ESTABLISHING IMPROVED PLATFORMS FOR DENGUE DIAGNOSIS AND HYBRIDOMA DEVELOPMENT USING DENGUE ENVELOPE DOMAIN III ANTIGEN MELVIN TAN LIK CHERN NATIONAL UNIVERSITY OF SINGAPORE 2010 ESTABLISHING IMPROVED PLATFORMS FOR DENGUE DIAGNOSIS AND HYBRIDOMA DEVELOPMENT USING DENGUE ENVELOPE DOMAIN III ANTIGEN Melvin Tan Lik Chern B.Sc. (Hons), Monash University, Australia A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF MICROBIOLOGY YONG LOO LIN SCHOOL OF MEDICINE NATIONAL UNIVERSITY OF SINGAPORE 2010 PUBLICATIONS AND PRESENTATIONS GENERATED DURING THE COURSE OF STUDY Publications: Tan, L.C.M., Chua, A.J.S., Goh, L.S.L., Pua, S.M., Cheong, Y.K. and Ng, M.L. (2010). A membrane chromatography method for the rapid purification of recombinant flavivirus proteins. Protein Expr Purif 74:129-37. - Provided at the back of thesis. Tan, L.C.M. & Ng, M.L. High throughput detection of dengue envelope domain III-specific antibodies by monovalent and tetravalent biotinstreptavidin indirect ELISA. Manuscript in preparation. Book Chapter: Tan, L.C.M and Ng M.L. Dengue envelope domain III protein: properties, production and potential applications in dengue diagnosis. In: Dengue virus: detection, diagnosis and control. Chapter 3. Editor: Basak Ganim and Adam Reis. ISBN: 978-1-60876-398-6. In press. - Provided at the back of thesis. International Conference Presentations (Oral): Tan, L.C.M and Ng M.L. (2008). Development and characterization of dengue virus serotypes to recombinant envelope Domain III proteins and antibodies as diagnostic reagents for a biotin-streptavidin enhanced indirect ELISA. Wilbio 5th International Meeting Bioprocess Technology: Asia Pacific, Singapore. Choi, J.H., Tan L.C.M., Ng M.L., Grotenbreg, G.M., Love, J.C. and Ploegh, H.L. (2009). Recent advancement of single cell assay as a platform for infectious disease research. International Conference on Materials for Advanced Technologies: GEM4/SMART Symposium on Infectious Diseases, Suntec City, Singapore. International Conference Presentations (Poster): Tan L.C.M. and Ng M.L. (2009). Immunogenicity and protective efficacy of dengue virus serotypes to recombinant envelope domain III proteins. 8th Asia Pacific Congress of Medical Virology, Hong Kong SAR, China. Tan L.C.M. and Ng M.L. (2009). Improved detection of dengue envelope domain III-specific antibodies by indirect biotin streptavidin ELISA. Emerging Infectious Diseases 2009, - 11 Dec 2009, Duke-NUS, Singapore. i Choi J.H., Tan L.C.M., Lee Y.H., Gijsbert M.J., Love J.C., Ng M.L. and Ploegh, H.L. (2009). Single cell assay for hybridomas and primary B lymphocytes secreting antibodies against dengue envelope domain III proteins. Emerging Infectious Diseases 2009, - 11 Dec 2009, Duke-NUS, Singapore. (Selected for colloquium) Goh L.S.L., Tan L.C.M., Chua A.J.S., Pua S.M., Cheong Y.K. and Ng M.L. (2009). A membrane chromatography method for the rapid purification of recombinant flavivirus proteins. Emerging Infectious Diseases 2009, - 11 Dec 2009, Duke-NUS, Singapore. Tan L.C.M., Choi J.H., Lee Y.H., Gijsbert M.J., Love J.C., Ng M.L. and Ploegh, H.L. (2010). Protein production and purification of dengue domain III proteins for all four serotypes and subsequent sortase labelling for single cell assay. 10th Nagasaki-Singapore Medical Symposium on Infectious Diseases, 15-16 April 2010, National University of Singapore, Singapore. Choi J.H., Tan L.C.M., Lee Y.H., Gijsbert M.J., Love J.C., Ng M.L. and Ploegh, H.L. (2010). High-throughput single cell assay: Screening by microengraving and isolating by automatic micromanipulator for hybridomas secreting antibodies against dengue envelope domain III proteins. 10th Nagasaki-Singapore Medical Symposium on Infectious Diseases, 15-16 April 2010, National University of Singapore, Singapore. Local Conference Presentations (Oral): Choi, J.H., Tan L.C.M., Ng M.L., Grotenbreg, G.M., Love, J.C. & Ploegh, H.L. (2009). Progress on single cell assay for B lymphocytes and hybridoma specific to dengue envelope domain III. Singapore - Massachusetts Institute of Technology Alliance for Research and Technology (Infectious Disease Interdisciplinary Research Group) Workshop-11 Jan 2010, Singapore. Local Conference Presentations (Poster): Choi J.H., Tan L.C.M., Lee, Y.H., Lai, Y.Q., Rafiq, N.B.M., Grotenbreg, G.M., Ng M.L. and Ploegh, H.L. (2009). Protein purification for immunization and labelling. Singapore - Massachusetts Institute of Technology Alliance for Research and Technology (Infectious Disease Interdisciplinary Research Group) Workshop-2009, Singapore. Choi J.H., Tan L.C.M., Lee Y.H., Ng, M.L., Love, J.C. and Ploegh, H.L. (2009). Recent advancement of single cell assay for infectious disease research. Singapore - Massachusetts Institute of Technology Alliance for Research and Technology (Infectious Disease - Interdisciplinary Research Group) Workshop-2009, Singapore. ii Choi J.H., Tan L.C.M., Lee Y.H., Gijsbert M.J., Love J.C., Ng M.L. and Ploegh, H.L. (2010). Advanced single cell assay (1) For hybridomas secreting antibodies against dengue envelope domain III protein serotype 1. Singapore Massachusetts Institute of Technology Alliance for Research and Technology (Infectious Disease - Interdisciplinary Research Group) Workshop-11 Jan 2010, Singapore. Choi J.H., Tan L.C.M., Lee Y.H., Gijsbert M.J., Love J.C., Ng M.L. and Ploegh, H.L. (2010). Advanced single cell assay (2) For hybridoma and primary lymphocyte secreting multi-specific