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 in Preclinical 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 Standard Curve
• 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 Scale Discovery
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- Meso Scale
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 -
Multiplex Luminex 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 and cons
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 drug development 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
