificity of the diagnosis of toxocariasis. Toward this goal, we developed an IgG4 enzyme-linked immunosorbent assay (ELISA) involving three recombinant antigens: rTES-30USM (previously produced), rTES-26, and rTES-120. The latter two antigens were produced by reverse transcription-PCR cloning; subcloned into glutathione S-transferase (GST)-tagged and His-tagged prokaryotic expression vectors, respectively; and expressed in Escherichia coli. The recombinant proteins were subsequently purified by affinity chromatography using GST and His-Trap resins. The diagnostic potential of each purified recombinant antigen was tested with various immunoglobulin classes (IgG, IgM, and IgE) and IgG subclasses. The IgG4 ELISA was determined to have the highest specificity and was further evaluated using a panel of serum samples. The rTES-26 IgG4 ELISA showed 80.0% (24/30 samples positive) sensitivity, and both the rTES-30USM IgG4 ELISA and rTES-120 IgG4 ELISA had 93.0% (28/30) sensitivity. Combined use of rTES-120 and rTES-30 IgG4 ELISA for the diagnosis of toxocariasis provided 100% sensitivity. The specificities of rTES-26, rTES-30USM, and rTES-120 antigens were 96.2%, 93.9%, and 92.0%, respectively. These results indicate that the development of a diagnostic test using the three recombinant antigens will allow for more-accurate detection of toxocariasis.
Leukemia can be a result of genetic changes associated with protein tyrosine kinase activity such as in MPL W515L and BCR/ABL genes. However, the current conventional treatment of leukemia produces severe side effects that urge the approach to use natural products. A medicinal mushroom, Lignosus rhinocerus shows potential as an anti-cancer treatment. To investigate the efficacy and mechanism of action of the L. rhinocerus cultivar (TM02®) extract on leukemogenic tyrosine kinase cell lines, a cold-water extract (CWE) was produced by using TM02® sclerotia powder at 4°C. The carbohydrate and protein contents were found to be 77.24% and 1.75% respectively. In comparison to the normal Ba/F3 cell, the CWE TM02® shows significant effects on exhibiting proliferation of Ba/F3 expressed MPL W515L and BCR/ABL, possibly due to the presence of phenolic compounds and antioxidant properties of TM02®, which contribute to act on various signaling pathways, and the reported apoptotic activity of CWE TM02®. In contrast, CWE TM02® significantly exhibited high scavenging activity of both Ba/F3 expressed MPL W515L and BCR/ABL. At concentrations of 125 μg/mL and 500 μg/mL of CWE TM02® decreased 49.5% and 67.5% of cell migration activity of Ba/F3 expressed MPL W515L and BCR/ABL respectively. Therefore, we postulate that CWE TM02® has the capability to mediate the migration route of the leukemogenic tyrosine kinase cell lines.