Loading...
Lala Lajpat Rai University of Veterinary & Animal Sciences, Hisar
Browse
9 results
Search Results
ThesisItem 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 Open Access New approaches for post- vaccinated surveillance of FMD control programme in Haryana(LUVAS, 2017) Jain, Reenu; Kadian, S.K.Foot-and-mouth disease (FMD) is economically significant disease of cloven hoofed animals and often produces a persistent infection following clinical or sub-clinical disease in either naïve or vaccinated ruminants. The present study was undertaken with an aim to find new test for the carrier animal detection The non-structural protein ELISA (NSP-ELISA) was used to detect the antibodies against3AB3 and 2B NSPs of FMDV in 800serum samples of vaccinated buffaloes collected from eight districts, 100 from each district of Haryana. Randomly collected 30 oro-pharyngeal fluid and 30 nasal samples were tested for the presence of FMDV specific IgA antibody. OPF samples were also tested for the detection of viral RNA. A rapid, sensitive and specific assay by using gold nano particle for the detection of FMDV specific antibodies was also developed in the present study. Out of 800 serum samples tested, the percentage positivity by r2B assay was found to be 2.25% whereas by 3AB3 assay it was 8%. Out of thirty OPF samples tested, only one OPF sample was found positive by one-step qRT-PCR. Six nasal fluid samples and five OPF samples were found positive for the presence of FMDV serotype O specific IgA antibodies where as in case of FMDV serotype A specific IgA , four nasal and three OPF samples were found positive.ThesisItem Restricted A study on surface immunogens of biofilm-associated Staphylococcus aureus in mice(LUVAS, 2008) Sharma, Krishan; Ajit SinghStaphylococci are the most important of all the pathogens causing clinical and subclinical bovine mastitis worldwide. In past two decades, role of ‘biofilms’ in persistence of these organisms in the host has clearly been established, but how immune system sees the biofilm-forming organisms in the infected udder remains largely unanswered. The present study was therefore conducted with the objectives to determine the immunogens specifically associated with biofilm-forming S. aureus phenotype and to assess their role in protection against biofilm-forming pathogen in the mouse model of mastitis. S. aureus isolate from a clinical bovine mastitis case was grown as planktonic and biofilm producing bacteria under different conditions. Biofilm production by S. aureus isolate was confirmed by appearance of black colonies on Congo red agar plate, Gram staining, biofilm quantitation and scanning electron microscopy. Extracellular polysaccharide (EPS) and cell membrane (CM) -xxviiantigens were extracted from the biofilm phenotype and CM antigens from the planktonic. Polypeptide profiles were determined by SDS-polyacrylamide gel electrophoresis. Several up- and down-regulated proteins in biofilm-forming S. aureus CM profile were visible. Different groups of mice (n=8/group) were inoculated with these antigens using Freund’s incomplete adjuvant (FIA) and CpG motifs-containing chromosomal DNA as adjuvant. Sequential sera samples at day 0, 14, 28, 42, 56 were collected and kinetics of humoral immune response was determined by indirect ELISA. Antigen profiles of CM were determined by immunoblotting using sera collected above. Mice immunized twice four-weeks apart were challenged with 106 CFUs of biofilm-forming S. aureus by intra-mammary route after >8 weeks in all groups for determining protection levels. Antibody (Ab) levels in biofilm-associated CM-CpG group were higher at most days than those in CM-FIA group. IgM titre, but not IgG rose in EPS groups. Planktonic CM-CpG induced lower Ab levels than CM-FIA did. Biofilm-associated CM immunogens induced lower Ab levels than planktonic CM, irrespective of adjuvant used. However, these differences did not correlate with protection against challenge. In immunoblotting, seven major antigens specific to biofilm CM were revealed. Mice immunized with biofilm-associated CM