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Antibody based assay Pitfall and practical issue 2012 05 30 Seok-Hyung Kim

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Antibody based assay 1. The chemical basis for Ab-reaction 2. How to choose good antibody 3. How to reduce non-specific reaction

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Structure of Antibody Heavy chain :Variable region + constant region (isotype ) => class of antibody Light chain : Variable region + constant region (kappa / lambda chain)

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Structure of antibody

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Beta pleated sheet containing two anti-Parallel beta strands

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Immunoglobulin fold

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Structure of Mouse IgG2a

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Structure of a whole antibody

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Computer simulation of an antibody-antigen Interaction between antibody and influenza Virus antigen(a globular protein) Ab-Ag interaction

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Ag contact area : flat undulating face 650 900 A (15 22 amino acid) small antigen : antigen binding site is generally smaller and appear more like a deep pocket in which ligand is largely buried

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Unbound Fab fragment Bound Fab fragment Solvent accessible surface of an anti-hemagglutinin Fab fragment

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Flexibility of the Fab and Fc regions

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Maturation of an antibody response is governed by modulations in flexibility of antigen combining site (immunity 2000 13: 611-620) Pliable germline antigen combining site epitope templated structural rigidity maturation

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Result (1) Temperature dependence of antigen affinities of antibodies from primary and secondary responses 25 -> 35C : IgM : affinity 3 100 folds decrease IgG : No difference ; Qualitative difference

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Table 1 Temperatur e dependanc e Model synthetic peptide antigen : PS1CT3

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Temperature differentially affects antigen association rates of primary and secondary mAbs

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Result(2) The cause of contradictory Effects of Temperature on Antigen Association Rates between Primary and Secondary Responses : Change of Entropy G= H-T S Enthalpy( H) : Heat change Entrophy( S) : net conformational, stereochemical structural perturbations

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Covalent bond : not used Hydrogen bond : important for Ag-Ab Ionic bond : infrequently used Van derwaals bond : frequently used but not important Hydrophobic interaction : important for Ag-Ab Chemical bond used in Ag-Ab interaction (1)

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Result (2) Primary Ab(IgM) : enthalpy diriven entropy constrained Secondary Ab : entropy driven Enthalpy

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Result(3) Germ line antibody 7cM(PS1CT3), 36-65(Ars), BBE6.12H3(NP) 37C : high degree of cross reactivity 4C : no cross reactivity Mature antibody Cys18(PS1CT3), P16.7(Ars), Bg110-2(NP) 37C, 4C : no cross reactivity

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Discussion(1) Germ line antibody affinity at high temperature cross reactivity at high temperature => multiple conformational state > induced fit trasition from one conformation to another

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Discussion (2) Entropic constraint of germline Ab. : Free germline paratope exist in an equilibrium between multiple conformational states, only subset of which are capable of binding to the Ag

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Molecular dynamics and free energy calculations applied to affinity maturation In antibody 48G7 Increasing the rigidity of the antibody structure further optimizes the binding affinity of the antibody for the hapten (PNAS 1999 96: 14330)

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rms fluctuations of the germ line and mature antibody hapten complexes. rms fluctuations are defined as rms deviations of the structure at a given time from the average structure of the MD simulation (PNAS 1999 96: 14330)

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Structural Insights into the Evolution of an Antibody Combining Site Many germline antibodies may indeed adopt multiple configurations with antigen binding, together with somatic mutation, stabilizing the configuration with optimum complementarity to antigen (Science 1997 : 276; 1665)

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Conclusion Flexibility Rigidity Germline Ab Versatile Low affinity Screening &recognition Temperature sensitive Polyspecific Multiple configuration Secondary Ab Specific High affinity Response Cross-reactive

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1. Immunohistochemisty 2. Flow cytometric analysis 3. Immunoprecipitation (IP, ChIP) 4. ELISA Applications of Antibody 1. Immunoblotting (Western blotting) 3D conformationLinear form Types of antigen (epitope)

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Antibody based assay 1. The chemical basis for Ab-reaction 2. How to choose good antibody 3. How to reduce non-specific reaction

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How to choose good antibody A good antibody? : High affinity : Entropy driven antibody A good antibody : low risk-low return : generally expensive (DAKO, Novo) : restriction in variety

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How to choose good antibody A bad antibody : High risk-high return : generally less expensive (santa cruz) : much less restriction in variety : but require highly skillful expert.

