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Ponnanna N.M.,Research and Development Center Indian Immunologicals Ltd Rakshapuram | Sriraman R.,Research and Development Center Indian Immunologicals Ltd Rakshapuram | Verma R.R.,Research and Development Center Indian Immunologicals Ltd Rakshapuram | Kasa B.,Research and Development Center Indian Immunologicals Ltd Rakshapuram | And 5 more authors.
Current Trends in Biotechnology and Pharmacy | Year: 2012

The study aimed to develop a standard ELISPOT protocol for the evaluation of T cell responses in mice to HPV 16 E7. C57BL/6 female mice had been immunized with the HPV 16 E7 recombinant S. typhi Ty21a, a candidate therapeutic vaccine for HPV associated cancer. Acommercial ready-to-use (RTU) IFNγELISPOT kit (supplied with pre-standardized capture and detection antibodies) was chosen; hence the effort in this study lay on the standardization of non-kit components (appropriate splenocyte and antigen concentration). The optimum splenocyte concentration determine by the assay using normal, non-immunized, mice splenocytes (stimulated with con A) was 0.5 million cells/well/ 10Oμl (373.3± 30.6 SFCs/million). Affinity purified recombinant protein (GST-E7) was chosen for stimulation of splenocytes. Despite using an RTU kit, initial assays were plagued with high background that hindered interpretation of data. Apparently, the heterogeneous immunogen (recombinant bacteria), the choice of heterogeneous antigen for stimulation (recombinant protein) and most importantly, the enzyme-conjugate/chromogenic substrate combination influenced the background. Streptavidin-ALP and its substrate (BCIP/NBT) but not the kit provided, Avidin-HRP and its substrate (TMB) showed vivid spots, reduced background and improved readability on development of the assay. Changing the enzyme-substrate combination enabled determination of the appropriate concentration (1.0μg/well) of GST-E7 for antigenic stimulation of splenocytes (290.67±8.1 SFCs/million cells). The ALP enzyme/BCIP NBT substrate seems a more ideal developing reagent for ELISPOT assays involving heterogeneous immunogens (S. typhiTy21a) or/ and stimulating antigens (recombinant whole proteins). The protocol with optimized splenocyte and recombinant antigen concentrations is of acceptable precision as determined in the intra assay %CV ranging from (7.3 to 14.1) and inter assay % CV of nearly 13%. Source


Ponnanna N.M.,Research and Development Center Indian Immunologicals Ltd Rakshapuram | Sriraman R.,Research and Development Center Indian Immunologicals Ltd Rakshapuram | Verma R.R.,Research and Development Center Indian Immunologicals Ltd Rakshapuram | Swapna E.,Research and Development Center Indian Immunologicals Ltd Rakshapuram | And 6 more authors.
Current Trends in Biotechnology and Pharmacy | Year: 2012

Immunotherapy is intensely being considered for either a stand-alone or supplemental treatment of HPV associated pre-cancers and cancers. The aim of the study was to enquire whether S. typhiTy21 a engineered to express the HPV 16 E7 (Ty21a-E7) elicits T cell response to the oncoprotein. The codon optimized synthetic HPV16 E7 gene was cloned in an expression vector under a constitutive promoter (Ptac). Female mice of strain C57BL/6 were immunized intra nasally with Ty21 a-E7. The cell mediated immune response was evaluated employing the IFNγ-Elispot assay. Splenocytes were stimulated with the affinity purified recombinant proteins, either GST-E7 or GST expressed in E. coli BL21. The HPV 16 E7 specific IFN secreting-spot forming cells (SFCs) obtained on stimulation with purified GST-E7 in the Elispot assay was 240 (±6.92) cells/million splenocytes, significantly higher (p< 0.01) than the number for splenocytes stimulated with GST (67.3 ±26.40 cells/million). The antigen specific T cells were found to be predominantly of the CD4+ T cell type. The results show E7 specific CMI response in the mouse model of study. To our knowledge this is the first study reporting CMI response in mice to an HPV oncoprotein delivered by S. typfriTy21a- the live-attenuated, oral, typhoid vaccine strain. Source

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