with both adjuvants withstood challenge showing 100% protection, whereas planktonic CM immunized group with both adjuvants also withstood challenge showing nearly 100% protection. ELISPOT assay detected the presence of B lymphocyte clones secreting specific antibodies against various antigens used in the study. Based on these findings, use of biofilm-associated S. aureus surface immunogens as vaccine for control of mastitis was envisaged.ThesisItem Restricted A study on antigenic profiles of vaccine strain and field isolates of pasteurella multocida B:2(LUVAS, 2008) Saini, Deepak; Kadian, S.K.Haemorrhagic septicaemia (H.S.) is an acute infectious disease of domestic animals, particularly bovines caused by Pasteurella multocida serotypes B:2 and E:2. P. multocida is a gram-negative, coccobacillus, bipolar staining, capsulated organism which is diverse and complex with respect to antigenic variation, host predilection and pathogenesis. To date only a few potential antigens of P. multocida have been clearly defined. The various tests developed for investigation of the H.S. are still provisional and open to improvement. Extensive and in depth studies are still required for the development of a diagnostic test which can differentiate vaccinated animals from diseased ones serologically. Hence the present study was undertaken with an objective to determine total polypeptide profiles of vaccine strain and some field isolates of P. multocida B:2 and to identify the immunoreactive antigens in these profiles by immunoblotting employing antisera of vaccinated animals. Five field isolates of P. multocida B:2 and vaccine strain (P52) were grown on blood agar and then brain heart infusion broth. Whole cell lysates were prepared and total polypeptides in different isolates and vaccine strain were resolved by SDS-PAGE in 12.5% resolving gel. The polypeptide profiles so obtained were electroblotted onto nitrocellulose membrane and the blots were developed with antisera from H.S. vaccinated cattle and buffaloes. On SDS-PAGE analyses of the whole cell lysates polypeptides of different molecular weights ranging from 6l to 4 kDa were obtained. Thirteen polypeptides were common to all the field isolates and vaccine strain with little variation. Out of the 13 common bands 7 were major (61, 53, 36, 25, 21, 15 and 7 kDa) and 6 were minor (48, 41.6, 30.0, 13.2, 5.6 and 4.8 kDa). Polypeptides of molecular weight 53 and 21 kDa were minor, 5.6 kDa was major and 41.6 kDa was absent in vaccine strain (P52). On immunoblotting with sera from vaccinated buffalo immune response was observed with all sera against polypeptides of 36 and 21 kDa (Major polypeptides) and 48, 30, 13.2 and 11.5 kDa (Minor polypeptides). With vaccine strain all sera gave immune response against 60 kDa polypeptide while variable response was observed against 21, 25 and 55.5 kDa polypeptides. Immune response against polypeptides of 36 and 25 kDa (Major polypeptides) and 61.3, 13.2 and 11.5 kDa (minor polypeptides) was observed on immunoblotting with sera from vaccinated cattle. With vaccine strain all sera gave response against 36, 30 and 25 kDa polypeptide while variable immune response was observed against 21, 25 and 68.7 kDa polypeptides on immunoblotting with cattle sera. Therefore, polypeptide of 36 kDa (major polypeptide) was found to be most strongly reacting with all antisera from H.S. vaccinated animals in all isolates. This study indicates that we can not rely upon a single polypeptide antigen to be used in diagnostic test for differentiating between the diseased and vaccinated animal serologically. Moreover, a number of antigens P. multocida B:2 should be considered for inclusion in safer and efficacious vaccine formulation(s) constructed by rDNA- based methods and a subunit vaccine using any one polypeptide antigen still requires extensive and in depth studies in future.ThesisItem Restricted Production of Staphylococcus aureus exoproteins-specific camel single domain antibodies from phage display library(LUVAS, 2009) Jangra, Pooja; Ajit SinghPhage display technology is one of the modern