antibodies against dengue envelope domain III proteins. Singapore - Massachusetts Institute of Technology Alliance for Research and Technology (Infectious Disease Interdisciplinary Research Group) Workshop-11 Jan 2010, Singapore. Choi J.H., Tan L.C.M., Lee Y.H., Love J.C., Ng M.L. and Ploegh, H.L. (2010). Pre-selective single cell assay for monoclonal antibodies specific to dengue envelope domain III: Selection of single cells specific to only one serotype or multi-serotypes. Singapore - Massachusetts Institute of Technology Alliance for Research and Technology (Infectious Disease Interdisciplinary Research Group) 3rd Annual Workshop- 7-8 July 2010, Singapore. Choi J.H., Tan L.C.M., Love J.C., Ng M.L. and Ploegh, H.L. (2010). Single cell assay for primary B lymphocytes specific to dengue envelope domain III. Singapore - Massachusetts Institute of Technology Alliance for Research and Technology (Infectious Disease - Interdisciplinary Research Group) 3rd Annual Workshop- 7-8 July 2010, Singapore. iii ACKNOWLEDGEMENTS I would like to express my sincere gratitude to Professor Ng Mah Lee for the immeasurable amount of support and guidance she has provided me throughout this study. Professor Ng‟s insights into this project and patience towards me have been a true blessing. I sincerely thank the members of the Flavivirus Laboratory: Boon, Bhuvana, Mun Keat, Han Yap, Xiao Ling, Li Shan, Shu Min, Samuel, Vincent, Adrian, Edwin, Terence, Kim Long, Anthony and Audrey for their friendship, expert technical advice on different techniques and constructive criticism. I would also like to thank the members of SMART-Single Cell Assay Group: Professor Hidde L. Ploegh, Dr Choi Jae Hyeok and Lee Ying Hui, for providing their state of the art technology and expert advice. I am grateful to my parents who never ceased loving and supporting me. This work is dedicated to my wife, Lydia, and my son, Benjamin (whose name means „Son of my right hand‟). Your presence alone is a constant joy and support for me. I thank Jesus for his great abundance of love, wisdom and grace. My heart is truly warmed. Thank you. iv TABLE OF CONTENTS Page Number Publications and presentations generated during the course of study …… . i Acknowledgements ……………………………………………… .……… iv Table of Contents ……….…………………………………………………. v Summary .………………………… ……………………………………… xi List of Tables ……………… ………………………….……….………… xiii List of Figures …………… …………………………….………………… xv Abbreviations ………….…………………………… .…………………… xx CHAPTER 1.0 LITERATURE REVIEW 1.1 Introduction ………………….………………………… … 1.2 Structural studies on the flavivirus envelope and DIII protein……………………………………………………… 1.3 Antagonistic activity of DIII protein ……… ………… .… 1.4 Neutralizing epitopes on DIII protein ………….……… … 1.5 Production of DENV rDIII protein ………… …………… 1.5.1 rDIII protein expression …………………………… 1.5.2 rDIII protein purification ……… .…………… … 11 1.5.2.1 Immobilized metal affinity chromatography (IMAC) …… .………………………… … 11 1.5.2.2 Membrane adsorber-based IMAC ….……… 13 1.6 DENV rDIII protein as a potential protein subunit vaccine 14 1.7 Potential applications of rDIII protein in dengue diagnosis 21 1.8 Microengraving as a potential tool for antibody generation 24 1.9 Project aims and hypothesis………………………… .…… 28 v 2.0 MATERIALS AND METHODS 2.1 Tissue culture and propagation of viruses ………………… 29 2.1.1 Cell culture ……………… ……………………… 29 2.1.2 Virus source and propagation .…………… …… 29 2.1.3 Plaque assay…… .………… ……………… …… 30 2.2 Bioinformatics and biostatistical analyses ………………… 31 2.3 Construction of DIII-expression plasmids ………………… 32 2.4 Transformation of E. coli competent cells for protein expression …………………….…………………………… 37 2.5 Construction of Series A and B DIII-expression plasmids . 37 2.6 Protein expression and purification ……….……………… 43 2.6.1 Pilot expression of rDIII protein …… …………… 43 2.6.2 Batch production of rDIII protein …………………. 43 2.6.3 Refolding of rDIII protein by dialysis ……………. 44 2.6.4 rDIII protein purification …………………………. 44 2.6.5 Isolation of E. coli inclusion bodies enclosing rDIIIprotein ……………………………… .…………… 45 2.6.6 Metal affinity membrane chromatography … ……. 46 2.6.7 Packed-bed chromatography …… ………………. 49 2.6.7.1 Purification under native conditions ….…… 49 2.6.7.2 Purification under denaturing conditions… . 49 2.6.8 Fast protein liquid chromatography (FPLC) .… 49 2.7 Protein storage and product analysis ………………….…… 50 2.8 Enzyme linked immunosorbent assay (ELISA) …………… 53 2.9 Immunization procedures …………………………….……. 56 vi 2.9.1 Immunization of mice with DENV rDIII proteins (serotypes 1-4) …………………………………… 57 2.9.2 Determining the effects of immunization of mice with different rDIII-protein-adjuvant formulations over a tenweek period ……………………………………… 57 2.9.3 Immunization of mice with rDIII proteins for hybridoma development (collaboration with SMART) ………………………………………… 58 2.10 Plaque reduction neutralization test (PRNT) … .…………. 58 2.11 Microengraving and micromanipulation of cells .……… . 61 2.11.1 In vitro treatment of splenocytes obtained from mice immunized with rDIII protein …………………… . 61 2.11.2 Treatment of splenocytes for microengraving studies performed on primary B cells …………………… . 62 2.11.3 Development of DIII-specific hybridomas …… .… 63 2.11.4 Preparation of cells for flow cytometry analyses … 64 2.11.5 Microengraving analysis of cells ……………… … 64 2.11.6 Robotic retrieval of cells by micromanipulation .…. 