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
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