21 3 5 1) HPLC GC/MS LC/MS/MS 2), 3) genomics, proteomics, metabolomics 1. PK/TK 2. 3. ELISA Enzyme-Linked ImmunoSorbent Assay ECL Electrochemi-luminescence SPR Surface Plasmon Resonance ECL ECLSulfo-Tag ELISA ECLELISA ECL Fig. 1 Table 1 ECL PK 212
1. Buffer %Serum Table 1 Comparative results of ECL and ELISA assay ECL ELISA Abs 1. Sensitivity&range (mu/ml in human serum) 2.74~6 5 ~ 4.1 1 1 EPO Conc. (mu/ml) (a) ELISA Matrix effect (human serum/buffer ratio at LLOQ) Time (h) Sample expenditure (μl) 2.74/2.74 = 1 4 ~ 5 25 5/12.5 = 4 6 ~ 7 Fig. 1 ECL Signal Buffer %Serum.1 1 1 EPO Conc. (mu/ml) (b) ECL Calibration curve of EPO using ELISA (a) and ECL (b) PK/PD REOPRO Remicade 4) 9) ELISAECLSPR Table 2 EMA 1) FDA 11) White paper 12) Table 2 Comparison of immunogenicity assay ECL ELISA SPR Biacore Characteristics Applied plate assay Popular plate assay Real-time flow-based detection High sensitive Established method Label-free Pros Wide range High sensitive Can detect Low-affinity Abs (early immune response) Can detect Low-affinity Abs Inexpensive equipment Characterize detected Abs High throughput High throughput Not yet established High background Not yet established Cons May not detect all low-affinity Abs? Limited in ability to detect low-affinity Abs Moderate Sensitivity Expensive equipment Low throughput relatively Sensitivity (detection of low-affinity Abs) 57 ± 37 ng/ml 26 ± 92 ng/ml 39 ng/ml 212
EMA 1) ECLSPR ECLSPR ECL HCP, Host Cell ProteinHCP SPRG-CSF Host Cell HCP Host Cell Protein 13) HCP PMDA HCP HCP HCP FcProteinA/GHCP Sulfo-Tag ProteinA/G Fig. 2HCP ECL 39 2. 1 5 ng/mlfda 11) 25 5 ng/ml Fig. 3 Fig. 2 Anti HCP antibody HCP-Biotin Streptavidin Streptavidin coated plate for ECL SULFO-TAG labeled Protein A/G Assay principle of detection of anti-hcp antibodies ECL Signal Fig. 3 1e6 1 1 Ab Conc.(ng/mL) Calibration curve of anti HCP antibodies in 1% human serum SPR SPR 1 G-CSF.3925 µg/ml.6 4.1 SPR QC 112 117QC 117 IgMIgAIgE IgG 212
Response ( = baseline) QC IgMIgAIgE QC IgG 93.5 RU QC 129 RU Fig. 4 QC IgG QCIgG RU 13 12 1 9 8 7 6 5 4 3 2 Fig. 4 QC sample blank sample QC sample Serum sample Anti-IgM Anti-IgA Anti-IgE 3.4 RU 4.6 RU 13.2 RU 225 RU blank sample 118 RU 3.4 RU 6.5 RU 13.7 RU 93.5 RU Sensorgrams of isotyping Anti-IgG 129 RU 2 5 8 1 14 17 2 s Time ( = baseline) FCM DNA T Jurkat cell V V FCM Fig. 5 PI-A 1 3 1 4 1 5 Q1 Q2 Live cells Q3 Q4 1 2 mrna SNPs PCR ECL LC-MS/MS FCM GC- MS 14) 17) FCM PI-A Fig. 5 1 5 1 4 1 3 1 2 1 2 1 3 1 4 1 5 Annexin V FITC-A Live cells (a) Control cells. Q1 Q3 Late apoptotic cells Early apoptotic cells 1 2 1 3 1 4 1 5 Annexin V FITC-A (b) Vinblastine treated cells. Apoptosis detection by flow cytometry of (a) control and (b) vinblastine treated cells Q2 Q4 212
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