68 3.0 RESULTS DEVELOPMENT OF FUNCTIONAL rDIII PROTEINS 3.1 Determination of growth kinetics of DENV in C6/36 and Huh7 cells ……………………………………………………… . 70 3.2 Bioinformatics analysis of envelope DIII proteins ……… . 72 3.3 Construction of DIII-expression vectors .………………… 80 3.4 Production of purified DENV rDIII proteins … .…………. 80 3.5 3.4.1 Pilot protein expression ……………………………. 80 3.4.2 Batch expression and purification of rDIII proteins . 87 Characterization of purified DENV rDIII proteins …… .… 93 vii 4.0 3.5.1 Determining the homologous and heterologous neutralizing potential of rDIII protein-specific antibodies ………………… ……………………… 97 3.5.2 Removal of N-terminal hexahistidine tag by thrombin cleavage reaction ……………… .……………… 101 RESULTS DENV rDIII PROTEIN-BASED DIAGNOSTIC ASSAYS 4.1 DENV rDIII proteins as diagnostic reagents ……………… 104 4.2 Development of monovalent biotin-streptavidin enhanced indirect ELISA [iELISA-(BS)] ……….…………………… 104 4.2.1 Preliminary test ………… .………………………. 104 4.2.2 Optimization of monovalent DENV rDIII iELISA(BS)………………………… …………………… 107 4.2.2.1 Exploring the suitability of various immunoplates for iELISA-(BS) ……….…………… 108 4.2.2.2 Determining the duration required for optimal coating of antigen on the immuno-plate … . 111 4.2.2.3 Determining the minimal concentration of antigen required for effective coating of immuno-plates ………………… ………….113 4.2.2.4 Determining the optimal volume of antigen required for coating of immuno-plate …… . 116 4.2.2.5 Determining the optimal time required for assay blocking ………….…………………………119 4.2.2.6 Determining the time required for optimal development of TMB substrate via a study comparing two different brands of TMB solutions ………………… …………….… 121 4.2.2.7 Determining the optimal concentration of streptavidin-HRP conjugate for DENV rDIII iELISA-(BS) …………………………….… 124 4.2.2.8 Determining the optimal concentration of secondary antibody-biotin conjugate for DENV rDIII iELISA-(BS) ……….……………… . 127 viii Substrate TMB One (Promega, USA) Ultrasensitive TMB (Chemicon, USA) rDIII Protein iELISA-(BS) DENV4 Development Time Mean Positive Value/ O.D. 650 nm Mean Negative Value/ O.D. 650 nm Mean Blank Value/ O.D. 650 nm Significance of Test* (p-value) 2.5 0.547 + 0.072 0.058 + 0.003 0.042 + 0.001 3.56E-03 1.001 + 0.089 0.072 + 0.005 0.044 + 0.001 1.48E-03 7.5 1.444 + 0.204 0.091 + 0.006 0.047 + 0.002 3.72E-03 10 1.740 + 0.287 0.101 + 0.008 0.050 + 0.002 5.01E-03 12.5 1.956 + 0.335 0.109 + 0.008 0.050 + 0.003 5.38E-03 15 2.089 + 0.364 0.113 + 0.008 0.051 + 0.002 5.54E-03 17.5 2.145 + 0.387 0.114 + 0.008 0.051 + 0.002 5.94E-03 20 2.206 + 0.402 0.115 + 0.009 0.052 + 0.002 6.03E-03 22.5 2.226 + 0.404 0.115 + 0.008 0.052 + 0.002 5.97E-03 25 2.239 + 0.414 0.115 + 0.008 0.052 + 0.002 6.20E-03 27.5 2.246 + 0.422 0.114 + 0.008 0.052 + 0.002 6.41E-03 30 2.235 + 0.421 0.114 + 0.009 0.052 + 0.002 6.42E-03 2.5 0.672 + 0.116 0.072 + 0.010 0.050 + 0.002 5.94E-03 1.043 + 0.138 0.088 + 0.011 0.056 + 0.003 3.31E-03 7.5 1.381 + 0.176 0.107 + 0.014 0.060 + 0.005 3.01E-03 10 1.677 + 0.219 0.118 + 0.017 0.064 + 0.006 3.14E-03 12.5 1.861 + 0.257 0.127 + 0.019 0.066 + 0.006 3.51E-03 15 1.995 + 0.266 0.134 + 0.021 0.067 + 0.006 3.25E-03 17.5 2.085 + 0.288 0.138 + 0.022 0.068 + 0.007 3.48E-03 20 2.144 + 0.302 0.140 + 0.023 0.070 + 0.007 3.61E-03 22.5 2.163 + 0.301 0.142 + 0.024 0.070 + 0.007 3.51E-03 25 2.190 + 0.298 0.142 + 0.025 0.071 + 0.008 3.33E-03 27.5 2.217 + 0.311 0.143 + 0.025 0.072 + 0.008 3.57E-03 30 2.224 + 0.322 0.144 + 0.026 0.073 + 0.008 3.78E-03 * Calculated according to Student’s T-Test to determine statistical significance of results (p-value < 0.05). 269 H. Optimization Test – Determining the optimal concentration of streptavidin-HRP conjugate for DENV rDIII iELISA-(BS) (i) Assay Protocol Reagents Multisorp (Nunc, USA) DENV rDIII proteins (serotypes - 4) PBST with % skimmed milk (Appendix 4B) DENV rDIII protein-specific anti-sera (serotypes - 4) Anti-IgG (H+L)-Biotin conjugate (eBioscience, USA) ELISA Plate Antigens Blocking Reagent Samples Conjugate Conjugate Strepavidin-HRP conjugate (Chemicon, USA) Substrate Stop Solution TMB one solution (Promega, USA) H2SO4 (Merck, Germany) Concentration N.A. 2.5 µg/ml Volume N.A. 50 µl Neat concentration 100 µl 1:1000 dilution 100 µl µg/ml 100 µl 1:1000, 1:3000,1:5000 and 1:7000 dilution Neat concentration 0.5 M concentration 100 µl 100 µl 100 µl (ii) Test Results rDIII Protein iELISA(BS) DENV DENV DENV DENV StrepavidinHRP Conjugate Dilution 1:1000 1:3000 1:5000 1:7000 1:1000 1:3000 1:5000 1:7000 1:1000 1:3000 1:5000 1:7000 1:1000 1:3000 1:5000 1:7000 Mean Positive Value/ O.D. 450 nm 3.067 + 0.070 2.620 + 0.071 2.474 + 0.256 1.847 + 0.376 2.748 + 0.171 2.477 + 0.339 2.365 + 0.321 1.994 + 0.371 2.641 + 0.491 2.331 + 0.525 2.096 + 0.553 1.715 + 0.578 1.654 + 0.348 1.445 + 0.483 1.478 + 0.441 0.962 + 0.072 Mean Negative Value/ O.D. 450 nm 0.437 + 0.043 0.383 + 0.022 0.377 + 0.030 0.279 + 0.021 0.619 + 0.023 0.531 + 0.039 0.440 + 0.038 0.327 + 0.009 0.469 + 0.049 0.418 + 0.073 0.376 + 0.010 0.314 + 0.037 0.545 + 0.002 0.480 + 0.027 0.466 + 0.051 0.399 + 0.046 Mean Blank Value/ O.D. 450 nm Significance of Test* (p-value) 0.101 + 0.004 0.071 + 0.003 0.066 + 0.002 0.057 + 0.004 0.113 + 0.011 0.103 + 0.014 0.083 + 0.001 0.067 + 0.007 0.082 + 0.014 0.075 + 0.007 0.063 + 0.010 0.050 + 0.004 0.081 + 0.011 0.069 + 0.013 0.078 + 0.002 0.068 + 0.003 2.33E-06 5.32E-05 2.23E-03 9.20E-03 9.12E-04 4.63E-03 4.20E-03 8.03E-03 7.91E-03 1.12E-02 1.64E-02 2.60E-02 3.59E-02 3.69E-02 2.80E-02 3.95E-04 * Calculated according to Student’s T-Test to determine statistical significance of results (p-value < 0.05). 