Babesia caballi (Nuttal, 1910) is one of the causative agents of equine piroplasmosis which causes economic losses to horse industry in China. There is an urgent need for rapid detection method for B. caballi infection in Xinjiang Province, China. To prepare monoclonal antibodies (mAbs) against Bc48 gene of B. caballi (Xinjiang local strains) and establish colloidal gold-immunochromatographic (ICT) assay for diagnosis of the disease, recombinant Bc48 was expressed and purified from Escherichia coli. With purified Bc48 as immunogen in mice, three hybridoma cells named 11F4, 1H2 and 7F4 secreting mAbs against Bc48 of B. caballi were obtained, which showed strong reaction with recombinant Bc48 and Bc48 gene transfected cells. Furthermore, colloidal gold labelled ICT assay based on purified Bc48 recombinant antigen and its mAb was developed. The ICT assay showed high sensitivity and specificity and no cross-reaction with Theileria equi (Laveran, 1901). Total of 56 horse serum samples collected from Xinjiang were tested by ICT and compared with the detection by commercial ELISA kit. The results showed that 32 out of 56 serum samples were positive by ICT and 33 were positive by ELISA. ICT assay had high coincidence (98%) to the reference ELISA kit. mAbs and ICT developed in this study could be provided as an efficient diagnosis tool for infection with B. caballi in horse in Xinjiang area.
- MeSH
- Babesia imunologie MeSH
- babezióza diagnóza parazitologie MeSH
- imunoanalýza metody veterinární MeSH
- koně MeSH
- monoklonální protilátky krev MeSH
- myši inbrední BALB C MeSH
- myši MeSH
- nemoci koní diagnóza parazitologie MeSH
- protilátky protozoální krev MeSH
- protozoální proteiny genetika imunologie MeSH
- rekombinantní proteiny genetika imunologie MeSH
- senzitivita a specificita MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Geografické názvy
- Čína MeSH
A chicken multiplex cytokine assay (Bio-Plex) to detect four different cytokines (IL-2, IL-12, IL-10, and interferon gamma) simultaneously in plasma samples was designed. Most standard curves range between 1 to 5 pg/mL and 5,000 pg/mL, except for IFNγ with the range of 50 to 25,000 pg/mL. Such a chicken multiplex assay proved to be fast and reliable, and comparable in sensitivity, accuracy, and reproducibility to conventional enzyme-linked immunosorbent assays. Comparison of the multiplex assay with the ELISA technique using the same clones of detection and capture antibodies resulted in correlation coefficients for all cytokines ranging from 0.95 to 0.99. Lower limit of detection and limit of quantification values were obtained for all tested cytokines by the Bio-Plex assay compared with ELISA. To reduce the risk of cross-reaction with other proteins, the Bio-Plex system was used, combining the principle of sandwich immunoassay with the Luminex bead-based technology. The cytokine standard recoveries for each cytokine varied between 86 and 118% in dynamic concentration ranges. A chicken multiplex cytokine assay (Bio-Plex) provided a more complete picture of differences between the Th1/Th2 cytokine profiles of the immunized via a new system of antigen delivery into chicken antigen-presenting cells and control groups. This multiplexed fluorescent-bead-based detection assay can be used as a quantitative or comparative tool for the study of the chicken ex vivo cellular immune response.
