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Immunoassay development
- 1. Immunoassays in PreclinicalDevelopmen Yu-Lu Ma Apr. 28th, 2009
- 2. Overview ■ About me ■Assay Types and Platforms ■ Enzyme-Linked Immunosorbent Assay(ELISA) ■ Meso Scale Discovery (MSD) ■ Multiplex-Luminex Technologies ■ Summary
- 3. My Background ■ M.S.in Biotechnology, Northeastern University ■ 3 years industry experience ■ Bioanalytical Assay Development ■ Hobbies ■ Cooking, gardening, hiking, golf
- 4. Assay Types inPreclinical and Clinical Studies Meso Scale Discovery
- 5. Pharmacokinetics (PK) Assay-ELISA •Measure drug exposure in biological matrices • Select capture and detection antibody specific • Endogenous Inhibitor or Structure-Similar Molecules • Detection : Enzyme-conjugated • Signal: Colormetric Antigen Dependent Antigen Independent Ag Capture antibody Drug Enzyme- linked Isotype Specific Antibody Drug Enzyme-linked Isotype Specific Antibody Substrate Addition Substrate Addition
- 6. PK Assay StandardCurve • Unknown sample interpolated from the standard curve • Hook Effect Cause Underestimation of drug exposure • Make sure sample in the detection range AAPS Journal. 2007; 9(2): E156-E163
- 7. Electrochemiluminescence (ECL) Platform-Meso ScaleDiscovery Ruthenium • Carbon coated plate, more surface area • Ruthenium-conjuated detector • Electro-current initiate reaction and emit light as signal • Detection: Ruthenylated conjugate • Signal: ECL counts (light) Meso Scale Discovery
- 8. Immunogenicity Assay- MesoScale Discovery • Measure Anti-drug antibody • Use drug as capture and detection reagents • Homogenous Incubation • Improve sensitivity in the presence of free drug Meso Scale Discovery Free drug Anti-Drug Antibody
- 9. Free Drug Tolerance •Six sets of standard curves spiked with various amount of free drug • No effect on assay for free drug up to 300 ng/ml Meso Scale Discovery
- 10. Pharmacodynamics (PD) Assays ■Evaluate what drug does to the body - Biomarker Research ■ Biomarker Definition- A factor that is objectively measured and evaluated as an indicator of normal biological processes, pathogenic processes, or pharmacologic responses to a therapeutic intervention (Biomarkers Definitions Working Group, 2001) ■ Example ■ Prostate Specific Antigen (PSA) for screening and monitoring prostate cancer; ■ HER2 overexpression for anti VEGF antibody responders ■ Potential use ■ Target Discovery and Validation ■ Lead Discovery and Optimization ■ Preclinical Studies ■ Clinical Trials
- 11. Biomarker Methodologies ■ Genomic ■Evaluate the over expression of certain gene(s) mutations, eg. qPCR. ■ Proteomic ■ Evaluate the protein levels in diseased v.s. normal states, eg. Luminex Technology
- 12. Biomarker Analysis - MultiplexLuminex Technology ■ Bead based assay on flow cytometer ■ Each bead is identified with dye ■ Capture antibody coated on beads to analyze cytokines, growth factors ■ Detection antibody labeled with Phycoerthrin (PE) ■ 100 beads available ■ Versitle functions ■ Proteomics ■ Cellular Signaling Pathway ■ Genotyping Invitrogen corp PE Anti IL-8 IL-8 VEGF PE Anti VEGF
- 13. Platform pros andcons Methods Pros Cons ELISA ■ Economical ■ Well established ■ High throughput- automation ■ Time consuming, laborious MSD ■ High matrix concentration okay ■ Increased surface area for detection ■ Robust ■ Prepare conjugates, introduce variables ■ Conjugating may mask epitope ■ Reagent and Vendor- specific ■ Single Read Luminex ■ Multiplex high throughput ■ High sensitivity ■ In house customize beads ■ Beads handling tricky ■ Cross reactivity
- 14. Summary ■ Successful drugdevelopment relies on well established analytical assays meet regulatory requirement ■ Monitoring each parameter in assay development is critical to problem solving ■ Early biomarker profiling contributes to successful drug discovery and development
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