270 I. Optimization Test – Determining the optimal concentration of secondary antibody-biotin conjugate for DENV rDIII iELISA-(BS) (i) Assay Protocol ELISA Plate Antigens Blocking Reagent Samples Conjugate Conjugate Substrate Stop Solution Reagents Multisorp (Nunc, USA) DENV rDIII proteins (serotypes - 4) PBST with % skimmed milk (Appendix 4B) DENV rDIII protein-specific anti-sera (serotypes - 4) Anti-IgG (H+L)-Biotin conjugate (eBioscience, USA) Strepavidin-HRP conjugate (Chemicon, USA) TMB one solution (Promega, USA) H2SO4 (Merck, Germany) Concentration N.A. 2.5 µg/ml Volume N.A. 50 µl Neat concentration 100 µl 1:1000 dilution 100 µl 8, 4, or µg/ml concentration 100 µl 1:5000 dilution 100 µl Neat concentration 0.5 M concentration 100 µl 100 µl (ii) Test Results rDIII Protein iELISA(BS) DENV DENV DENV DENV Secondary AntibodyBiotin Conjugate µg/ml 8 8 Mean Positive Value/ O.D. 450 nm Mean Negative Value/ O.D. 450 nm Mean Blank Value/ O.D. 450 nm Significa nce of Test* (p-value) 3.309 + 0.016 3.316 + 0.017 3.291 + 0.047 3.264 + 0.045 3.276 + 0.024 3.267 + 0.021 3.217 + 0.066 3.174 + 0.092 3.317 + 0.023 3.304 + 0.010 3.290 + 0.037 3.306 + 0.037 3.245 + 0.024 3.216 + 0.034 3.043 + 0.180 2.627 + 0.292 0.335 + 0.042 0.334 + 0.014 0.301 + 0.031 0.251 + 0.006 0.395 + 0.043 0.378 + 0.035 0.362 + 0.042 0.288 + 0.011 0.313 + 0.035 0.310 + 0.009 0.263 + 0.007 0.223 + 0.016 0.332 + 0.014 0.342 + 0.018 0.301 + 0.018 0.235 + 0.018 0.083 + 0.005 0.076 + 0.001 0.071 + 0.011 0.060 + 0.004 0.072 + 0.004 0.069 + 0.003 0.071 + 0.004 0.065 + 0.015 0.073 + 0.001 0.086 + 0.043 0.057 + 0.003 0.058 + 0.008 0.073 + 0.009 0.058 + 0.004 0.055 + 0.004 0.051 + 0.005 3.62E-06 2.37E-09 2.55E-07 2.90E-05 7.50E-07 1.89E-07 1.38E-06 1.42E-04 8.68E-08 1.42E-10 1.44E-05 1.37E-06 9.30E-08 4.76E-07 6.55E-04 2.40E-03 * Calculated according to Student’s T-Test to determine statistical significance of results (p-value < 0.05). 271 J. Determining optimal concentration of secondary antibody-biotin conjugate required for DENV iELISA-(N). (i) Assay Protocol ELISA Plate Antigens Blocking Reagent Samples Reagents Multisorp (Nunc, USA) DENV rDIII proteins (serotypes - 4) PBST with % skimmed milk (Appendix 4B) DENV rDIII protein-specific anti-sera (serotypes - 4) Conjugate Anti-IgG (H+L)- HRP conjugate (Millipore, USA) Substrate TMB One solution (Promega, USA) Stop Solution H2SO4 (Merck, Germany) Concentration N.A. 2.5 µg/ml Volume N.A. 50 µl Neat concentration 100 µl 1:1000 dilution 100 µl 4, 2, or 0.5 µg/ml concentration Neat concentration 0.5 M concentration 100 µl 100 µl 100 µl (ii) Test Results rDIII Protein iELISA-(N) DENV DENV DENV DENV Secondary AntibodyBiotin Conjugate µg/ml 0.5 0.5 0.5 0.5 Mean Positive Value/ O.D. 450 nm Mean Negative Value/ O.D. 450 nm Mean Blank Value/ O.D. 450 nm Significance of Test* (p-value) 3.324 + 0.029 3.281 + 0.029 3.175 + 0.100 2.538 + 0.279 3.225 + 0.060 3.124 + 0.138 2.978 + 0.166 2.170 + 0.378 2.884 + 0.306 2.217 + 0.319 1.682 + 0.498 0.967 + 0.226 3.241 + 0.069 2.984 + 0.292 2.585 + 0.374 1.666 + 0.343 1.442 + 0.036 0.946 + 0.080 0.592 + 0.030 0.352 + 0.014 1.282 + 0.051 1.028 + 0.052 0.710 + 0.031 0.439 + 0.012 1.303 + 0.014 0.935 + 0.015 0.647 + 0.023 0.416 + 0.008 1.599 + 0.102 1.097 + 0.037 0.790 + 0.015 0.495 + 0.025 0.162 + 0.012 0.106 + 0.012 0.076 + 0.008 0.059 + 0.004 0.097 + 0.019 0.121 + 0.017 0.122 + 0.015 0.088 + 0.005 0.103 + 0.010 0.088 + 0.014 0.065 + 0.005 0.061 + 0.003 0.090 + 0.002 0.075 + 0.010 0.086 + 0.014 0.085 + 0.016 2.22E-07 4.41E-05 8.89E-05 2.66E-03 1.22E-06 2.04E-04 6.47E-04 7.73E-03 6.08E-03 9.93E-03 3.45E-02 2.59E-02 2.87E-05 3.57E-03 7.04E-03 1.34E-02 * Calculated according to Student’s T-Test to determine statistical significance of results (p-value < 0.05). 272 APPENDIX Validation of DENV rDIII iELISA-(BS) platforms. A. Comparing the difference in P/N ratio between DENV rDIII iELISA-(BS) and iELISA-(N) platforms. (i) DENV rDIII iELISA-(BS) Assay Protocol Substrate Reagents Multisorp (Nunc, USA) DENV rDIII proteins (serotypes - 4) PBST with % skimmed milk (Appendix 4B) DENV rDIII protein-specific anti-sera (serotypes - 4) x per serotype Anti-IgG (H+L)-Biotin conjugate (eBioscience, USA) Strepavidin-HRP conjugate (Chemicon, USA) TMB one solution (Promega, USA) Stop Solution H2SO4 (Merck, Germany) ELISA Plate Antigens Blocking Reagent Samples Conjugate Conjugate (ii) Concentration N.A. 2.5 µg/ml Volume N.A. 50 µl Neat concentration 100 µl 1:1000 dilution 100 µl µg/ml concentration 100 µl 1:5000 dilution 100 µl Neat concentration 0.5 M concentration 100 µl Concentration N.A. 2.5 µg/ml Volume N.A. 50 µl Neat concentration 100 µl 1:1000 dilution 100 µl 100 µl DENV rDIII iELISA-(N) Assay Protocol Substrate Reagents Multisorp (Nunc, USA) DENV rDIII proteins (serotypes - 4) PBST with % skimmed milk (Appendix 4B) DENV rDIII protein-specific anti-sera (serotypes - 4) x per serotype Anti-IgG (H+L)- HRP conjugate (Millipore, USA) TMB One solution (Promega, USA) Stop Solution H2SO4 (Merck, Germany) ELISA Plate Antigens Blocking Reagent Samples Conjugate µg/ml concentration Neat concentration 0.5M concentration 100 µl 100 µl 100 µl 273 (iii) Determining the mean P/N ratios of DENV rDIII iELISA-(BS) and iELISA-(N) (serotypes - 4). Mean Positive Mean Negative value/ O.D. value/ O.D. 