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Control Control Control Control Good antibody / bad antibody

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Bad antibody : structurally more flexible 37 C : high degree of cross reactivity : multiple conformational state 4 C : no cross reactivity Good antibody : more rigid 37 C, 4 C : no cross reactivity Structural difference in good / bad antibody (1)

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Flexibility Rigidity Germline Ab Versatile Low affinity Temperature sensitive Polyspecific Multiple configuration Secondary Ab Specific High affinity cross-reactive Structural difference in good / bad antibody (2)

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Antibody based assay 1. The chemical basis for Ab-reaction 2. How to choose good antibody 3. How to reduce non-specific reaction

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- Polyspecificity (Multi-specificity) : unrelated specificities, which means interactions caused by different binding modes. - Cross-reactivity (Molecular mimicry) : interactions based on wild-type-derived key residues. Non-specific reactivity of Antibody (Unwanted reactivity)

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1. Unwanted reaction of Antibody 2. Non-specific reaction of detection kit 3. Non-opitimized buffer Causes of non-specific reactivity of Antibody based assay

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1.Selection of good Antibody 2. Optimization of antibody dilution 3. Simple but sensitive detection kit 4. Opitimization of buffer (ion concentration / blocking agent) Solution of non-specific reactivity of Antibody based assay

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Positive control Negative control

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Causes of background staining in immunohistochemistry 1. Non-specific interaction between SA-HRP and tissue : ionic interaction hydrophobic interaction 2. Endogenous biotin 3. Binding of SA-HRP to endogenous lectin 3. Non-specific interaction of 2ndary antibody

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SPECIFIC ANTIBODY NON-SPECIFIC ANTIBODY CONCENTRATION AMOUNT BOUND TITERING ANTIBODIES

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876543210 2 3 4 5 Dilution Signal to Noise TITER

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10 1 2 3 4 1 g S/N Ab 278 IC 5.8 isotype control antibody cytokeratin.3 g S/N Ab 100 IC 3.6 10 1 2 3 4.01 g S/N Ab 25.7 IC 2.6 number

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: 1 PBS NaCl : 150mM 1/10 PBS NaCl : 15mM Lymph node : L26(anti-CD20; B cell marker)

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The enhanced reactivity of endogenous biotin-like molecules by the antigen retrieval procedures and signal amplification with tyramine Seok Hyung Kim 1, Kyeong Cheon Jung 2, Young Kee Shin 1,4, Kyung Mee Lee 4, Young S. Park 1, Yoon La Choi 1, Kwon Ik Oh 1, Min Kyung Kim 1, Doo Hyun Chung 1, Hyung Geun Song 3,4 & Seong Hoe Park 1, * Histochemical journal 2002 34;97-103

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DAB :Horseradish Peroxidase (HRP) Bb : Streptavidin : Biotin Bb Schematic drawings of principle of false positive staining due to endogenous biotin

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(A)(B) : with Microwave heaing (A) ductal cell of mammary gland (B) gland of seminal vesicle (C)(D) : with heating under pressure (C) Neurons of cerebrum (D) thyrocyte of thyroid Figure 2. Immunostaining of normal human tissues using HRP-conjugated streptavidin only with microwave heating or heating under pressure as an antigen retrieval method.

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(A) No antigen retrieval (B) Heating under pressure (C) Signal amplification with biotinylated tyramine (D) Immunostaining with anti-biotin antibody Figure 3.. Immunostaining of normal human tissues using anti-biotin antibodies or signal amplification technique without antigen retrieval treatment.

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An Improved Protocol of Biotinylated Tyramine-based Immunohistochemistry Minimizing Nonspecific Background Staining Seok Hyung Kim 1, Young Kee Shin 2, Kyung Mee Lee 1,4, Jung Sun Lee 4, Ji Hye Yun 1, Journal of Histochemistry & Cytochemistry 2003 51;129-131

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B : Streptavidin :HorseradishPeroxidase (HRP) :Biotin B SecondaryAb Primary Ab B B B B :Biotinyl tyramide Schematic drawings of priciple of Tyramine Based signal amplified immunohistochemistry

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Figure 1. Background staining of a normal lymph node in various conditions. (A)SA-HRP DAB (B)B-T SA-HRP DAB (C) SA-HRP B-T SA- HRP DAB (D) 2 Ab SA-HRP B-T SA-HRP DAB

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Figure 2. Suppression of background staining induced by HRP-conjugated streptavidin by several kinds of blocking agents. (A)Bovine serum albumin (B)Goat globulin (C) Skim milk (D) Casein sodium salt (E) Trypton casein pepton

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Figure 3. Suppression of background staining induced by biotinyl goat anti-mouse antibody by several kinds of blocking agents.. (A)Bovine serum albumin (B)Goat globulin (C) Skim milk (D) Casein sodium salt (E) Trypton casein pepton

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Figure 4. Effects of washing buffer on suppression of background staining (A)Imidazole buffer (B)PBS (C) Tris buffer (D) Distilled water (E) Borate buffer (F) Citrate buffer

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Figure 5. Immunostaining of human lymph node tissues with anti-CD20 antibodies under various blocking conditions. (A)Conventional immunostaining (B)Tyramide-based immunostaining (C) Modified protocol of tyramide-based immunostaining