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Studies on endotoxin-neutralizing effects of three camelid single-domain antibody clones

KrishiKosh

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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