450nm 450nm rDIII iELISA-(BS) (n = per serotype) DENV 2.701 + 0.047 0.263 + 0.003 DENV 1.894 + 0.122 0.272 + 0.012 DENV 2.201 + 0.237 0.274 + 0.015 DENV 2.101 + 0.106 0.258 + 0.009 rDIII iELISA-(N) (n = per serotype) DENV 2.701 + 0.047 1.020 + 0.132 DENV 1.894 + 0.122 1.423 + 0.328 DENV 2.201 + 0.237 1.172 + 0.289 DENV 2.101 + 0.106 1.290 + 0.404 Significance of Test (p-value) Mean P/N ratio 4.94E-10 4.14E-07 5.50E-06 1.10E-07 10.285 + 0.021 6.930 + 0.055 8.053 + 0.106 8.176 + 0.047 6.17E-08 3.15E-05 6.71E-06 5.36E-04 3.321 + 0.060 2.484 + 0.147 2.973 + 0.131 2.535 + 0.350 Positive samples comprised anti-sera from mice immunized with DENV rDIII (serotypes 1, 2, or 4) proteins. Four groups of mice were immunized with rDIII protein - group per serotype, mice per group. Additionally, negative sera were obtained from PBS-immunized mice. 274 B. Comparing the difference in limit of detection (LOD) between DENV rDIII iELISA-(BS) and iELISA-(N) platforms. (i) DENV rDIII iELISA-(BS) Assay Protocol ELISA Plate Antigens Blocking Reagent DENV rDIII protein-specific anti-sera (serotypes - 4) x per serotype Samples Conjugate Conjugate Substrate Stop Solution (ii) Reagents Multisorp (Nunc, USA) DENV rDIII proteins (serotypes - 4) PBST with % skimmed milk (Appendix 4B) Anti-IgG (H+L)-Biotin conjugate (eBioscience, USA) Strepavidin-HRP conjugate (Chemicon, USA) TMB one solution (Promega, USA) H2SO4 (Merck, Germany) Concentration N.A. 2.5 µg/ml Volume N.A. 50 µl Neat concentration 100 µl 1:1000 to 100,000,000 dilution µg/ml concentration 100 µl 100 µl 1:5000 dilution 100 µl Neat concentration 0.5 M concentration 100 µl 100 µl DENV rDIII iELISA-(N) Assay Protocol ELISA Plate Antigens Blocking Reagent Samples Conjugate Substrate Stop Solution Reagents Multisorp (Nunc, USA) DENV rDIII proteins (serotypes - 4) PBST with % skimmed milk (Appendix 4B) DENV rDIII protein-specific anti-sera (serotypes - 4) x per serotype Anti-IgG (H+L)- HRP conjugate (Millipore, USA) TMB One solution (Promega, USA) H2SO4 (Merck, Germany) Concentration N.A. 2.5 µg/ml Volume N.A. 50 µl Neat concentration 100 µl 1:1000 to 100,000,000 dilution µg/ml concentration Neat concentration 0.5M concentration 100 µl 100 µl 100 µl 100 µl (iii) Summary table showing results for LOD for each serum tested on DENV rDIII iELISA-(BS) and iELISA-(N) platforms. Sample Sample Sample Sample Sample Sample Sample Sample Sample Sample Sample Sample Limit of Detection (dilution) iELISA-(BS) iELISA-(N) DENV 1:100,000 1:10,000 1:100,000 1:10,000 1:100,000 1:10,000 1:100,000 1:10,000 1:100,000 1:10,000 1:10,000 1:1000 DENV 1:10,000 1:1000 1:10,000 1:10,000 1:10,000 1:10,000 1:10,000 1:1000 1:10,000 1:10,000 1:10,000 1:10,000 Sample Sample Sample Sample Sample Sample Sample Sample Sample Sample Sample Sample Limit of Detection (dilution) iELISA-(BS) iELISA-(N) DENV 1:10,000 1:10,000 1:10,000 1:1000 1:10,000 1:10,000 1:100,000 1:1000 1:10,000 1:1000 1:10,000 1:10,000 DENV 1:10,000 1:10,000 1:10,000 1:10,000 1:10,000 1:10,000 1:10,000 1:10,000 1:10,000 1:10,000 1:10,000 1:1000 275 C. Assessing the specificity of DENV rDIII iELISA-(BS) platforms for detection of homotypic and heterotypic DENV rDIII protein-specific antibodies. (i) Assay Protocol ELISA Plate Antigens Blocking Reagent Samples Conjugate Conjugate Substrate Stop Solution Reagents Multisorp (Nunc, USA) DENV rDIII proteins (serotypes 1, 2, or 4) coated on each individual Multisorp plate, i.e. one protein per plate. PBST with % skimmed milk (Appendix 4B) DENV rDIII protein-specific anti-sera (serotypes - 4) x per serotype Anti-IgG (H+L)-Biotin conjugate (eBioscience, USA) Strepavidin-HRP conjugate (Chemicon, USA) TMB one solution (Promega, USA) H2SO4 (Merck, Germany) Concentration N.A. Volume N.A. 2.5 µg/ml 50 µl Neat concentration 100 µl 1:1000 dilution 100 µl µg/ml concentration 100 µl 1:5000 dilution 100 µl Neat concentration 0.5 M concentration 100 µl 100 µl Positive samples comprised anti-sera from mice immunized with DENV rDIII (serotypes 1, 2, or 4) proteins. Four groups of mice were immunized with rDIII protein - group per serotype, mice per group. Additionally, negative sera were obtained from PBS-immunized mice. (ii) Test Results DENV rDIII Protein Coated DENV DENV DENV DENV DENV rDIIIspecific Anti-Sera Tested (n = 6) DENV DENV DENV DENV DENV DENV DENV DENV DENV DENV DENV DENV DENV DENV DENV DENV Mean Positive Value/ O.D. 450 nm Mean Negative Value/ O.D. 450 nm Mean Blank Value/ O.D. 450 nm Significanc e of Test* (p-value) 3.132 + 0.196 2.258 + 0.223 1.975 + 0.967 2.307 + 0.488 1.034 + 0.360 1.845 + 0.442 1.274 + 0.798 1.718 + 0.283 1.129 + 0.342 1.509 + 0.647 1.975 + 0.584 1.536 + 0.772 2.843 + 0.399 2.685 + 0.275 2.500 + 0.702 3.023 + 0.303 0.104 + 0.015 0.119 + 0.017 0.109 + 0.003 0.126 + 0.011 0.101 + 0.006 0.110 + 0.011 0.104 + 0.005 0.093 + 0.012 0.095 + 0.006 0.099 + 0.021 0.100 + 0.010 0.111 + 0.004 0.110 + 0.006 0.115 + 0.007 0.116 + 0.009 0.122 + 0.004 0.078 + 0.002 0.089 + 0.005 0.077 + 0.008 0.083 + 0.012 0.076 + 0.004 0.074 + 0.002 0.070 + 0.009 0.075 + 0.004 0.083 + 0.012 0.082 + 0.011 0.089 + 0.014 0.124 + 0.014 0.076 + 0.009 0.075 + 0.002 0.082 + 0.027 0.074 + 0.003 8.71E-23 5.24E-19 2.67E-07 6.48E-13 3.77E-09 5.26E-12 9.39E-06 8.12E-15 3.47E-10 4.54E-08 1.35E-10 4.85E-07 5.79E-16 2.75E-18 5.95E-11 2.14E-18 * Calculated according to Student’s T-Test to determine statistical significance of results (p-value < 0.05). 