Development of a novel competitive ELISA based on nanobody-horseradish peroxidase fusion protein for rapid detection of antibodies against avian hepatitis E virus.

Avian hepatitis E virus (avian HEV) increases poultry mortality and decreases egg production, leading to huge economic losses worldwide. However, there is no effective serological test for avian HEV. Researchers previously created a testing platform using the nanobody (Nb)-horseradish peroxidase (HRP) fusion protein as an ultrasensitive probe to develop competitive ELISA (cELISA) to detect antibodies against different animal viruses. In this study, a rapid and reliable cELISA was developed to test for antibodies against avian HEV using the same platform. Six anti-avian HEV capsid protein nanobodies were selected from an immunized Bactrian camel using phage display technology. The avian HEV-Nb49-HRP fusion protein was expressed and used as a probe for developing a cELISA assay to test for avian HEV antibodies. The cut-off value of the developed cELISA was 22.0%. There was no cross-reaction with other anti-avian virus antibodies, suggesting that the cELISA had good specificity. The coefficients of variation were 0.91% to 4.21% (intra-assay) and 1.52% to 6.35% (inter-assay). Both cELISA and indirect ELISA showed a consistency of 86.7% (kappa = 0.738) for clinical chicken serum samples, and coincidence between cELISA and Western blot was 96.0% (kappa = 0.919). The epitope recognized by Nb49 was located in aa 593-604 of the avian HEV capsid protein, and the peptide (TFPS) in aa 601-604 was essential for binding. The novel cELISA is a saving cost, rapid, useful, and reliable assay for the serological investigation of avian HEV. More importantly, the peptide TFPS may be crucial to immunodominant antigen composition and protection.

Strontium stimulates alkaline phosphatase and bone morphogenetic protein-4 expression in rat chondrocytes cultured in vitro.

The trace element strontium has a significant impact on cartilage metabolism. However, the direct effects of strontium on alkaline phosphatase (ALP), a marker of bone growth, and bone morphogenetic protein-4 (BMP-4), which plays a key role in the regulation of bone and cartilage development, are not entirely clear. In order to understand the mechanisms involved in these processes, the chondrocytes were isolated from Wistar rat articular cartilage by enzymatic digestion and cultured under standard conditions. They were then treated with strontium at 0.5, 1.0, 2.0, 5.0, 20.0 and 100.0 µg/mL for 72 h. The mRNA abundance and protein expression levels of ALP and BMP-4 were measured using real-time polymerase chain reaction (real-time PCR) and Western blot analysis. The results showed that the levels of expression of ALP and BMP-4 in chondrocytes increased as the concentration of strontium increased relative to the control group, and the difference became significant at 1.0 µg/mL strontium (P<0.05). These results indicated that strontium could be involved in cartilage development via regulating ALP and BMP-4 expression.