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Antigens to Detect Or Inhibit Antibodies to Knops (Kn) Blood Group System Antigens

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Antigens to Detect Or Inhibit Antibodies to Knops (Kn) Blood Group System Antigens PRODUCTION OF SOLUBLE RECOMBINANT COMPLEMENT RECEPTOR 1 (CR1) ANTIGENS TO DETECT OR INHIBIT ANTIBODIES TO KNOPS (KN) BLOOD GROUP SYSTEM ANTIGENS WENDY ETHERIDGE A thesis submitted in partial fulfilment of the requirements of the University of the West of England, Bristol for the Professional Doctorate in Biomedical Science Faculty of Applied Sciences, University of the West of England, Bristol October 2014 ABSTRACT The purpose of this study was to produce a reagent to use in investigation of antibodies directed against the Knops blood group system antigens. A novel reagent based on sr-proteins was produced and used in a new test to inhibit these antibodies. Current investigation of patients with alloantibodies directed against Knops blood group system antigens can be a difficult, time-consuming process and the provision of blood for transfusion of these patients can often be delayed. This is because these antibodies are hard to identify and the most commonly found anti-Knops antibodies react with most reagent or donor cells that they are tested with because the corresponding Knops antigens are found at high frequency in most populations. The presence of Knops related antibodies can mask underlying antibodies that are clinically significant. The Knops antigens are carried on Complement Receptor 1 (CR1) located on the red blood cell membrane. Two DNA constructs encoding different parts of CR1 termed long homologous repeat (LHR) C and D were used to transfect human embryonic kidney (HEK293) cells. The cells were grown in different culture systems. Cell culture supernatant containing soluble recombinant (sr)-LHRC or sr-LHR-D was harvested and purified by affinity gel chromatography. The production and purification processes were optimised in terms of protein yield and cost. i The resulting purified sr-LHR-C and sr-LHR-D proteins were used to create a novel reagent containing both proteins. This reagent was used in a new inhibition test based on an indirect antiglobulin technique using commercial gel cards. Using the reagent all examples of previously identified Knops antibodies were inhibited. In addition once these antibodies had been inhibited, underlying antibodies were then detected and identified in some samples. For the first time Knops specific antibodies can be detected and identified using one unique test. Any underlying clinically significant antibodies will be rapidly identified if present due to inhibition of the KN antibodies. Introduction of the inhibition test into nine NHSBT patient testing laboratories will reduce the time taken for investigation of these patients and make provision of blood for patients a safer, faster process. ii CONTENTS ABSTRACT ................................................................................................................. i CONTENTS .............................................................................................................. iii LIST OF TABLES ..................................................................................................... vii LIST OF FIGURES .................................................................................................. viii ABBREVIATIONS ...................................................................................................... x ACKNOWLEDGEMENTS ........................................................................................ xiii 1.0 INTRODUCTION ................................................................................... 1 1.1 Blood group antigens .............................................................................. 1 1.2 KN blood group antigens ........................................................................ 8 1.3 Antibody detection and identification .................................................... 9 1.4 Haemolytic transfusion reactions ......................................................... 15 1.5 Haemolytic disease of the foetus and newborn ................................... 18 1.6 Clinical significance of antibodies........................................................ 19 1.7 KN blood group antibodies ................................................................... 20 1.8 Recombinant antigens........................................................................... 24 1.9 Complement receptor 1 ......................................................................... 27 1.10 Sr-protein expression systems ............................................................. 30 1.11 Previous work on soluble CR1 .............................................................. 34 1.12 Aims and objectives .............................................................................. 38 1.13 Assay validation..................................................................................... 39 2.0 MATERIALS AND METHODS ............................................................ 40 2.1 DNA techniques ................................................................................ 40 2.1.1 Preparation of recombinant plasmid DNA for transfection................. 40 2.1.2 Agarose gel electrophoresis ................................................................. 41 2.1.3 Spectrophotometric analysis of p3xFLAG-LHR-C plasmid DNA ........ 41 2.1.4 3xFLAG-LHR-C and 3xFLAG-LHR-D plasmid DNA sequencing ......... 42 2.1.5 Transfection of HEK293 cells with p3XFLAG-CMV-LHR-C or p3XFLAG- CMV-LHR-D ............................................................................................ 43 2.2 Cell culture techniques ..................................................................... 44 2.2.1 Cloning of stably transfected HEK293 cells producing sr-LHR proteins ................................................................................................................ 44 2.2.2 Freezing cloned stably transfected cells for liquid nitrogen storage . 45 2.2.3 Recovery of cells from liquid nitrogen storage ................................... 46 2.2.4 Production of sr-LHR-C and sr-LHR-D in HEK293 cells ...................... 46 2.2.5 Transfected HEK293 cell culture in T175 flasks .................................. 47 2.2.6 Transfected HEK293 cell culture in spinner flasks .............................. 48 2.2.7 Transfected HEK293 cell culture in Hollow Fiber Bioreactors ............ 49 iii 2.3 Protein detection and purification techniques ............................... 52 2.3.1 ELISA for sr-LHR-C and sr-LHR-D protein detection........................... 52 2.3.2 Immunopurification of sr-3X-FLAG fusion proteins using anti-FLAG agarose affinity gel column .................................................................. 53 2.3.2.1 Acid elution .................................................................................................. 53 2.3.2.2 3XFLAG elution ............................................................................................ 54 2.3.3 Bicinchoninic acid protein assay .................................................... 54 2.3.4 SDS-PAGE analysis of sr-proteins ....................................................... 55 2.3.5 Semi-dry electro blotting (western blotting) from SDS-PAGE gels .... 55 2.3.6 Development of western blots .............................................................. 56 2.3.7 Protein staining of SDS-PAGE gels and western blots ....................... 57 2.3.8 Mass spectrometry analysis of sr-proteins .......................................... 57 2.4 Patient testing ................................................................................... 58 2.4.1 Clinical material ..................................................................................... 58 2.4.2 Ethical considerations ........................................................................... 58 2.4.3 Antigenicity ELISA ................................................................................. 59 2.4.4 Monoclonal antibody-specific immobilization of erythrocyte antigens assay ...................................................................................................... 60 2.5 Patient testing using serological techniques ................................. 60 2.5.1 Red cell reagents in CellStab ................................................................ 60 2.5.2 Column indirect antiglobulin test .................................................... 61 2.5.3 Inhibition of KN antibodies ................................................................... 62 2.6 Statistical methods used in this study ............................................ 62 3.0 SR-LHR-C AND SR-LHR-D PROTEIN PRODUCTION IN HEK293 CELLS ............................................................................................... 63 3.1 Introduction ....................................................................................... 63 3.2 Materials and Methods ...................................................................... 64 3.2.1 Primer design ......................................................................................... 64 3.2.2 Development of an ELISA for detection of sr-3XFLAG tagged LHR-C and LHR-D protein ................................................................................. 65 3.2.2.1 ELISA development method one ............................................................... 65 3.2.2.2 ELISA development method two................................................................ 67 3.2.2.3 ELISA development method three ............................................................. 68 3.2.3 Comparison of different cell culture
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