276 D. Development of tetravalent DENV rDIII iELISA-(BS). (i) Assay Protocol ELISA Plate Antigens Blocking Reagent Samples Conjugate Conjugate Substrate Stop Solution Reagents Multisorp (Nunc, USA) DENV rDIII proteins (serotypes 1, 2, and 4) mixed in 1:1:1:1 proportion. PBST with % skimmed milk (Appendix 4B) DENV rDIII protein-specific anti-sera (serotypes - 4) x per serotype Anti-IgG (H+L)-Biotin conjugate (eBioscience, USA) Strepavidin-HRP conjugate (Chemicon, USA) TMB one solution (Promega, USA) H2SO4 (Merck, Germany) Concentration N.A. Volume N.A. 2.5 µg/ml 50 µl Neat concentration 100 µl 1:1000 dilution 100 µl µg/ml concentration 100 µl 1:5000 dilution 100 µl Neat concentration 0.5 M concentration 100 µl 100 µl Positive samples comprised anti-sera from mice immunized with DENV rDIII (serotypes 1, 2, or 4) proteins. Four groups of mice were immunized with rDIII protein - group per serotype, mice per group. Additionally, negative sera were obtained from PBS-immunized mice. (ii) Test Results DENV rDIIIspecific AntiSera Tested (n = 6) DENV DENV DENV DENV Mean Positive Value/ O.D. 450 nm 3.264 + 0.115 3.190 + 0.081 2.961 + 0.397 3.210 + 0.104 Mean Negative Value/ O.D. 450 nm 0.105 + 0.015 Mean Blank Value/ O.D. 450 nm Significance of Test* (p-value) 0.087 + 0.020 1.42E-39 4.01E-30 7.60E-14 9.95E-29 * Calculated according to Student’s T-Test to determine statistical significance of results (p-value < 0.05). 277 E. Establishing the suitability of monovalent and tetravalent DENV rDIII iELISA-(BS) for HTS. (i) Assay Protocol ELISA Plate Antigens Blocking Reagent Samples Conjugate Conjugate Substrate Stop Solution (ii) Reagents Multisorp (Nunc, USA) DENV rDIII proteins (serotypes - 4) PBST with % skimmed milk (Appendix 4B) DENV rDIII protein-specific antiserum (serotypes - 4) (i.e. only per serotype) Anti-IgG (H+L)-Biotin conjugate (eBioscience, USA) Strepavidin-HRP conjugate (Chemicon, USA) TMB one solution (Promega, USA) H2SO4 (Merck, Germany) Concentration N.A. 2.5 µg/ml Volume N.A. 50 µl Neat concentration 100 µl 1:1000 dilution 100 µl µg/ml concentration 100 µl 1:5000 dilution 100 µl Neat concentration 0.5 M concentration 100 µl 100 µl Test Results (O.D. 450 nm spectrophotometric reading) DENV Anti-serum Negative Anti-serum 10 11 12 13 14 15 16 17 18 19 20 21 22 3.448 3.407 3.455 3.355 3.346 3.329 3.354 3.332 3.343 3.351 3.354 3.336 3.338 3.354 3.338 3.367 3.355 3.331 3.356 3.333 3.336 3.404 0.217 0.231 0.168 0.182 0.183 0.188 0.199 0.217 0.129 0.134 0.144 0.151 0.106 0.107 0.108 0.109 0.088 0.095 0.103 0.104 0.158 0.166 Monovalent DENV2 rDIII iELISA-(BS) Sample Sample Monovalent DENV1 rDIII iELISA-(BS) DENV Anti-serum Negative Anti-serum 10 11 12 13 14 15 16 17 18 19 20 21 22 3.297 3.324 3.349 3.243 3.222 3.234 3.242 3.264 3.250 3.231 3.229 3.260 3.187 3.230 3.238 3.219 3.226 3.227 3.208 3.221 3.218 3.203 0.111 0.114 0.101 0.101 0.103 0.104 0.108 0.109 0.091 0.091 0.092 0.093 0.074 0.078 0.078 0.078 0.070 0.071 0.072 0.072 0.094 0.100 278 23 24 25 26 27 28 29 30 3.361 3.334 3.365 3.344 3.343 3.366 3.343 3.338 0.234 0.250 0.251 0.277 0.112 0.114 0.123 0.129 23 24 25 26 27 28 29 30 DENV Anti-serum Negative Anti-serum 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 2.899 2.786 2.694 3.221 3.247 3.099 3.354 3.323 3.100 3.245 3.132 3.225 3.260 3.348 3.293 3.206 3.376 3.315 3.218 3.264 3.208 3.117 3.273 3.047 3.078 3.044 2.963 3.122 3.249 3.195 0.096 0.096 0.088 0.091 0.091 0.093 0.093 0.096 0.079 0.080 0.080 0.081 0.071 0.072 0.072 0.074 0.070 0.070 0.070 0.070 0.083 0.087 0.104 0.106 0.109 0.118 0.074 0.078 0.078 0.079 0.115 0.130 0.130 0.133 0.082 0.082 0.083 0.089 Monovalent DENV4 rDIII iELISA-(BS) Sample Sample Monovalent DENV3 rDIII iELISA-(BS) 3.144 3.230 3.216 3.262 3.244 3.285 3.257 3.293 DENV Anti-serum Negative Anti-serum 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 3.359 3.368 3.413 3.317 3.297 3.312 3.297 3.296 3.303 3.316 3.291 3.288 3.309 3.290 3.296 3.307 3.350 3.313 3.330 3.370 3.411 3.317 3.312 3.330 3.313 3.345 3.302 3.293 3.299 3.287 0.112 0.113 0.102 0.105 0.105 0.106 0.107 0.111 0.094 0.095 0.098 0.098 0.086 0.087 0.091 0.091 0.079 0.081 0.082 0.083 0.099 0.102 0.117 0.120 0.122 0.123 0.091 0.092 0.092 0.093 279 Sample Tetravalent DENV iELISA-(BS) DENV Anti-serum DENV Anti-serum DENV Anti-serum DENV Anti-serum Negative Anti-serum 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 3.068 3.071 3.074 3.113 3.142 3.149 3.152 3.161 3.167 3.181 3.212 3.215 3.287 3.282 3.268 3.269 3.259 3.266 3.261 3.277 3.273 3.291 3.274 3.260 3.257 3.240 3.252 3.248 3.243 3.249 3.100 3.109 3.110 3.120 3.121 3.122 3.158 3.164 3.172 3.175 3.177 3.179 3.183 3.187 3.189 3.194 3.196 3.203 3.205 3.214 3.217 3.225 3.230 3.238 3.240 3.242 3.245 3.247 3.249 3.261 3.128 3.148 3.154 3.232 3.233 3.234 3.237 3.237 3.239 3.239 3.240 3.241 3.242 3.244 3.246 3.246 3.248 3.249 3.250 3.252 3.253 3.253 3.256 3.257 3.257 3.258 3.260 3.261 3.271 3.273 3.098 3.171 3.172 3.176 3.186 3.187 3.191 3.192 3.198 3.201 3.205 3.206 3.209 3.210 3.210 3.211 3.212 3.213 3.214 3.218 3.223 3.227 3.228 3.237 3.237 3.237 3.241 3.243 3.253 3.257 0.068 0.070 0.071 0.080 0.080 0.080 0.082 0.083 0.084 0.086 0.090 0.090 0.090 0.091 0.091 0.092 0.092 0.092 0.094 0.094 0.094 0.096 0.096 0.096 0.097 0.097 0.097 0.098 0.098 0.100 280 F. Detection of DIII-specific antibodies in human sera using human IgM and IgG tetravalent rDIII protein-based iELISA-(BS) (i) Patient sera database Patient A B C D E F G H I J K L M N O P Q R S T