Antibody based assay –Pit-fall 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

Computer simulation of an antibody-antigenInteraction between antibody and influenzaVirus antigen(a globular protein)

Ab-Ag interaction

Ag contact area : flat undulat-ing 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)

1. Immunohistochemisty

2. 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-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 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-reaction

2. What is a good antibody?

3. How to reduce non-specific reaction

4. How to validate the antibody

- 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

AM

OU

NT B

OU

ND

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

Sig

nal to

Nois

eTITER

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

nu

mb

er

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

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 re-ceptor (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 anti-bodies

• 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 an-tibody?

• 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

26 kDa

17 kDa

34 kDa

43 kDa

56 kDa

72 kDa

43 kDa

ab50887(abcam Co. mouse

monoclonal, clone : Twist2C1a)

Ab50583(abcam Co.

Rabbit polyclonal)

siRNAtreated

SNU484 (gastric cancer cell line)siRNAtreated

siRNAtreated

siRNAtreatedWT WT WT WT

Actin

H81(santa-cruz Co.

Rabbit polyclonal)

4119S(Cell signaling Co.

Rabbit polyclonal)

Validation of anti-Twist1 antibody

Validation of anti-Twist1 antibody