ABSTRACT
Owing to the threat of cadmium (Cd2+) to public health, it is an urgent demand to develop effective, sensitive, and rapid methods for the detection of cadmium. In this study, indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) and immunochromatographic test strips (ICTS) were established for the determination of Cd2+ based on the obtained mAb with high specificity and high affinity (Kaff = 3.0 × 109 L/moL). The linear range of ic-ELISA detection was 0.03-1.11 ng/mL and 50% inhibitive concentration (IC50) of cadmium ion was determined to be 0.15 ng/mL. The visual limit of detection (vLOD) of the AuNS-based strip was 0.375 ng/mL. The vLOD of AuNF-based strip using higher intensity reporter determined to be 0.03 ng/mL, which was enhanced 12 times compared to the traditional strip. In summary, the developed immunoassays based on mAb shows great potential for monitoring the cadmium ion in environmental samples.
Subject(s)
Antibodies, Monoclonal , Cadmium , Enzyme-Linked Immunosorbent Assay , Immunoassay , Limit of DetectionABSTRACT
BACKGROUND: Mitogen-activated protein kinase (MAPK) signaling pathway plays an important role in response to viral infection. The aim of this study was to explore the function and mechanism of MAPK signaling pathway in enterovirus 71 (EV71) infection of human rhabdomyosarcoma (RD) cells. METHODS: Apoptosis of RD cells was observed using annexin V-FITC/PI binding assay under a fluorescence microscope. Cellular RNA was extracted and transcribed to cDNA. The expressions of 56 genes of MAPK signaling pathway in EV71-infected RD cells at 8 h and 20 h after infection were analyzed by PCR array. The levels of IL-2, IL-4, IL-10, and TNF-α in the supernatant of RD cells infected with EV71 at different time points were measured by ELISA. RESULTS: The viability of RD cells decreased obviously within 48 h after EV71 infection. Compared with the control group, EV71 infection resulted in the significantly enhanced releases of IL-2, IL-4, IL-10 and TNF-α from infected RD cells (p < 0.05). At 8 h after infection, the expressions of c-Jun, c-Fos, IFN-i, MEKK1, MLK3 and NIK genes in EV71-infected RD cells were up-regulated by 2.08-6.12-fold, whereas other 19 genes (e.g. AKT1, AKT2, E2F1, IKK and NF-κB1) exhibited down-regulation. However, at 20 h after infection, those MAPK signaling molecules including MEKK1, ASK1, MLK2, MLK3, NIK, MEK1, MEK2, MEK4, MEK7, ERK1, JNK1 and JNK2 were up-regulated. In addition, the expressions of AKT2, ELK1, c-Jun, c-Fos, NF-κB p65, PI3K and STAT1 were also increased. CONCLUSION: EV71 infection induces the differential gene expressions of MAPK signaling pathway such as ERK, JNK and PI3K/AKT in RD cells, which may be associated with the secretions of inflammatory cytokines and host cell apoptosis.
Subject(s)
Humans , Enterovirus A, Human/genetics , Mitogen-Activated Protein Kinases/genetics , Rhabdomyosarcoma/virology , Cytokines/genetics , Enzyme-Linked Immunosorbent Assay , Enterovirus A, Human/enzymology , Enterovirus A, Human/physiology , Gene Expression Regulation, Neoplastic , Mitogen-Activated Protein Kinases/physiology , Polymerase Chain Reaction , Rhabdomyosarcoma/enzymology , Rhabdomyosarcoma/genetics , Signal Transduction/genetics , Signal Transduction/physiology , Time Factors , Tumor Cells, Cultured , Up-Regulation , Virus ReplicationABSTRACT
BACKGROUND: Mitogen-activated protein kinase (MAPK) signaling pathway plays an important role in response to viral infection. The aim of this study was to explore the function and mechanism of MAPK signaling pathway in enterovirus 71 (EV71) infection of human rhabdomyosarcoma (RD) cells. METHODS: Apoptosis of RD cells was observed using annexin V-FITC/PI binding assay under a fluorescence microscope. Cellular RNA was extracted and transcribed to cDNA. The expressions of 56 genes of MAPK signaling pathway in EV71-infected RD cells at 8h and 20h after infection were analyzed by PCR array. The levels of IL-2, IL-4, IL-10, and TNF-α in the supernatant of RD cells infected with EV71 at different time points were measured by ELISA. RESULTS: The viability of RD cells decreased obviously within 48h after EV71 infection. Compared with the control group, EV71 infection resulted in the significantly enhanced releases of IL-2, IL-4, IL-10 and TNF-α from infected RD cells (p<0.05). At 8h after infection, the expressions of c-Jun, c-Fos, IFN-ß, MEKK1, MLK3 and NIK genes in EV71-infected RD cells were up-regulated by 2.08-6.12-fold, whereas other 19 genes (e.g. AKT1, AKT2, E2F1, IKK and NF-κB1) exhibited down-regulation. However, at 20h after infection, those MAPK signaling molecules including MEKK1, ASK1, MLK2, MLK3, NIK, MEK1, MEK2, MEK4, MEK7, ERK1, JNK1 and JNK2 were up-regulated. In addition, the expressions of AKT2, ELK1, c-Jun, c-Fos, NF-κB p65, PI3K and STAT1 were also increased. CONCLUSION: EV71 infection induces the differential gene expressions of MAPK signaling pathway such as ERK, JNK and PI3K/AKT in RD cells, which may be associated with the secretions of inflammatory cytokines and host cell apoptosis.
