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xix | |
| Abbreviations |
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xxvii | |
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Preparation of rodent monoclonal antibodies by In vitro somatic hybridization |
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1 | (24) |
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Introduction and strategy |
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1 | (2) |
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Choice of host for immunization and myeloma for cell fusion |
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3 | (1) |
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4 | (1) |
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Preparation of antigen for immunization |
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5 | (2) |
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5 | (1) |
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Antigens expressed on live cells |
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5 | (1) |
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6 | (1) |
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7 | (1) |
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7 | (2) |
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Generation of immune spleen cells |
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7 | (1) |
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Immunization via the Peyer's patches of rats |
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7 | (2) |
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Growth of myeloma cell lines |
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9 | (1) |
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Preparation of cells for fusion |
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10 | (1) |
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11 | (1) |
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Screening hybridoma culture supernatants for specific antibody |
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12 | (6) |
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Antigen coated multiwell plates |
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12 | (4) |
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16 | (1) |
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17 | (1) |
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18 | (1) |
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Characterization and use of the antibodies obtained |
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19 | (6) |
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20 | (1) |
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20 | (1) |
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Specific immunoprecipitation |
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21 | (2) |
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23 | (2) |
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Preparation of recombinant antibodies from immune rodent spleens and the design of their humanization by CDR grafting |
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25 | (33) |
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25 | (1) |
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Preparation of mouse spleen |
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26 | (2) |
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Isolation of total RNA from spleen |
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28 | (2) |
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30 | (1) |
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Reverse transcriptase reaction |
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31 | (2) |
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Primary PCR of antibody genes |
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33 | (4) |
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37 | (2) |
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Assembly of VH and VK gene fragments with linker DNA |
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39 | (2) |
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Assembly of single chain Fv antibody fragments |
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41 | (2) |
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Reamplification of assembled scFv DNA |
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43 | (1) |
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Restriction enzyme digestion of assembled scFv |
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44 | (2) |
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Purification of pHEN-1 vector by equilibrium centrifugation in CsCI ethidium bromide gradients |
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46 | (3) |
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Restriction digestion of the phage display vector, pHEN-1 |
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49 | (1) |
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Ligation of pHEN-1 and insert antibody scFv |
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50 | (2) |
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Preparation of electroporation competent E. coli TG1 strain cells |
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52 | (1) |
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53 | (2) |
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Analysis of recombinant clones from the library |
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55 | (3) |
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57 | (1) |
| Appendix |
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58 | (405) |
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The design of the humanized antibody |
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58 | (9) |
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59 | (5) |
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64 | (3) |
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Selection of antibodies from phage libraries of immunoglobulin genes |
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67 | (24) |
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67 | (1) |
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Preparation and storage of phage library stocks |
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68 | (1) |
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68 | (1) |
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69 | (1) |
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Maintenance of bacterial stocks and titration of phage preparations |
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69 | (2) |
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Selection of phage libraries on purified, immobilized antigen |
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71 | (3) |
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Immobilization of antigen on immunotubes |
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71 | (1) |
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72 | (1) |
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Storage and rescue of the phagemid population after selection |
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72 | (1) |
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Choice of number of selection rounds |
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73 | (1) |
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Selection of phage libraries on biotinylated antigen |
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74 | (2) |
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74 | (1) |
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Selections using biotinylated antigen |
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75 | (1) |
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76 | (2) |
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77 | (1) |
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78 | (1) |
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Screening the output of cell surface selections |
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78 | (1) |
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78 | (5) |
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Proximity selection using an existing antibody |
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80 | (1) |
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Proximity selection using natural ligands |
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81 | (2) |
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83 | (1) |
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Screening of selected phage |
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83 | (4) |
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83 | (4) |
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87 | (1) |
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Soluble scFv production and purification |
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87 | (4) |
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89 | (2) |
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ARM complexes for in vitro display and evolution of antibody combining sites |
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91 | (20) |
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91 | (2) |
