1. Bioinorganic and Immunoassay Group, Seoul Women’s University
Immunoassay V
Bioinorganic and Immunoassay Group, Seoul Women’s University

2. 학습한 내용 Ag Ab Ag-Ab reaction Immunoassay Designs
Coupling Method : Functional chemistry of Proteins
Bioconjugation with protein ( or small chemicals)
Monitoring methods of Labels
RIA(125I) / EIA(HRP, AKP, G6PDH) / FIA (FITC)
BLIA (Aequorin)
Heterogeneous IA / Homogeneous IA

3. 학습 할 내용
Immunoassay performance Measures (Assay Optimization / Standard curve generation / Assay Performance Evaluation / stability study)
Rapid Kit (On-site screening test)
Aptamer
DA / TC assay
Applications and a New Trend in immunoassay

4. References for this lecture
Chapter 9. Separation system in “The Immunoassay Handbook”
hCG MAIA cloneTM
FPIA : TDx autoanalyzer; Abbott Labs

5. Heterogeneous IA / Homogeneous IA

6. Heterogeneous IA Homogeneous IA 1. Choice of solid phase
2. General principles of protein binding to plastic surfaces
3. Coating conditions
4. ProLinker
5. Magnetic solid phase - hCG assay
Homogeneous IA
1. EMIT (Enzyme Multiplied Immunoassay Technique)
2. FPIA (Fluorescence Polarization ImmunoAssay)
3. EFC (Enzyme Fragment Complementation)

7. Heterogeneous IA 1. Choice of Solid Phase Beads Test tubes Wells
Microparticles
Magnetic microparticles
membranes

8. Various Micro-Well Plates
96 Wells
384 Wells
1536 Wells

9. High-Throughput Well Sensor

10. 2. General Principles of Protein binding to Plastic surfaces
Capture protein : Ab or appropriate Ag
Direct binding: conformational change
reduce the affinity for the analyte
Spacer Protein : Avidin , Protein A/G and ProLinkers

11. Passively adsorb onto glass or plastic surfaces
5-95 %
Binding of proteins to plastic can be very strong, primarily through hydrophobic forces.
Hydrophobic sites stick to the plastic.
Large proteins tends to bind more strongly.

12. 3. Coating conditions
The concentration of the protein in the coating solution has an effect on the speed of the coating process.
- 10~100ug/mL are typically used.
The amount of protein that can be bound is affected by the size of the protein and the extent of the coverage of the plastic surface.
- 0.5~1ug of IgG / cm2
Optimum binding pH of most antibodies
- at neutral or slightly alkaline pH
- for protein in general a pH near the pI is most effective.
Temperature can have a significant effect on binding rate.
Time is a key variable in the coating process.

13. Most current systems use microparticles - to increase available surface area
Have a common capture protein coated onto the plastic - Avidin or 2nd Ab or Protein A/G
Remaining available binding sites are blocked off by washing with buffer containing BSA. - nonspecific binding
Polystyrene - an ideal material
- cheap
- easily molded into a wide variety of shapes.
Covalent attachment of proteins
A uniform monolayer on the plastic

14. Solid supports
Membrane filters made with nitrocellulose, nylon or polyvinylidene difluoride
Polystylene film
Aldehyde or amine slides
Glass slides coated with poly-L-lysine, poly-acrylamide, BSA-N-hydroxysuccinmide
Glass slides coated with gold film and various derivatives of cellulose, collagen, dextran or SAM molecules

15. 4. Two Types of ProteoChip Base Plate - ProLinker
Type A
Type B
ProLinkerTM
Au film

16. ProLinker Supermolecular = calixarene + crown-5
Crown / ammonium cation ( Fc part)
Molecular recognition : self-assembled monolayer (SAM)
A type : - aldehyde , aminated slide glass
B type : - SH, Au coated glass slide

18. Protein Immobilization Process
STEP1
Formation of
crown SAMs
STEP2
Protein
Monolayer
STEP3
protein-
protein
interaction
Immobilized
capture proteins
Analyte
proteins
Au substrate
or glass substrate
Ammonium
Functions

19. 5. Magnetic solid phase - hCG assay
125I-Ab1(w)
125I-Ab2(ß)
Ab3(ß)-F
Sheep anti-F antiserum - █
hCG(w)
hCG(ß)
125I-Ab1(w) / hCG(w) / Ab3(ß)-F / Sheep anti-F antiserum -█
125I-Ab2(ß) / hCG(ß) / Ab3(ß)-F / Sheep anti-F antiserum - █

20. Homogeneous Immunoassay
1. EMIT (Enzyme Multiplied Immunoassay Technique)
2. FPIA (Fluorescence Polarization ImmunoAssay)
3. EFC (Enzyme Fragment Complementation)

21. 1. EMIT (Enzyme Multiplied Immunoassay Technique)
Enzyme activity
Limited E E
[Antigen]
Antigen-Enzyme conjugate
< Free antigen E E ×
% Inhibition
Limited
[Antigen]
Antigen-Enzyme conjugate
> Free antigen

22. 2. FPIA (Fluorescence Polarization ImmunoAssay)

24. 3. EFC (Enzyme Fragment Complementation)
To restore an enzyme function by supplying a correct or missing polypeptide.
Involves noncovalent, intracistronic interaction of polypeptide chains.
-galactosidase
(-D-galactosidase galactohydolyase), EC

25. Enzyme Fragment Complementation
 -D-galactosidase galactohydrolylase
a tetramer of identical monomers.
each monomer (1021 amino acid residues) is a single polypeptide chain (MW 116,349 per monomer)
Formation of an active -D-galactosidase by interaction of
enzymatically inactive fragment EA (enzyme acceptor )
an inactive fragment, ED (enzyme donor)
EA is formed by mutation in the  region
ED is a peptide containing amino acids lacking in the  acceptor, EA.
-Complementation reaction
Essentially irreversible (KD =107 M-1)
multistep mechanism

26. The Engineering of Inactive Deletion Mutantsp z y a
DNA
E. coli lac operon
transcription
RNA
translation
Substrate
Protein 1
1021
active ( 
-galactosidase)
enzyme
deletion
Product
Enzyme Donor (ED)
inactive
enzyme
Enzyme Acceptor (EA)
Substrate
Product EA
Fragment
Complementation + ED
enzyme
active 
-galactosidase

27. Design of EA & ED Enzyme Enzyme acceptor fragment donor fragment
Fused to a leader sequence
Localizable to intracellular compartments
Complements to both enzyme donor or enzyme donor protein fusions
In vitro or in vivo for protein detection
EA/ED complementation forms active enzyme
Large signal generation
Small changes readily detected
Enzyme
donor fragment
Easily fused to many proteins
Generic label
Rapidly degraded, when expressed alone
Low baseline signal
Stable when fused
High maximum signal
Small, non toxic
No protein perturbation
Tolerant to detergents
Complements in cell lysis conditions

28. DiscoveRx Core Technology: Enzyme Fragment Complementation (EFC)
No signal
Amplified signal
104-fold amplification of signal
15 yrs R&D, 30+ patents
Small size innocent label  minimal perturbation
High sensitivity  endogenous expression
No need to over express  no inherent cell toxicity
Unique Enzyme System

29. HitHunter™ In Vitro Assay Method
Label Ligand to ED (small biological label, 90 amino acids)
Binding protein: receptor, antibody or enzyme
EA/Substrate = detection