U V W X Y Z AA AB AC AD (ii) Serotype 1 1 1 1 1 1 1 1 1 2 2 3 3 - Day sera 05K0872DK1 05K2887DK1 05K3288DK1 05K3318DK1 05K3905DK1 05K3908DK1 05K3933DK1 05K3934DK1 05K4138DK1 05K4172DK1 05K4173DK1 05K4174DK1 05K4175DK1 05K4456DK1 05K4468DK1 05K4604DK1 05K4605DK1 05K4606DK1 08K3126DK1 07K1907DK1 07K1913DK1 07K3598DK1 08K3111DK1 05K0843DK1 05K2933DK1 05K3909DK1 05K4141DK1 05K4176DK1 06K2270DK1 05K3329DK1 Day sera 05K0872DK2 05K2887DK2 05K3288DK2 05K3318DK2 05K3905DK2 05K3908DK2 05K3933DK2 05K3934DK2 05K4138DK2 05K4172DK2 05K4173DK2 05K4174DK2 05K4175DK2 05K4456DK2 05K4468DK2 05K4604DK2 05K4605DK2 05K4606DK2 08K3126DK2 07K1907DK2 07K1913DK2 07K3598DK2 08K3111DK2 05K0843DK2 05K2933DK2 05K3909DK2 05K4141DK2 05K4176DK2 06K2270DK2 05K3329DK2 Day 21 sera 05K0872DK3 05K2887DK3 05K3288DK3 05K3318DK3 05K3905DK3 05K3908DK3 05K3933DK3 05K3934DK3 05K4138DK3 05K4172DK3 05K4173DK3 05K4174DK3 05K4175DK3 05K4456DK3 05K4468DK3 05K4604DK3 05K4605DK3 05K4606DK3 08K3126DK3 07K1907DK3 07K1913DK3 07K3598DK3 08K3111DK3 05K0843DK3 05K2933DK3 05K3909DK3 05K4141DK3 05K4176DK3 06K2270DK3 05K3329DK3 Assay Protocol ELISA Plate Antigens Blocking Reagent Samples Conjugate Reagents Multisorp (Nunc, USA) DENV rDIII proteins (serotypes 1, 2, or 4) coated on each individual Multisorp plate, i.e. one protein per plate. PBST with % skimmed milk (Appendix 4B) DENV patient sera (tested in duplicates) Anti-(human)IgM-Biotin conjugate (BD Pharmigen, USA) Concentration N.A. Volume N.A. 2.5 µg/ml 50 µl Neat concentration 100 µl 1:100 dilution 100 µl µg/ml concentration 100 µl 281 Conjugate Substrate Stop Solution or Anti-(human)IgG-Biotin conjugate (BD Pharmigen, USA) Strepavidin-HRP conjugate (Chemicon, USA) TMB one solution (Promega, USA) H2SO4 (Merck, Germany) 1:5000 dilution 100 µl Neat concentration 0.5 M concentration 100 µl 100 µl (iii) Mean Test Results for Tetravalent DENV rDIII iELISA-(BS) against IgM or IgG. Tetravalent DENV rDIII iELISA(BS) IgM Day Day Day 21 A 0.128 0.049 3.204 B 0.163 1.284 3.232 C 0.157 1.287 3.243 D 0.223 0.704 0.536 E 0.235 3.215 3.248 F 0.268 0.907 1.230 G 0.152 0.613 2.476 H 0.272 3.202 1.242 I 0.182 0.476 2.855 J 0.249 1.349 1.187 K 0.359 0.943 3.204 L 0.218 3.261 2.862 M 0.123 1.275 3.228 N 0.108 0.475 0.773 O 0.203 1.408 3.158 P 0.155 1.496 2.388 Q 1.111 2.759 1.534 R 0.174 0.411 3.205 S 0.240 0.602 0.473 T 0.114 0.120 0.472 U 0.155 0.457 0.340 V 0.255 0.491 1.449 W 0.194 0.998 0.674 X 0.187 0.276 3.127 Y 0.130 1.224 0.198 Z 0.109 0.462 0.254 AA 0.137 0.487 0.415 AB 0.176 0.751 0.930 AC 0.307 0.317 0.634 AD 0.154 0.302 0.434 Neg 0.225 Blank 0.116 Cut- off for borderline positive: Neg x 1.5 = 0.338 Cut- off for positive: Neg x = 0.45 Patient Tetravalent DENV rDIII iELISA-(BS) IgG Day Day Day 21 0.370 0.050 3.041 0.457 0.765 3.259 1.264 1.242 3.286 0.695 3.249 3.269 0.373 0.684 3.266 1.369 2.253 3.299 0.497 0.866 3.148 0.759 3.203 2.738 2.990 3.188 3.281 0.305 0.433 3.174 0.363 0.536 3.284 0.822 1.571 3.242 0.735 1.236 3.277 1.082 1.140 3.072 1.996 2.419 3.234 0.525 0.878 3.219 3.156 3.037 2.955 3.137 3.115 3.263 1.080 3.280 3.261 1.885 2.500 3.201 2.263 3.268 3.288 0.308 0.429 1.622 1.670 1.981 1.667 2.619 2.734 2.785 1.235 3.265 3.289 1.288 2.079 1.693 1.970 2.820 2.516 0.507 0.912 1.661 0.596 0.535 1.058 2.473 3.161 3.184 0.701 0.084 Cut- off for borderline positive: Neg x 1.5 = 1.052 Cut- off for positive: Neg x = 1.402 282 (iv) Test Results - Panbio Units for Panbio IgM Capture ELISA and IgG Indirect ELISA. Patient A B C D E F G H I J K L M N O P Q R S T U V W X Y Z AA AB AC AD Pos Neg Calibrator Calibration factor Panbio Units* Panbio IgM Capture ELISA Panbio IgG Indirect ELISA Day Day Day 21 Day Day Day 21 3.97 49.27 49.67 1.52 33.30 38.41 5.58 48.28 49.34 1.33 2.21 34.46 2.59 40.27 49.53 1.97 2.17 35.53 2.95 37.44 23.86 27.67 37.82 38.07 7.93 48.88 49.81 2.43 33.74 38.69 5.99 44.54 48.13 16.36 36.19 38.92 3.97 27.96 49.20 6.66 16.83 38.05 9.94 47.08 39.37 36.33 38.52 38.57 3.27 8.06 49.32 1.26 1.45 35.11 3.98 24.60 42.26 2.29 3.07 34.88 5.95 19.35 49.66 1.90 2.34 36.21 17.09 49.41 49.26 0.76 1.87 35.68 8.30 47.61 49.40 1.31 1.33 35.91 9.99 52.27 55.72 33.70 34.93 35.80 4.51 42.54 48.56 11.32 35.84 37.42 4.30 45.88 48.93 0.93 1.24 33.50 4.57 61.67 55.87 8.18 34.09 35.66 31.85 34.89 49.09 3.16 2.86 29.34 5.14 25.62 22.33 34.58 39.00 39.13 7.33 22.92 42.74 29.72 37.18 38.49 2.78 40.90 24.83 34.43 39.41 39.31 4.10 4.39 43.98 2.82 2.64 33.60 5.91 65.35 62.07 1.49 3.54 31.34 3.35 6.40 56.82 2.25 3.33 34.93 4.11 43.31 18.71 10.02 39.31 38.79 3.50 53.72 38.85 14.57 19.87 34.94 6.05 47.49 56.19 9.97 34.00 35.45 3.33 18.21 49.76 1.93 2.57 35.54 4.65 29.51 48.51 0.98 2.96 30.69 4.22 50.22 71.52 1.17 4.23 35.20 O.D. 450 nm Spectrophotometric Reading 1.514 1.999 0.166 0.695 0.506 1.171 1.30 0.68 Panbio Units Calculation: Panbio Unit = 10 x Sample absorbance / (Absorbance of Calibrator x Calibration Factor) Interpretation of results: Negative: < Borderline: – 11 Positive: > 11 * Mean O.D. 450 nm absorbance reading for samples were calculated as Panbio units according to the formula provided by the manufacturer. 283 G. Effect of E. coli extract on reduction of background for human tetravalent IgG DENV rDIII iELISA-(BS). (i) Assay Protocol ELISA Plate Antigens Blocking Reagent Sample Conjugate Conjugate Substrate Stop Solution (ii) Reagents Multisorp (Nunc, USA) DENV1 rDIII protein PBST with % skimmed milk (Appendix 4B) plus 0. 