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Ribosome display methodology |
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93 | (12) |
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93 | (1) |
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Primer design and single chain antibody (VH/K) construction for ARM display |
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93 | (3) |
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Generation of ARM complexes by coupled transcription/translation in vitro |
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96 | (2) |
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Antigen selection of ARM complexes |
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98 | (2) |
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Recovery and amplification of DNA from antigen-selected ARM complexes |
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100 | (3) |
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Further ARM cycles and cloning |
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103 | (1) |
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Analysis of clones encoding antibodies by ARM display |
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104 | (1) |
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105 | (2) |
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105 | (1) |
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Selection of DB3R VH/K from libraries |
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106 | (1) |
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107 | (1) |
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107 | (1) |
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107 | (1) |
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107 | (4) |
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109 | (2) |
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Human monoclonal antibodies to blood group antigens |
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111 | (14) |
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111 | (1) |
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General equipment and reagents required |
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112 | (1) |
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113 | (1) |
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Preparation of lymphocytes |
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113 | (1) |
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EBV transformation of B cells |
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114 | (3) |
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115 | (1) |
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EBV transformation of B cells and growth of B-LCL |
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116 | (1) |
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Selection of antigen-specific B cells by rosetting |
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117 | (1) |
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Fusion of B-LCL with murine myeloma cells (P3X63Ag8.653) |
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118 | (2) |
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120 | (1) |
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121 | (1) |
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Cryopreservation of cells |
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122 | (3) |
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123 | (2) |
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Laboratory based methods for small scale production of monoclonal antibodies |
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125 | (24) |
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125 | (1) |
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125 | (4) |
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125 | (2) |
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127 | (2) |
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129 | (2) |
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Tissue culture plates and flasks |
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129 | (1) |
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Specialized (scale-up) culture systems |
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130 | (1) |
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131 | (4) |
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131 | (2) |
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133 | (1) |
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134 | (1) |
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Dynamic (non-stirred) culture systems |
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135 | (2) |
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135 | (1) |
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136 | (1) |
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Perfusion (high cell density) systems |
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137 | (6) |
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Hollow fibre (Acusyst) culture |
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138 | (1) |
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138 | (1) |
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Membrane culture systems (miniPERM) |
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139 | (1) |
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140 | (2) |
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Fluidized bed bioreactors |
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142 | (1) |
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Harvesting and concentration |
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143 | (2) |
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Harvesting and clarification |
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143 | (1) |
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144 | (1) |
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145 | (4) |
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146 | (3) |
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Isolation and purification of monoclonal antibodies from tissue culture supernatant |
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149 | (32) |
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Objectives of antibody purification |
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149 | (1) |
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Essential information about the antibody |
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150 | (7) |
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Problems with purifying antibodies from culture supernatant |
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157 | |
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Low concentration of antibody in culture supernatant compared with serum |
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151 | (1) |
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Potential contamination by bovine IgG |
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152 | (1) |
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Equipment for antibody purification |
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153 | (3) |
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Equipment for chromatography |
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154 | (2) |
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156 | (2) |
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158 | (4) |
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Reuse of affinity columns |
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159 | (1) |
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Choice of affinity ligand |
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160 | (2) |
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Immunoaffinity purification |
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162 | (1) |
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Ion exchange chromatography |
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162 | (4) |
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Cation exchange chromatography |
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163 | (2) |
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Anion exchange chromatography |
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165 | (1) |
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Immobilized metal affinity chromatography (IMAC) |
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166 | (2) |
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Size exclusion chromatography (SEC) |
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168 | (1) |
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Hydrophobic interaction chromatography |
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169 | (3) |
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Other chromatographic methods |
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172 | (1) |
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173 | (1) |
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Purification of antibody fragments |
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173 | (1) |
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173 | (1) |
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Analysis of purity and activity |