technologies for production of antibodies for their potential applications in diagnosis, prevention and treatment of human and animal diseases. Aim of the present study was to isolate and characterize Staphylococcus aureus exoproteins-specific dAb clones from a phage display library of LPS-immunized Indian desert camel. Total exoproteins were harvested from S. aureus culture supernatant, polypeptide profile obtained by SDS-PAGE, and SAE- p39, SAE-p27 and SAEp23 extracted from the gel. Several major bands were visible in the polypeptide profile. S. aureus exoprotein Ags-specific dAb clones were selected from the phage display library of LPS-immunized Indian desert camel by three rounds of panning. The Ag-specific dAb-displaying phages were enrichment at the end of the three rounds as revealed by phage titration, Phage-ELISA and VHH-PCR. The expression of soluble dAb.HA clones by infecting amber non-suppressor WK6 with Ag-specific phages was confirmed by SDS-PAGE and blotting. The four high-expressing VHH clones were subcloned into pHEN6c vector and the resulting 14 subclones expressed as soluble dAb.6xHis as analyzed by SDSPAGE. Only four of the 14 clones were found positive by indirect ELISA. Three of these Ag-specific clones, viz., dAb.6xHis Cl-7-1, Cl-7-5 and Cl-9-2 were expressed in WK6 and purified by Ni-chelate chromatography using Ni-TED columns or Ni-NTA resin followed by ultrafiltration through 30 kDa membrane. The three purified dAb.6xHis clones bound to total exoproteins and SAE-p39 Ags, but not SAE-p27 and SAE-p23 as detected by both indirect ELISA and immunoblotting. S. aureus β-hemolysin in the culture supernatant was neutralized completely by dAb.6xHis Cl-7-5, partially by dAb.6xHis Cl-7-1 and not at all by dAb.6xHis Cl-9-2. The dAb.6xHis Cl-7-5 resisted thermal inactivation at all six temperatures from 50°C to 99°C for neutralizing β- hemolysin. These findings have revealed that dAb Cl-7-5 has the potential for diagnostic and therapeutic applications in future.ThesisItem Restricted Development of monoclonal antibodies against west nile virus and its seroprevalence in equines(LUVAS, 2010) Gupta, Akil Kumar; Kadian, S.K.West Nile Virus (WNV) infection is a self-limiting, non-fatal mild febrile illness occasionally causing encephalitis in humans, horses, domestic and wild birds. The status of WNV infection in horses is not known. In the present study, out of 1098 equine serum samples tested for WNV and JEV antibodies, 39 (3.55 %) tested positive for WNV and 92 (8.37%) tested positive for JEV antibodies in HI. Out of 37 WNV-positive samples by HI, 34 were also positive by VNT and 3 were found negative, giving a sero-prevalence of 3.14 % by VNT. For production of WNV-specific murine monoclonal antibodies, BALB/c mice were immunized with partially purified inactivated WNV antigen. On fusion of spleen cells with SP2/0 myeloma cells, 57 hybrids showing good growth were observed microscopically were found positive for WNV antibodies on screening by ELISA. Six of these hybrids (1H11, 2B2, 3C8, 4A5, 5A1 and 5A10) were selected, cloned, amplified and cryopreserved. MAbs secreted by clones 1H11E7 & 5A1E11 as ascites had titer of 6,400 and 12,800 and belonged to IgG2b and IgM isotype, respectively. On western blotting, both mAbs were found to be directed against 53 kDa E-protein and mAb 5A1E11 also cross-reacted with JEV E-protein. On the other hand, 1H11E7 mAb reacted only with WNV antigen, indicating it to be WNV specific. A blocking ELISA developed using mAb 1H11E7 had sensitivity and specificity of 97.14% and 75.86%, respectively in comparison to VNT. In the present study, six clones secreting mAbs against WNV were developed and B-ELISA for determining seroprevalence of WNV was developed using one of mAb, 1H11E7ThesisItem 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.ThesisItem Restricted Functional characterization of a lipopolysaccharide-binding single domain antibody clone selected from phage display library(LUVAS, 2015) Banerjee, Somesh; Ajit SinghGram–ve sepsis and endotoxemia are leading causes of morbidity and mortality