2013 03 20 Seok-Hyung Kim

1. Antibody based assay –Pitfall and practical issue 2013 03 20Seok-Hyung Kim

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

3. Structure of Antibody • Heavy chain :Variable region + constant region (isotype ) => class of antibody • Light chain : Variable region + constant region (kappa / lambda chain)

4. Structure of antibody

8. Beta pleated sheet containing two anti-Parallel beta strands

9. Immunoglobulin fold

10. Structure of Mouse IgG2a

11. Structure of a whole antibody

13. Ab-Ag interaction Computer simulation of an antibody-antigen Interaction between antibody and influenza Virus antigen(a globular protein)

14. 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

15. Solvent accessible surface of an anti-hemagglutinin Fab fragment Bound Fab fragment Unbound Fab fragment

16. Flexibility of the Fab and Fc regions

17. 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

18. 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

19. Model synthetic peptide antigen : PS1CT3 Table 1 Temperature dependance

20. Temperature differentially affects antigen association rates of primary and secondary mAbs

21. Result(2) The cause of contradictory Effectsof 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

22. 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

23. Result (2) • Primary Ab(IgM) : enthalpy diriven entropy constrained • Secondary Ab : entropy driven Enthalpy란 면에선 불리

24. 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

25. 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

26. 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

27. 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)

28. 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)

29. Structural Insights into the Evolution of an Antibody Combining Site Many germline antibodies may indeed adopt multipleconfigurations with antigen binding, together with somatic mutation,stabilizing the configuration with optimum complementarity toantigen (Science 1997 : 276; 1665)

30. 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)

31. 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

32. 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

33. How to choose good antibody • A bad antibody : High risk-high return : generally less expensive (santacruz) : much less restriction in variety : but require highly skillful expert.

34. Good antibody / bad antibody 역가가 낮은 항체 Control Control 측정값 측정값 항체를 저농도로 사용시 항체를 고농도로 사용시 역가가 높은 항체 Control Control 측정값 측정값 항체를 저농도로 사용시 항체를 고농도로 사용시

35. 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

36. Structural difference in good / bad antibody (2) Flexibility Rigidity Germline Ab Versatile Low affinity Temperature sensitive Polyspecific Multiple configuration Secondary Ab Specific High affinity cross-reactive

37. 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

38. 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.

39. 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

40. Solution of non-specific reactivity of Antibody based assay Selection of good Antibody 2. Optimization of antibody dilution 3. Simple but sensitive detection kit 4. Opitimization of buffer (ion concentration / blocking agent)

41. Positive control Negative control

42. 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

43. TITERING ANTIBODIES SPECIFIC ANTIBODY AMOUNT BOUND NON-SPECIFIC ANTIBODY CONCENTRATION

44. 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 3 0.003 µg s/n = 3.5 0.001 µg s/n = 3.2 auto

45. 5 4 3 2 0 1 2 3 4 5 6 7 8 TITER Signal to Noise Dilution

46. 1 2 3 4 1 2 3 4 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 1 1 2 2 3 3 4 4 isotype control antibody number 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

47. 1PBS NaCl : 150mM 1/10  PBS NaCl : 15mM Lymph node : L26(anti-CD20; B cell marker) 면역조직화학의 주요문제 :비특이적 배경염색의 원인

48. 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

49. : Streptavidin :Horseradish Peroxidase (HRP) : Biotin Bb DAB Bb Bb Bb Schematic drawings of principle of false positive staining due to endogenous biotin

50. (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.