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-TS • 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 1PBS 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