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174 | (5) |
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Antibody concentration and purity |
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174 | (3) |
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177 | (2) |
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179 | (2) |
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179 | (1) |
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180 | (1) |
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Antibody production in plants |
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181 | (26) |
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181 | (1) |
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Expressing recombinant proteins in plants |
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181 | (5) |
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182 | (1) |
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182 | (3) |
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Modified viruses for transient expression in plants |
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185 | (1) |
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Glycosylation of recombinant proteins in transgenic plants |
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185 | (1) |
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186 | (10) |
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187 | (1) |
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Agrobacterium tumefaciens-mediated transformation |
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188 | (4) |
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Principles of particle bombardment |
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192 | (4) |
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Plant transformation techniques |
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196 | (5) |
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Agrobaderium-mediated transformation of tobacco |
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196 | (3) |
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Transformation of wheat by micro-projectile bombardment |
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199 | (2) |
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Screening regenerated plantlets for immunoglobulin chain production |
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201 | (1) |
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Self and cross-fertilization of transgenic plants |
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202 | (1) |
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The overall advantages in expressing antibodies in plants |
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202 | (5) |
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203 | (4) |
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Radiolabelling of monoclonal antibodies |
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207 | (30) |
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207 | (1) |
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The choice of radionuclide |
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207 | (1) |
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In vitro applications of radiolabelled antibodies |
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207 | (10) |
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208 | (9) |
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In vivo applications of radiolabelled antibodies |
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217 | (17) |
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218 | (1) |
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219 | (4) |
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223 | (8) |
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231 | (1) |
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2 | (233) |
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Other therapeutic radionuclides |
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233 | (1) |
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Antibody fragments and genetically engineered constructs |
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234 | (1) |
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Quality control of radiolabelled antibodies |
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234 | (3) |
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235 | (2) |
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Non-radioactive antibody probes |
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237 | (10) |
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237 | (1) |
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238 | (1) |
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General aspects of labelling |
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238 | (2) |
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238 | (1) |
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239 | (1) |
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239 | (1) |
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Purification and storage of the labelled antibody |
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239 | (1) |
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240 | (2) |
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Labelling with fluorescein |
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242 | (1) |
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243 | (1) |
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Labelling with digoxigenin (DIG) |
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244 | (1) |
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Evaluation of labelled monoclonal antibodies |
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244 | (3) |
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246 | (1) |
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Immunogold probes for light and electron microscopy |
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247 | (18) |
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247 | (2) |
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Pre-embedding labelling for SEM and TEM |
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249 | (2) |
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251 | (4) |
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Progressive lowering of temperature embedding (PLT) |
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255 | (1) |
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256 | (2) |
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Immunocytochemistry of resin sections |
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258 | (3) |
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261 | (1) |
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262 | (3) |
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262 | (3) |
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Characterization of cellular antigens using monoclonal antibodies |
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265 | (32) |
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265 | (1) |
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Initial characterization of monoclonal antibodies |
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266 | (1) |
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Preparation of cell lysates |
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266 | (4) |
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Detection of cellular antigens by immunoblotting |
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270 | (13) |
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Separation of proteins by SDS-PAGE |
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270 | (3) |
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Electrotransfer of proteins from gels to membranes |
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273 | (4) |
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Blocking non-specific binding sites on the membrane |
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277 | (1) |
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Incubation with the primary monoclonal antibody |
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277 | (1) |
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Detection of secondary antibodies and quantitation of signals |
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277 | (5) |
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Troubleshooting problems encountered during immunoblotting |
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282 | (1) |
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Analysis of the electrophoretic properties of polypeptides by 2D PAGE |
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283 | (4) |
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Analysis of subcellular localization of proteins |
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287 | (1) |
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Immunofluorescence staining |
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287 | (1) |
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Subcellular fractionation |
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287 | (1) |
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Resolution of oligomeric complexes by non-denaturing PAGE |
