Standardization of an ELISPOT protocol for the evaluation of Human papillomavirus 16 (HPV16) E7 specific T cell response in mice immunized with the E7 recombinant Salmonella typhi strain Ty21a (S.typhi Ty21a)

Order of Publishing in Issue: 
12
Volume :6
Issue :4
October, 2012
Page No: 
479-488
Authors: 
Ponnanna NM[1], Rajan Sriraman [1], Ravi Ranjan Verma [1], Balaji Kasa[1], Kota Sathish [1], Maroudam V [1], Bala Obulapathi [1], M Lakshmi Narasu2 and VA Srinivasan1*
Address: 
[1] Research & Development Centre Indian Immunologicals Limited Rakshapuram, Gachibowli, Hyderabad 500032, Andhra Pradesh, India
Address: 
[2] Center for Biotechnology, Jawaharlal Nehru Technological University, Hyderabad

Abstract

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. A commercial ready-to-use (RTU) IFN? ELISPOT 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/ 100μl (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 enzymesubstrate 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. typhi Ty21a) 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%.

Keywords: 
RTU ELISPOT kit, enzymeconjugate and substrate combination, Recombinant protein, SFCs
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