Transcript Antibody based assay

Antibody based assay –
Pitfall and practical issue
2013 03 20
Seok-Hyung Kim
Antibody based assay
1. The chemical basis for Ab-reaction
2. What is a good antibody?
3. How to reduce non-specific reaction
4. How to validate the antibody
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
Ab-Ag interaction
Computer simulation of an antibody-antigen
Interaction between antibody and influenza
Virus antigen(a globular protein)
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
Solvent accessible surface of an anti-hemagglutinin
Fab fragment
Unbound Fab fragment
Bound Fab fragment
Flexibility of the Fab and Fc regions
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
maturation
epitope templated structural
rigidity
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
Model synthetic
peptide antigen
: PS1CT3
Table 1
Temperature
dependance
• Temperature differentially affects antigen association
rates of primary and secondary mAbs
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) :
• Entrophy(S) :
Heat change
net conformational, stereochemical
structural perturbations
Chemical bond used in Ag-Ab
interaction (1)
•
•
•
•
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
Result (2)
• Primary Ab(IgM) : enthalpy diriven
entropy constrained
• Secondary Ab : entropy driven
Enthalpy란 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
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)
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 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)
Applications of Antibody
Types of antigen (epitope)
3D conformation
Linear form
1. Immunohistochemisty
2. Flow cytometric analysis
3. Immunoprecipitation
(IP, ChIP)
4. ELISA
1. Immunoblotting
(Western blotting)
Antibody based assay
1. The chemical basis for Ab-reaction
2. What is a good antibody?
3. How to reduce non-specific reaction
4. How to validate the antibody
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 expert.
Good antibody / bad antibody
역가가 낮은 항체
Control
Control
측정값
측정값
항체를 저농도로 사용시
항체를 고농도로 사용시
역가가 높은 항체
Control
Control
측정값
항체를 저농도로 사용시
측정값
항체를 고농도로 사용시
Structural difference in good / bad
antibody (1)
• 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 (2)
Flexibility
Germline Ab
Versatile
Low affinity
Temperature sensitive
Polyspecific
Multiple configuration
Rigidity
Secondary Ab
Specific
High affinity
cross-reactive
Antibody based assay
1. The chemical basis for Ab-reaction
2. What is a good antibody?
3. How to reduce non-specific reaction
4. How to validate the antibody
Non-specific reactivity of Antibody
(Unwanted reactivity)
- 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.
Causes of non-specific reactivity of
Antibody based assay
1. Unwanted reaction of Antibody
2. Non-specific reaction of detection kit
3. Non-opitimized buffer
Solution of non-specific reactivity of
Antibody based assay
1. Selection of good Antibody
2. Optimization of antibody dilution
3. Simple but sensitive detection kit
4. Opitimization of buffer
(ion concentration / blocking agent)
Positive control
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
AMOUNT BOUND
TITERING ANTIBODIES
SPECIFIC
ANTIBODY
NON-SPECIFIC
ANTIBODY
CONCENTRATION
3 µg
s/n = 2.5
1 µg
s/n = 2.1
0.3 µg
s/n = 2.4
0.1 µg
s/n = 4.1
0.03 µg
s/n = 4.8
0.01 µg
s/n = 4.6
0.001 µg
s/n = 3.2
auto
3
0.003 µg
s/n = 3.5
5
Signal to Noise
TITER
4
3
2
0
1
2
3
4
5
Dilution
6
7
8
isotype
control
number
antibody
1
10
2
10
3
10
4
10
1
10
2
10
3
10
4
10
1
10
2
10
3
10
4
10
cytokeratin
1 µg S/N
Ab 278
IC
5.8
.3 µg S/N
Ab 100
IC
3.6
.01 µg S/N
Ab 25.7
IC
2.6
면역조직화학의 주요문제
:비특이적 배경염색의 원인
1PBS
NaCl : 150mM
1/10  PBS
NaCl : 15mM
Lymph node : L26(anti-CD20; B cell marker)
The enhanced reactivity of endogenous
biotin-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
Schematic drawings of principle of false positive
staining
due to endogenous biotin
: Streptavidin
:Horseradish Peroxidase (HRP)
Bb
: Biotin
DAB
Bb
Bb
Bb
(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
Staining
Seok Hyung Kim1, Young Kee Shin2 , Kyung Mee Lee1,4,
Jung Sun Lee4, Ji Hye Yun1,
Journal of Histochemistry & Cytochemistry
2003 51;129-131
Schematic drawings of priciple of Tyramine
Based signal amplified immunohistochemistry
: Streptavidin
:HorseradishPeroxidase (HRP)
B
B
:Biotin
:Biotinyl tyramide
B
B
Secondary
Ab
B
Primary Ab
B
(A) SA-HRP  DAB
(B) B-T  SA-HRP  DAB
(C) SA-HRP  B-T  SAHRP
 DAB
(D) 2’ Ab  SA-HRP  B-T
 SA-HRP  DAB
Figure 1. Background staining of a normal lymph node
in various conditions.
(A) Bovine serum
albumin
(B) Goat globulin
(C) Skim milk
(D) Casein sodium salt
(E) Trypton casein
pepton
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
Figure 3. Suppression of background staining
induced by biotinyl goat anti-mouse antibody
by several kinds of blocking agents..
(A) Imidazole buffer
(B) PBS
(C) Tris buffer
(D) Distilled water
(E) Borate buffer
(F) Citrate buffer
Figure 4. Effects of washing buffer
on suppression of background staining
(A) Conventional
immunostaining
(B) Tyramide-based
immunostaining
(C) Modified protocol
of tyramide-based
immunostaining
Figure 5. Immunostaining of human lymph node tissues
with anti-CD20 antibodies under various blocking conditions.
Antibody based assay
1. The chemical basis for Ab-reaction
2. What is a good antibody?
3. How to reduce non-specific reaction
4. How to validate the antibody
Nonspecific antibodies
• Double knockout mice for M2 & M3 suptypes of
muscarinic receptors (Joistsch et al. 2009)
 All of 16 Abs stains these K/O mice.
• Commercial antibodies are used widely to quantify
and localize the alpha1-adrenergic receptor (AR)
subtypes, alpha1A, alpha1B, and alpha1D.
(Jensen et al. 2009)
 All of 10 Abs fail to stain the positive control.
• Of 20 monoclonal Abs  7 : non-specific, 5 : no
reactivity for positive control. (Spicer et al.)
Hence, the present data demonstrate the unpleasant fact that reliable
Immunohistochemical localisation of MR subtypes with antibodies is
the exception rather than the rule.
Non-reproducible antibodies
• Two lot number of same antibody
(monoclonal 3D4 Met Ab)
=> opposite staining pattern and
R2 value of 0.038
• Anti-VEGF (monoclonal clone: VG-1)
=> not reproducible even with same lot of Ab
(R2 = 0.016)
How to validate the antibody?
• Western blotting  IHC
• Positive control
The confirmed cell line or tissue
Gene transfection to cell lines
• Negative control
The confirmed cell line or tissue
Knock down of gene in cell lines
Validation of anti-Twist1 antibody
SNU484 (gastric cancer cell line)
WT
siRNA
treated
siRNA
WT treated
siRNA
WT treated
siRNA
WT treated
72 kDa
56 kDa
43 kDa
34 kDa
26 kDa
17 kDa
Actin
43 kDa
ab50887
Ab50583
H81
4119S
(abcam Co. mouse
monoclonal,
clone : Twist2C1a)
(abcam Co.
(santa-cruz Co.
(Cell signaling Co.
Rabbit polyclonal)
Rabbit polyclonal)
Rabbit polyclonal)
Validation of anti-Twist1 antibody