Browsing by Author "Rawat, Jagveer"
Now showing 1 - 4 of 4
Results Per Page
Sort Options
ThesisItem Open Access A Study Of Aspects of lmmunoregulation In Murine Mesocest-aides corti Infection(Veterinary Immunology Department of Veterinary Pathology University of Liverpool Liverpool UK., 1999) Rawat, Jagveer; Dixon, John B.ThesisItem Open Access Characterization Of Lymphoid Cells And Monocytes Of Buffalo (Bubalus Bubalis)(College of Veterinary Sciences Chaudhary Charan Singh Haryana Agricultural University Hisar, 1989) Rawat, Jagveer; Goel, M. CThesisItem Open Access A study of fine structural details of a lipopolysaccharide-binding single domain antibody selected from phage display library(LUVAS, 2017) Yadav, Vikas; Rawat, JagveerGram negative sepsis caused by endotoxin incurs huge economic burden in human and animal health sector, globally. Various rational therapies have been attempted in past decades with futile outcomes. Endotoxin neutralisation have been attempted with antibodies targeting the core and O polysaccharide regions as epitope with some success in experimental studies. Recombinant single domain antibodies produced by phage display technology provide newer therapeutic candidates to target challenging epitopes with improved affinities. The objective of present study were to determine the affinity of a dAb clone 26 with LPS and also to biophysically characterize the dAb clone 26. In the present study, LPS was extracted and purified from an isolate of E.coli (ATCC25922). The dAb was expressed with C-terminal 6xHis tag in VHH clone 26- pET303 transformed BL21(DE3) under IPTG induction and monitored with SDS-PAGE profile. The dAb clone 26 was purified with Ni-NTA affinity chromatography. The silver stained dAb clone26 band was subjected to MALDI-TOF/MS analysis after the in-gel trypsin digestion and the partial sequence was obtained. The affinity of dAb clone 26 with LPS was analysed in SPR experiment which revealed a binding constant KA of 8.1x106M-1. Further, the dAb clone 26 was subjected to co-crystallisation with grid screening approach in a hanging drop vapour diffusion set up in 96 different conditions with pH and precipitant(Ammonium sulphate and PEG-3350,6000& 8000) as variables. The cocrystallisation resulted in crystal hits in three different condition- one each in ammonium sulphate, PEG3350 and PEG8000. In conclusion, the sequence of dAb clone 26 has been verified whereas the affinity and crystallisation experiments have scope of further optimisation.ThesisItem Restricted A study on immune responses in mice against polyacrylamide nanoparticles trapped monophosphoryl lipid A as Toll like receptor 4 ligand and inactivated peste des petits ruminants virus(LUVAS, 2014) Dhamu, Nitin; Rawat, JagveerModern delivery systems are being developed with aims of improving traditional vaccines, prolonging duration of host immunity, designing mucosal vaccines, etc. This study presents the synthesis of polyacrylamide nanoparticle (PAA-NPs) and exploring their utility as the delivery system for peste des petits ruminants virus antigens (PPRV-Ags) and monophosphoryl lipid A (MPL) adjuvant. Size, charge and polydispersity of PAA-NPs before and after adsorption of the PPRV-Ags and MPL were examined by DLS and TEM. PAA-NPs with adsorbed PPRV-Ags & MPL were of 269 nm size by DLS (80 nm - 200 nm by TEM), -31.8 ± 6.48 mV charge and 0.437 polydispersity index. The kinetics of antibody response in different groups (11 in all, n=7/group) of Swiss albino mice inoculated with the experimental vaccine and controls were examined. Each mouse was inoculated twice at 4-week interval and the serum samples were collected bi-weekly up to 42 days. IgG levels in serum samples from each group were determined by indirect ELISA. Statistically significant increase in IgG levels was observed in NP + MPL + PPRV (NMV) group on day 42 as compared to those in other groups (p<0.01). This observation indicated that PAA-NPs adsorbed with the inactivated PPRV antigens and MPL elicit a robust humoral immune response. For study of innate immune stimulation, the serum samples were collected 90 minutes after single intramuscular injection in the PAA-NPs-MPL and three controls groups of Swiss albino mice (n=7/group). TNF-α levels were measured by sandwich ELISA, using anti-TNF-α monoclonal antibodies. TNF-α levels in PAA-NPs-MPL group were significantly higher than those in MPL group and the negative controls (p<0.01). This study concluded that PAA-NPs adsorbed PPRV-Ags and MPL induced strong innate as well as IgG immune responses, and suggested that PAA-NPs could be developed as a delivery platform for vaccinal Ags and adjuvants.