Studies on endotoxin-neutralizing effects of three camelid single-domain antibody clones
KrishiKosh
View Archive InfoField | Value | |
Title |
Studies on endotoxin-neutralizing effects of three camelid single-domain antibody clones
|
|
Creator |
Gupta, Akhil Kumar
|
|
Contributor |
Ajit Singh
|
|
Subject |
Sepsis, Endotoxin, PET303/CT vector, DAb, ELISA, TNFα
|
|
Description |
Gram–ve sepsis causes high morbidity and mortality in human and animals worldwide. LPS, also called endotoxin, released from G–ve bacteria has a predominant role in sepsis through excessive production of proinflammatory mediators. Attempts to design rational therapies against endotoxemia and sepsis continue, and the present study represents an approach to directly neutralize endotoxin by single-domain antibody clones selected from the phage display library of LPS- immunized Indian desert camel. Four dAb.HA clones (Cl- 16, Cl-18, Cl-23 and Cl-26) selected as LPS-binders from the above-mentioned phage display library were used. The clones were initially expressed as dAb.HA products in WK6 cells, and confirmed by SDS-PAGE and immunoblotting. But the level of expression was later increased by sub-cloning into pET303/CT vector/ BL21(DE3) host system. The dAb.6xHis clones were purified by Ni-NTA chromatography and confirmed by SDS-PAGE and immunoblotting. The nucleotide sequences of the four clones were determined and the bioinformatics revealed their typical VHH features. In indirect ELISA, dAbCl-26 showed the highest reactivity with LPS as well as lipid A, followed by Cl-18 and then Cl-23. The affinity constants (Ka) were: 4.28 x108/M of Cl-26, 2.18 x108/M of Cl-18, and 2.19x108/M of Cl-23. All the dAb clones were thermo-stable. The dAbCl-26 and Cl-18 decreased the LPS-induced expression of TNFα, IL-1 and MHC II genes in buffalo leukocytes and TNFα (not by Cl-18), IL-1β, IL-6, CD80 and MHC-II genes in murine macrophages, but dAbCl-23 increased buffalo TNFα and MHC-II and all the above murine genes as measured by RT-qPCR. The dAbCl-26 and dAbCl-18 decreased, but dAbCl-23 increased LPS-induced TNFα levels in Swiss albino mouse model. In conclusion, dAbCl-26 was found to be the best of the three clones tested for LPS-neutralization, but needs to be further investigated for developing it as a drug for treatment of endotoxaemia in animals. |
|
Date |
2016-08-17T09:12:21Z
2016-08-17T09:12:21Z 2014 |
|
Type |
Thesis
|
|
Identifier |
http://krishikosh.egranth.ac.in/handle/1/72677
|
|
Language |
en
|
|
Format |
application/pdf
|
|
Publisher |
LUVAS
|
|