Development of a high-throughput flow cytometric neutralization assay to screen for human enterovirus A71 (EVA71) neutralizing antibodies.

The conventional method for screening neutralizing antibodies to human enterovirus A71 (EVA71) (microneutralization assay) is time consuming and requires an expert to perform manual evaluation. An automated neutralization assay could shorten the testing time, improve reproducibility, and provide automatic analysis. This study aimed to develop a high-throughput flow cytometric neutralization assay to screen for EVA71 neutralizing antibodies, and to develop quality control materials to ensure accurate testing. The results of this study demonstrate the high potential viability of the proposed flow cytometric method. Compared to the standard method, the flow cytometric method was shown to require a smaller sample volume, provide a much faster turnaround time, provide a rapid result for interpreting the neutralizing antibody level, and allow for possible quantification of results. The observed drawbacks of the proposed method include higher cost per test, longer hands-on time, and lower sensitivity in low titer conditions, which could lead to false negative results. The developed quality control materials were demonstrated to be effective and storable for 1 month. These results pave the way for the optimization and implementation of an automated neutralization assay to screen for neutralizing antibodies not only against EVA71, but also against other viruses in the enterovirus genus.

Combined effects of curcumin and doxorubicin on cell death and cell migration of SH-SY5Y human neuroblastoma cells.

Neuroblastoma is the most common cancer of the sympathetic nervous system in children. Here, the influences of curcumin on survival, apoptosis, migration, and its combined effects with doxorubicin were investigated in SH-SY5Y cells by cell survival assay, flow cytometry, migration assays, and RT-PCR. Curcumin inhibited SH-SY5Y cell growth and induced apoptosis in dose- and time-dependent manners. This apoptotic induction relied on the upregulation of p53 and p21. Moreover, the treatment of curcumin for 24 h significantly suppressed cell migration, together with the downregulation of matrix metalloproteinase-2 (MMP-2) and upregulation of tissue inhibitor of metalloproteinases-1 (TIMP-1). The combination of curcumin augmented the anticancer activity of doxorubicin and significantly induced apoptosis. Pretreatment with curcumin increased the fraction of doxorubicin-induced apoptotic cells from 21.76 ± 0.50 to 57.74 ± 2.68%. Co-treatment with doxorubicin plus curcumin further inhibited 3D tumor migration. Altogether, the results suggest that curcumin suppresses growth and migration of SH-SY5Y cells and enhances the anticancer activity of doxorubicin. The addition of curcumin to therapeutic regimens may be promising for the treatment of neuroblastomas if a number of problems related to its in vivo bioavailability can be resolved. Graphical abstract ᅟ.