0.25, 0.5 or mg/ml of E. coli extract. Serum from Patient O Day 21 (Serum number: 05K4468DK3) Anti-(human)IgG-Biotin conjugate (BD Pharmigen, USA) Strepavidin-HRP conjugate (Chemicon, USA) TMB one solution (Promega, USA) H2SO4 (Merck, Germany) Concentration N.A. 2.5 µg/ml Volume N.A. 50 µl Neat concentration 100 µl 1:100 dilution 100 µl µg/ml concentration 100 µl 1:5000 dilution 100 µl Neat concentration 0.5 M concentration 100 µl 100 µl Test Results E. coli Extract added µg/ml 0.25 0.5 Mean Positive Value/ O.D. 450 nm 3.277 + 0.087 3.365 + 0.103 3.329 + 0.088 3.235 + 0.004 Mean Negative Value/ O.D. 450 nm 0.967 + 0.013 0.398 + 0.016 0.385 + 0.011 0.309 + 0.006 Significance of Test* (p-value) 3.55E-04 2.98E-04 2.48E-04 1.21E-09 * Calculated according to Student’s T-Test to determine statistical significance of results (p-value < 0.05). 284 [...]... 138 Table 4.11 Z-factor calculations for monovalent DIII iELISA-(BS) and tetravalent DIII iELISA-(BS) ………………………… … 143 Table 4.12 PRNT50 results obtained for DENV3 or 4 DIII-specific antibodies against respective viruses ……………………… 152 Table 4.13 Comparison of tetravalent DENV rDIII iELISA-(BS) IgM and IgG platforms against Commercial Panbio IgM and IgG serological assays ………………………………… ……… 158 Table... glycoprotein; and seven non-structural (NS) proteins: NS1, NS 2a, NS2b, NS3, NS 4a, NS4b and NS5 (Fig 1. 1A) (Clyde et al., 2006; Mackenzie et al., 2004) The envelope protein comprises 3 regions: Domain I, Domain II and Domain III (Fig 1.1B) Domain I is the central domain, Domain II is the dimerization and fusion domain, while Domain III (DIII) is an immunoglobulin-like receptor binding domain (Mukhopadhyay et al.,... encephalitis virus (TBEV) and West Nile virus (WNV) Dengue fever is categorized by rapid onset of fever, severe headache, retroorbital pain, gastrointestinal discomfort and rashes (Martina et al., 2009) Minor haemorrhagic manifestations such as petechiae formation may be observed Patients suffering from DHF generally experience severe haemorrhage due to increased vascular leakage and thrombocytopenia,... improved platforms for dengue diagnosis and hybridoma development using dengue envelope Domain III antigen …………………………… ……………………… 228 xix ABBREVIATIONS < Less than ºC Degrees Celsius μg Microgram μl Microlitre % Percent xg Times gravitational force ADE Antibody-dependent enhancement bp Base pairs BALB/C Bagg Albino Strain C BCIP/NBT 5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium BHK Baby hamster... ……… 295 x Summary Dengue is a mosquito-transmitted viral disease of global importance By exploiting the immunogenic properties of dengue virus (DENV) envelope recombinant Domain III (rDIII) proteins, several innovative platforms were established for research and diagnosis of dengue infection In this study, a metal affinity membrane chromatography method for rapid purification of DENV rDIII proteins... is an important immunogen for the development of a prospective protein subunit vaccine and also a prospective diagnostic reagent for improved clinical diagnosis of dengue infection Although to date, there is no DIII protein-based diagnostic assay available commercially, many in-house tests have demonstrated the possibility of using DIII protein as a reagent to serologically detect presence of DIII-specific... DIII-specific antibodies in dengue patient sera (Pattnaik et al., 2007; Tripathi et al., 2008) 2 A B Fig 1.1 (A) Translation of viral RNA yields a single polypeptide that is further processed by cellular and viral proteases, generating three structural and seven non-structural proteins Picture adapted from Whitehead et al., 2005 (B) Structure of the envelope protein dimer of a mature dengue virus particle Domain. .. illustrated in red, Domain II in yellow and Domain III in blue The envelope protein is modelled from DENV3 and illustrated here according to a two-fold symmetry axis (top and side-view) Picture adapted from Modis et al., 2005 3 1.2 Structural studies on flavivirus envelope and DIII protein A huge step forward in the field of flavivirus research was made when the structure of flavivirus major envelope. .. Validation of DENV rDIII iELISA-(BS) platforms 272 Appendix 8 A membrane chromatography method for the rapid purification of recombinant flavivirus proteins Protein Expr Purif 74:129-37……………………………… 285 Appendix 9 Book Chapter: Dengue envelope domain III protein: properties, production and potential applications in dengue diagnosis In: Dengue virus: detection, diagnosis and control Chapter 3 Uncorrected... of the spatial arrangement between the glycans on the envelope proteins This caused hidden epitopes to be exposed and further attacked by neutralizing antibodies, leading to increased viral neutralization (Lok et al., 2008) DENV envelope- specific MAbs are able to block virus entry into cells (Crill & Roehrig, 2001) MAbs that are specific against Domains I (IB4C-2, 4A5 C-8, 2B 3A- 1 and 9A4 D-1) and II (6B6C-1, . NATIONAL UNIVERSITY OF SINGAPORE 2010 ESTABLISHING IMPROVED PLATFORMS FOR DENGUE DIAGNOSIS AND HYBRIDOMA DEVELOPMENT USING DENGUE ENVELOPE DOMAIN III ANTIGEN i PUBLICATIONS AND. ESTABLISHING IMPROVED PLATFORMS FOR DENGUE DIAGNOSIS AND HYBRIDOMA DEVELOPMENT USING DENGUE ENVELOPE DOMAIN III ANTIGEN MELVIN TAN LIK CHERN NATIONAL UNIVERSITY. cell assay: Screening by microengraving and isolating by automatic micromanipulator for hybridomas secreting antibodies against dengue envelope domain III proteins. 10 th Nagasaki-Singapore