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287 | (2) |
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Detection of cellular antigens by immunoprecipitation |
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289 | (6) |
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Analysis of physiological interactions by immunoprecipitation |
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292 | (1) |
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Immunoprecipitation under disruptive conditions |
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292 | (1) |
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Troubleshooting high background signals |
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292 | (3) |
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295 | (1) |
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Further applications of monoclonal antibodies in protein characterization |
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296 | (1) |
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296 | (1) |
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296 | (1) |
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297 | (22) |
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297 | (1) |
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297 | (4) |
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297 | (1) |
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Selecting an assay standard |
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297 | (2) |
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299 | (1) |
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Sample preparation and dilution |
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299 | (1) |
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300 | (1) |
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Solid support and separation options |
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301 | (2) |
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301 | (1) |
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Precipitation from solution |
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302 | (1) |
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303 | (1) |
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Labelling and detection of antibody or antigen |
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303 | (6) |
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303 | (2) |
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305 | (1) |
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Labelling with lanthanide fluorophores |
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306 | (1) |
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307 | (1) |
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308 | (1) |
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309 | (10) |
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Competitive binding immunoassay |
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309 | (2) |
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311 | (4) |
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315 | (1) |
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316 | (2) |
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318 | (1) |
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Immunoaffinity chromatography of macromolecules |
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319 | (22) |
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319 | (1) |
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320 | (10) |
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320 | (1) |
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320 | (1) |
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321 | (8) |
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Monitoring of coupling efficiency |
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329 | (1) |
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329 | (1) |
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330 | (1) |
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330 | (1) |
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331 | (3) |
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334 | (1) |
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334 | (1) |
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334 | (1) |
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335 | (1) |
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335 | (1) |
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335 | (1) |
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Analysis of purified antigen |
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336 | (1) |
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337 | (4) |
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338 | (3) |
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Immunochemical detection of BrdUrd labelled nuclei for monitoring cell kinetics |
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341 | (14) |
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341 | (1) |
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Basic concepts in cell kinetics |
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341 | (2) |
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Background to the BrdUrd flow cytometry technique |
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343 | (2) |
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In vitro BrdUrd incorporation |
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345 | (1) |
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345 | (1) |
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346 | (3) |
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346 | (2) |
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348 | (1) |
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349 | (1) |
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Examples of data and analysis |
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350 | (2) |
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352 | (3) |
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352 | (3) |
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Immunofluorescence microscopy |
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355 | (16) |
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355 | (1) |
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355 | (8) |
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Fluorescent labelling of antibodies |
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356 | (2) |
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Fixation, permeabilization, and staining of cells |
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358 | (5) |
|
Microscopy and imaging equipment |
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363 | (5) |
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Microscopes and associated hardware |
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363 | (2) |
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Imaging detectors for 3D sectioning microscopy |
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365 | (2) |
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Data analysis, image processing, and data presentatio |
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367 | (1) |
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Immunolabelling and visualization in living cells |
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368 | (2) |
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Labelling by microinjection of fluorescent antibodies |
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368 | (1) |
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Microscope set-up for live cell IFM |
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369 | (1) |
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Examples of IFM in fixed cells |
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370 | (1) |
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370 | (1) |
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FACS analysis of clinical haematological samples in transplantation for cancer |
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371 | (20) |
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371 | (1) |
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Fluorescence activated cell analysis |
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371 | (6) |
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371 | (2) |
|
Choice of antibodies, reagents, and general maintenance of the FACS analyser |
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373 | (1) |
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374 | (2) |
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Preparation of clinical samples |
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376 | (1) |
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376 | (1) |
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Calibration of the analyser |
|
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377 | (1) |
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Assessment of the progenitor cell content in bone marrow (BM) and peripheral blood stem cell (PCSC) harvests |
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377 | (6) |
|