in animals and humans worldwide, and attempts have continued to design rational therapies. Endotoxin- neutralizing polyclonal and monoclonal antibodies has been produced, but they are not free from problems. Recombinant single-domain antibodies (dAbs), derived from camelid and shark ‘heavy chain antibodies’ by phage display technology, are of small size, stable and soluble, which make them suitable for in vivo applications. The objectives of this study were to test the cross-reactive binding of a dAb clone to LPS from five genera of G-ve microorganisms and also to test its ability to neutralize E. coli endotoxin in chicken embryo model. The phage display library of LPS-immunized Indian desert camel was constructed to select and partially characterize some LPS-binding dAb clones. One of these clones, designated as dAb clone 26 was used in the present study. LPS was extracted and purified from Escherichia coli, Salmonella Typhimurium, Pseudomonas aeruginosa, Klebsiella spp. and Pasteurella multocida B:2. The dAb was expressed with C-terminal 6xHis tag in VHH clone 26- pET303 transformed BL21(DE3) under IPTG induction. The expression of 17 kDa dAb.6xHis clone 26 in whole cell lysate was confirmed by SDS-PAGE and immunoblotting. The dAb.6xHis clone 26 was purified by Ni-chelate chromatography under denaturing conditions. The renatured dAb.6xHis clone bound to lipid A and purified LPS from five genera in indirect ELISA, and also protected in a dose-dependent manner LPS-induced death of the 10-day chicken embryos. In conclusion, dAb specific to lipid A and cross-reactive to LPS from diverse bacteria neutralized endotoxin in chicken embryo model.ThesisItem Restricted Investigation into lectin pathway of complement system of water buffalo (Bubalus bubalis)(LUVAS, 2015) Daljeet Singh; Kadian, S.K.The components of the lectin pathway are among the least characterized of the complement activation pathways in domestic animal species, including water buffalo (Bubalus bubalis). Mannan binding lectin (MBL) is the initiator of the lectin pathway and plays a major role in innate defense against pathogens, involving recognition of arrays of carbohydrates on microbial surfaces. In serum, it exists in multimeric forms of MBL-A and MBL-C types, each organized from 28 kDa-32kDa subunits in different species. The concentration of MBL in serum varies widely, from 5 ng/mL to >5 μg/mL, in different individuals of mammalian species and can be influenced by inflammation, infection, pregnancy, etc. The present study was undertaken with the objectives to isolate MBL from water buffalo (Bubalis bubalus L.) and to estimate levels in serum from apparently healthy animals of the Murrah breed. Peripheral blood serum from 50 animals was pooled to process for isolation and purification of MBL. PEG6000 precipitation followed by mannose-Sepaharose affinity chromatography with D-mannose eluent resulted in partially purification of MBL, along with several proteins such as conglutinin, IgM, IgG, etc. as contaminants. The conglutinin was removed by affinity chromatography in mannose-Sepharose column with N-acetyl glucosamine as eluent. Subsequently, IgM was removed by anti-IgM-Sepharose affinity column and IgG removed by protein G-Sepharose affinity column. Oligomers of MBL were detected by SDS-PAGE under non-reducing conditions. The finally purified 28 kDa MBL in SDS- reducing polyacrylamide gel showed a 70 kDa contaminant band. The 28 kDa MBL was extracted from the gel slice and used for raising monospecific anti-MBL antisera in rabbits and guinea pigs. Protein G affinity purified IgG fractions from rabbit and guinea pig anti-MBL antisera were used in ‘sandwich ELISA’ to estimate MBL concentrations in sera from heifers (n=25) and multiparous females (n=25) of the Murrah breed. Concentration of MBL were found to be in the range from 14ng-1.5μg/mL (Median= 75.4 ng/mL) and 13ng-1.7μg/mL (Median= 66.8 ng/mL) in apparently healthy heifers and multiparous females, respectively. In conclusion, MBL was isolated from water buffaloes and the normal levels in the Murrah breed were estimated.