Concept of Ag-Ab immunological technique

1. Concept of Ag-Ab immunological technique • Using the characteristic of high affinity and specificity of antibody, we can detect or quantitative the antigen. Keep in mind that the antibody is protein, can also be recognized as an antigen. The major principle to determine the antigen-antibody interaction is to separate the bound form of antigen-antibody complex from the free form of either antigen or antibody.

2. CNBr  Antibodies • Analytical Techniques • Utilizing Antibodies: • flow cytometry • gel electrophoresis • immunoprecipitation (IP) • immunoblotting • microscopy • immunofluorescence (IFA) • electron microscopy • ELISA • antibodies bind proteins with high specificity and affinity • affinity chromatography • analytical techniques

8. Western Blotting

9. Western Blotting • Protein denature (SDS) • SDS-PAGE gel electrophoresis • Blotting (transfer) • Blocking (BSA) • Staining with Coomasie or Ponceau S (checking) • 1° Ab serum (probe) • Washes • 2 ° Ab serum • Washes • Color development Why do proteins stick to the membrane? Hydrophobic & charge interactions.

10. Vertical Gel Electrophoresis

11. Prep and Run Samples

13. Example of Western Blot Result • Blot interpretation • Lane 1, HIV+ serum (positive control) • Lane 2, HIV- serum (negative control) • Lane A, Patient A • Lane B, Patient B • Lane C, Patient C

14. Enzyme-Mediated Detection

16. TUGAS: Komposisidanfungsidarimasing-masingbahanpada transfer buffer Methodesecararinci western blotting

17. bead protein A primary antibody Immunopreciptation: Identification of protein-protein interactions Steps: 1. Attach antibody to beads via protein A 2. Lyse cells to release antigen and its binding partners 3. Mix cell lysate + antibody-coated beads (antibody binds antigen) 4. Purify antigen and its binding partners by centrifugation

18. Immunoprecipitation • affinity purification based on isolation of Ag-Ab complexes • analyze by gel electrophoresis • initially based on centrifugation of large supramolecular complexes • [high] and equal amounts • isolation of Ag-Ab complexes • fixed S. aureus • protein A-agarose • protein G-agarose • Bacterial proteins that bind IgG (Fc): • protein A (Staphylococcus aureus) • protein G (Streptococcus) • binds more species and subclasses

19. agarose Typical IP Protocol • 1. Solubilize antigen • usually non-denaturing • SDS + excess of TX100 • 2. Mix extract and Ab • 3. Add protein G-agarose, etc • 4. Extensively wash • 5. Elute with sample buffer • 6. SDS-PAGE • 7. Detection • protein stain • radioactivity G

20. Radiolabeling of Proteins • carried out before IP • metabolic (amino acids or other precursors + cells) • chemically (eg., iodination) • IP and SDS-PAGE • detect by autoradiography or fluorography following electrophoresis • also provides information about synthesis, post-translational events, etc.

21. Western Blot vs Immunoprecipitation • Experimental Design • eg., synthesis (IP) • Ag concentration • IP better for low abundance proteins • Ag solubility • Western for insoluble proteins • Ab recognition • conformational dependent epitopes • 4o structure

22. Basics of Immunohistochemistry 03/2005

23. Immunofluorescence Microscopy

24. What is Immunohistochemistry?

25. CELLULAR ANTIGENS Adhesion Sensory Metabolic

27. Outline of Procedure Fixwholemount, embed and section tissue (or treat as ”” preparation – small specimens only, such as cultured cells)Wash sections in physiological buffer, e.g. PBSIncubate with protein solution (BSA or normal serum) to reduce non-specific binding of antibody to specimen (”blocking”- important!)Incubate with antibody specific to antigen in question (”primary antibody”). Include positive and negative controls (!)Wash in physiological bufferApply suitable detection system (see below)Mount specimens and analyse microscopically

28. Fixing and Sectioning of Tissue Common fixation methods are chemical fixation (e.g. paraformaldehyde) or cryofixation (i.e. rapid freezing). Fixation serves to preserves tissue structure and properties of antigenTissues can be embedded in wax or resins for sectioning, or cut in the frozen state in a cryostat.The method chosen for fixation and sectioning are dependent on the properties of the tissue, antigen and the antibody used in the procedure

29. Immunohistochemical Detection Methods Through fluorescent substances (fluorophores) –”Immunofluorescence technique”Through enzymatic conversion (often by horseradish peroxidase, HRP) of a soluble substrate (chromogen) into a non-soluble and colourful reaction product - ”Immunoenzyme technique”

30. Immunofluorescence Fluorescence or confocal microscope necessary for analysis of specimensHigh structural resolution possibleAdvanced image reconstruction (3D) and signal quantification possibleMultiple labelling easyLimited shelf life of labelled specimensMethod of choice for labelling of live cells

31. Direct Immunofluorescence Method Easy application, only few stepsNot very sensitiveNot very versatile, as primary antibodies need to be directly labelled with fluorophore

32. Fluorochrome-conjugated Primary Antibody Antigen expressing Cell

33. Indirect Immunofluorescence Method More steps involvedMore sensitiveMore versatile, as only secondary antibodies need to be labelled and different combinations of fluorophores are possible in multiple labelling experiments

34. Fluorochrome-conjugated Secondary Antibody (anti species X) Primary Antibody (from species X) Antigen expressing Cell

35. Indirect Immunfluorescence UsingBiotinylated secondary Antibody Biotin (Vitamin B) binds with high affinity to Avidin – thus good linker systemVery high sensitivityEndogenous biotin may be present in tissue – risk of background

36. Fluorochrome-conjugated Streptavidin Biotinylated Secondary Antibody (anti species X) Primary Antibody (from species X) Antigen expressing Cell

37. Multiple Immunofluorescence

38. Multiple Labelling of a Tissue Section

39. Live Labelling of Cultured Cells

40. Enzymatic detection methods Brightfield microscope sufficient for analysis of specimensSuitable for tissue analysis at low magnificationResolution of subcellular structures not as good as with fluorescence methods, but can be combined with electron microscopyUnimited shelf life of labelled specimensSubstrate reagents often toxic/carcinogenic

41. Immunoezyme Labelling of Tissue Section

42. ABC (Avidin Biotin Complex) - Method Biotin (Vitamin B) binds with high affinity to Avidin – thus good linker systemExtremely high sensitivityEndogenous biotin my be present in tissue – risk of background

43. Substrate-chromogen solution Enzyme-conjugated Avidin-Biotin Complex Biotinylated Secondary Antibody (anti species X) Primary Antibody (species X) Antigen expressing Cell

44. Common Problems Non-specific binding of primary or secondary antibodies to tissue sample; e.g. through ionic or hydrophobic interactions or binding of antibodies to free amino groups: „Background staining“ Cross-reactivity of antibodies with unrelated antigens present in tissue sample

45. Good Resources Dako On-line Immunohistochemistry Handbook (http://www.dakousa.com/ihcbook/hbcontent.htm) NIH Immunohistochemistry Protocols (http://dir.niehs.nih.gov/dirlep/immuno.html)