2012 05 30 seok-hyung kim
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Antibody based assay –Pit-fall and practical issue
2012 05 30
Seok-Hyung Kim
Antibody based assay
1. The chemical basis for Ab-reaction2. How to choose good antibody3. How to reduce non-specific reaction
Structure of Antibody • Heavy chain :Variable region + constant region (isotype ) => class of antibody • Light chain : Variable region + constant region (kappa / lambda chain)
Structure of antibody
Beta pleated sheet containing two anti-Parallel beta strands
Immunoglobulin fold
Structure of Mouse IgG2a
Structure of a whole antibody
Computer simulation of an antibody-antigenInteraction between antibody and influenzaVirus antigen(a globular protein)
Ab-Ag interaction
Ag contact area : flat undulating face
• 650 – 900 A (15 – 22 amino acid)• small antigen : antigen binding site is gener-
ally smaller and appear more like a deep pocket in which ligand is largely buried
Unbound Fab fragment Bound Fab fragment
Solvent accessible surface of an anti-hemagglutinin Fab fragment
Flexibility of the Fab and Fc regions
Maturation of an antibody re-sponse is governed by modula-
tions in flexibility of antigen com-bining site
(immunity 2000 13: 611-620)
Pliable germline antigen combining site
epitope templated structural rigidity
maturation
Result (1)• Temperature dependence of antigen
affinities of antibodies from primary and secondary responses
• 25 -> 35’C : IgM : affinity 3 – 100 folds decrease IgG : No difference ; Qualitative difference
Table 1 Tempera-ture depen-dance
Model syn-thetic peptide antigen : PS1CT3
• Temperature differentially affects antigen association rates of primary and sec-ondary mAbs
Result(2) The cause of contradictory Effects of Temperature on
Antigen Association Rates between Primary and Secondary Responses : Change of Entropy
G= H-TS • Enthalpy(H) : Heat change
• Entrophy(S) : net conformational, stereochemical structural perturbations
• 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 in-teraction (1)
Result (2)
• Primary Ab(IgM) : enthalpy diriven entropy constrained • Secondary Ab : entropy driven Enthalpy 란 면에선 불리
Result(3)
• Germ line antibody 7cM(PS1CT3), 36-65(Ars), BBE6.12H3(NP)
37’C : high degree of cross reactivity 4’C : no cross reactivity
• Mature antibody Cys18(PS1CT3), P16.7(Ars), Bg110-2(NP)
37’C, 4’C : no cross reactivity
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
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
Molecular dynamics and free energy cal-culations 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)
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)
Structural Insights into the Evolution of an Antibody Com-
bining Site
Many germline antibodies may in-deed adopt multiple configurations with antigen binding, together with somatic mutation, stabilizing the configuration with optimum com-plementarity to antigen
(Science 1997 : 276; 1665)
ConclusionFlexibility Rigidity
Germline AbVersatileLow affinityScreening &recognitionTemperature sensitivePolyspecificMultiple configuration
Secondary AbSpecificHigh affinityResponseCross-reactive
1. Immunohistochemisty2. Flow cytometric analysis 3. Immunoprecipitation (IP, ChIP)4. ELISA
Applications of Anti-body
1. Immunoblotting (Western blotting)
3D conformation Linear form
Types of antigen (epitope)
Antibody based assay
1. The chemical basis for Ab-reaction2. How to choose good antibody3. How to reduce non-specific reaction
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
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 ex-pert.
항체를 저농도로 사용시
Control
측정값
Control
측정값
항체를 고농도로 사용시
역가가 낮은 항체
Control
측정값
Control
측정값
역가가 높은 항체
항체를 저농도로 사용시 항체를 고농도로 사용시
Good antibody / bad antibody
• 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)
Flexibility RigidityGermline AbVersatileLow affinityTemperature sensitivePolyspecificMultiple configuration
Secondary AbSpecificHigh affinitycross-reactive
Structural difference in good / bad antibody (2)
Antibody based assay
1. The chemical basis for Ab-reaction2. How to choose good antibody3. How to reduce non-specific reaction
- 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 An-tibody
(Unwanted reactivity)
1. Unwanted reaction of Antibody
2. Non-specific reaction of detection kit
3. Non-opitimized buffer
Causes of non-specific reac-tivity of Antibody based as-
say
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 reac-tivity of Antibody based as-
say
Positive con-trol
Negative control
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
SPECIFIC ANTIBODY
NON-SPECIFIC ANTIBODY
CONCENTRATION
AMO
UN
T BO
UN
D
TITERING ANTIBODIES
3
3 µgs/n = 2.5
1 µgs/n = 2.1
0.3 µgs/n = 2.4
0.1 µgs/n = 4.1
0.03 µgs/n = 4.8
0.01 µgs/n = 4.6
0.003 µgs/n = 3.5
0.001 µgs/n = 3.2
auto
8765432102
3
4
5
Dilution
Sign
al t
o N
oise
TITER
101 102 103 104101 102 103 104101 102 103 104
1 µg S/NAb 278IC 5.8
isotypecontrol
antibody
cytokeratin.3 µg S/NAb 100IC 3.6
101 102 103 104
.01 µg S/NAb 25.7IC 2.6
num
ber
면역조직화학의 주요문제: 비특이적 배경염색의 원인
1PBSNaCl : 150mM
1/10 PBSNaCl : 15mM
Lymph node : L26(anti-CD20; B cell marker)
The enhanced reactivity of endogenous bi-otin-like molecules by the antigen retrieval procedures and signal amplification with
tyramine
Seok Hyung Kim1, Kyeong Cheon Jung2 , Young Kee Shin1,4, Kyung Mee Lee4, Young S. Park1, Yoon La Choi1, Kwon Ik
Oh1, Min Kyung Kim1, Doo Hyun Chung1, Hyung Geun Song3,4 & Seong Hoe Park1,*
Histochemical journal 2002 34;97-103
DAB
:Horseradish Peroxidase (HRP)
Bb
: Streptavidin
: Biotin
Bb BbBb
Schematic drawings of principle of false positive stain-ing
due to endogenous biotin
(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.
(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.
An Improved Protocol of Biotinylated Tyramine-based Immunohistochemistry
Minimizing Nonspecific Background Stain-ing
Seok Hyung Kim1, Young Kee Shin2 , Kyung Mee Lee1,4, Jung Sun Lee4, Ji Hye Yun1,
Journal of Histochemistry & Cytochemistry 2003 51;129-131
B
: Streptavidin
:HorseradishPeroxidase (HRP)
:Biotin
B
SecondaryAb
Primary Ab
B
B
B
B :Biotinyl tyramide
Schematic drawings of priciple of TyramineBased signal amplified immunohistochemistry
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
Figure 2. Suppression of background staining induced by HRP-conjugated streptavidin
by several kinds of blocking agents.
(A)Bovine serum albu-min
(B) Goat globulin
(C) Skim milk
(D) Casein sodium salt
(E) Trypton casein pep-ton
Figure 3. Suppression of background staining induced by biotinyl goat anti-mouse antibody
by several kinds of blocking agents..
(A)Bovine serum albu-min
(B) Goat globulin
(C) Skim milk
(D) Casein sodium salt
(E) Trypton casein pep-ton
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
Figure 5. Immunostaining of human lymph node tissues with anti-CD20 antibodies under various blocking conditions.
(A) Conventional im-munostaining
(B) Tyramide-based
immunostaining
(C) Modified protocol
of tyramide-based
immunostaining
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