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

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

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

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

Heterogeneous IA / Homogeneous IA

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)

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

Various Micro-Well Plates 96 Wells 384 Wells 1536 Wells

High-Throughput Well Sensor

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

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.

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.

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

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

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

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

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

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 - █

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

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

2. FPIA (Fluorescence Polarization ImmunoAssay)

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 3.2.1.23

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

The Engineering of Inactive Deletion Mutants p 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

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

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

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