Rationale for measuring the progenitor cell content of BM and PBSC harvests for transplantation after intensive therapy for cancer |
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377 | (1) |
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378 | (1) |
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Collecting peripheral blood stem cell (PBSC) harvests for CD34+ quantitation |
|
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378 | (1) |
|
Collecting bone marrow (BM) harvests for CD34+ quantitation |
|
|
379 | (3) |
|
Quantitation of purified CD34+ cells from PBSC harvests from patients with chronic lymphocytic leukaemia |
|
|
382 | (1) |
|
Analysis of lymphocyte subsets in peripheral blood and bone marrow harvests from unrelated donors |
|
|
383 | (8) |
|
T cell depletion of paediatric matched (MUD) and unmatched (U-UD) bone marrow (BM) harvests from unrelated donors |
|
|
383 | (1) |
|
Analysis of lymphocyte subsets after transplantation or autologous rescue |
|
|
384 | (1) |
|
Measurement of intracellular cytokines in mononuclear cells (MNC) and T cells after transplantation or autologous rescue |
|
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384 | (5) |
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389 | (2) |
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Immunocytochemical staining of cells and tissues for diagnostic applications |
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391 | (20) |
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391 | (1) |
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Tissue and cell substrates |
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391 | (1) |
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391 | (1) |
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391 | (1) |
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Fixation and processing for paraffin wax embedded tissues |
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392 | (1) |
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392 | (1) |
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392 | (1) |
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393 | (1) |
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393 | (2) |
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393 | (1) |
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394 | (1) |
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395 | (1) |
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Immunocytochemical methodology |
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395 | (7) |
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396 | (2) |
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398 | (4) |
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402 | (3) |
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402 | (2) |
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404 | (1) |
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405 | (2) |
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406 | (1) |
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407 | (4) |
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407 | (1) |
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407 | (1) |
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External quality assurance scheme (EQA) |
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407 | (1) |
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407 | (1) |
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Suggested Further Reading |
|
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407 | (1) |
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408 | (3) |
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Detection of chemically modified DNA in lymphocytes of patients undergoing chemotherapy |
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411 | (20) |
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411 | (3) |
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Types of modifications to which antibodies can be produced |
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412 | (1) |
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Applications of antibodies against modified DNA |
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412 | (1) |
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Merits of raising antibodies against modified mono/dinucleotides versus modified polymeric DNA |
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412 | (2) |
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Requirements for detection of low frequencies of DNA modifications |
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414 | (1) |
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Production of appropriate antibodies |
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414 | (1) |
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414 | (1) |
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415 | (1) |
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ELISA techniques for quantification of DNA modifications |
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415 | (11) |
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416 | (2) |
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418 | (1) |
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419 | (7) |
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Staining for adducts in individual cells |
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426 | (5) |
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Staining techniques: conventional versus agarose embedded DNA |
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426 | (1) |
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Staining agarose embedded DNA |
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427 | (2) |
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429 | (2) |
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Monoclonal antibody therapy in organ transplantation |
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431 | (18) |
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431 | (1) |
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The challenge of antibody therapy in humans |
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432 | (3) |
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Immunological tolerance versus immunosuppression |
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432 | (1) |
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Depletion versus non-depletion |
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433 | (1) |
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Avoiding the antiglobulin response |
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434 | (1) |
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Can monoclonal antibodies ever be used to induce tolerance in humans? |
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434 | (1) |
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Animal models of transplantation and mAb therapy: defining the problem |
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435 | (1) |
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Which mAbs for organ transplantation |
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436 | (13) |
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437 | (3) |
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Monoclonal antibodies to CD4 and CD8 |
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440 | (2) |
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Monoclonal antibodies to CD2S (IL-2 receptor) |
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442 | (1) |
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Blockade of co-stimulation through CD4O and CD28 pathways |
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443 | (1) |
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444 | (1) |
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445 | (1) |
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445 | (4) |
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Monoclonal antibody therapy In rheumatoid arthritis |
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449 | (14) |
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449 | (1) |
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Pathogenesis of rheumatoid arthritis |
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449 | (1) |
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Treatment strategies in rheumatoid arthritis |
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450 | (1) |
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Therapeutic monoclonal antibodies |
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|
451 | (1) |
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Monoclonal antibodies in rheumatoid arthritis |
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|
452 | (6) |
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Anti-cytokine mAbs in rheumatoid arthritis |
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|
452 | (2) |
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Anti-adhesion molecule mAbs |
|
|
454 | (1) |
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455 | (3) |
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458 | (5) |
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|
459 | (1) |
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|
459 | (4) |
| List of suppliers |
|
463 | (6) |
| Index |
|
469 | |
Other versions by this Author