CIHRT Exhibit P-1764 Page 1 IMMUNOHISTOCHEMISTRY ACCURATE LOCALIZATION OF TISSUE OR CELLULAR CONSTITUENTS WITH ANTIBODIES

Transcription

1 CIHRT Exhibit P-1764 Page 1 IMMUNOHISTOCHEMISTRY ACCURATE LOCALIZATION OF TISSUE OR CELLULAR CONSTITUENTS WITH ANTIBODIES

2 CIHRT Exhibit P-1764 Page 2 FUNCTIONAL ROLE OF ANTIBODIES Identify the tissue of origin of a metastatic tumour Provide data for therapy Measure tumour antigen levels

3 CIHRT Exhibit P-1764 Page 3 TUMOUR ORIGIN CARCINOMA - Pan keratin SARCOMA - Vimentin, Desmin NEUROENDOCRINE Synaptophysin LYMPHOMA - CD 45

4 CIHRT Exhibit P-1764 Page 4 THERAPY Hormone Receptors Estrogen and Progesterone Tamoxifen Therapy HER 2-NEU2 HERCEPTIN GIST CD 117 Glivex

5 CIHRT Exhibit P-1764 Page 5 TUMOUR ANTIGEN LEVELS CA125 - Ovarian Carcinoma CEA - Colon & Gastric Cancer PSA,PSAP - Prostate Cancer

6 CIHRT Exhibit P-1764 Page 6 Antigen Substance that can induce a detectable immune response Epitope -structural part of antigen.

7 CIHRT Exhibit P-1764 Page 7 Antibody Immunoglobulins that are produced as a result of the introduction of an antigen.

8 CIHRT Exhibit P-1764 Page 8 IgG IMMUNOGLOBULIN MOLECULE 2 Identical Heavy (H) chains Constant Fragment 2 Identical Light (L) chains Either kappa or lambda Variable Fragment Heavy chain Light chain Inter and Intra chain disulfide bonds provide the stability and structure

9 CIHRT Exhibit P-1764 Page 9 Fc portion carries the specific antigenic determinants to which ab raised to that particular IgG can bind

10 CIHRT Exhibit P-1764 Page 10 The primary binds to the antigen in the tissue and then acts as an antigen for a second antibody The secondary ab binds to the anitgenic sites on the Fc portion of the primary ab molecule

11 CIHRT Exhibit P-1764 Page 11

12 CIHRT Exhibit P-1764 Page 12 Polyclonal Antibodies Immunochemically dissimilar, react with various epitopes. Injected with ag and titre measured. Once a high titre is achieved animal bled and serum purified.

13 CIHRT Exhibit P-1764 Page 13 Monoclonal Antibodies Immunochemically identical,clones of plasma cells directed to one epitope. Injected with ag,, animal sacrificed. B-lymph B fused + myeloma cell = Hybrid Myeloma cell.

14 CIHRT Exhibit P-1764 Page 14 ANTIBODY CHARACTERISTICS AFFINITY AVIDITIY

15 CIHRT Exhibit P-1764 Page 15 AFFINITY THE BINDING SITES FIT WELL WITH THE ANTIGENIC SITES ON ITS SPECIFIC AG DO NOT WANT THE AB TO BIND TO OTHER AG

16 CIHRT Exhibit P-1764 Page 16 AVIDITY BINDING STRENGTH/STICKINESS DEPENDS UPON THE NUMBER OF FITTING SITES BETWEEN THE AG AND THE AB

17 CIHRT Exhibit P-1764 Page 17 STAINING METHODS Direct Method Two-Step Indirect Method PAP Method Avidin and Biotin Method

18 CIHRT Exhibit P-1764 Page 18 REQUIREMENTS FOR IHC PRESERVATION OF THE ANTIGEN IN THE TISSUE SPECIFIC AND SENSITIVE STAINING EFFICIENT LABELLING AND DETECTION

19 CIHRT Exhibit P-1764 Page 19 PRESERVATION IS ACHIEVED BY FIXATION WANT THE ANTIGEN MADE INSOLUBLE BUT AVAILABLE FOR DETECTION FORMALIN CROSS LINKING FIXATIVE THE LONGER A PIECE OF TISSUE IS LEFT IN FORMALIN THE GREATER THE DEGREE OF CROSS LINKING

20 CIHRT Exhibit P-1764 Page 20 DIRECT Method Enzyme labelled primary antibody reacts with the antigen in the tissue.

21 CIHRT Exhibit P-1764 Page 21 TWO-STEP INDIRECT Unconjugated primary antibody binds to the antigen. Enzyme labelled secondary antibody reacts with the primary antibody

22 CIHRT Exhibit P-1764 Page 22 PAP Method Soluble enzyme immune complex. Primary antibody and the animal specific

23 CIHRT Exhibit P-1764 Page 23 Avidin-Biotin Method Avidin has a strong affinity for biotin. Primary antibody is applied, then a biotinylated secondary then the avidin-biotin biotin-enzyme complex.

24 CIHRT Exhibit P-1764 Page 24 CONTROLS REAGENT Primary Detection System Chromogen TISSUE Positive Negative Internal

25 CIHRT Exhibit P-1764 Page 25 REAGENT CONTROLS Positive and Negative validation of staining technique assessment of handling and fixation standardization of methods and results amongst laboratories education for performance and interpretation

26 CIHRT Exhibit P-1764 Page 26 TISSUE CONTROLS POSITIVE Processed identically to the specimen Contains the target protein NEGATIVE Processed identically to the specimen Does not contain the relevant tissue marker INTERNAL Built in control

27 CIHRT Exhibit P-1764 Page 27 PRETREATMENTS Proteolytic digestion HIER aka Heat Induced Epitope Retrieval

28 CIHRT Exhibit P-1764 Page 28 Proteolytic Digestion Pepsin Proteinase K Formalin fixes by forming cross-linking methylene bridges digestion compensates for the impermeable nature of the non-coagulant fixative the enzyme etches the tissue allowing the epitope to be exposed.

29 CIHRT Exhibit P-1764 Page 29 PanKeratin without Pepsin

30 CIHRT Exhibit P-1764 Page 30 PanKeratin with Pepsin

31 CIHRT Exhibit P-1764 Page 31 HIER Heating provides the energy to rupture the hydroxyl bonds and releases tissue-bound calcium ions which break the fixative bond permanently exposing the epitope. Efficiency of HIER is a function of time, temperature, ph and the chemical composition of the buffer.

32 CIHRT Exhibit P-1764 Page 32 PGP 9.5 Pretreatment Pepsin 37º C 10 min. No staining observed

33 CIHRT Exhibit P-1764 Page 33 PGP 9.5 Pretreatment HIER Citrate Buffer ph º C

34 CIHRT Exhibit P-1764 Page 34 PGP 9.5 Pretreatment HIER Tris-HCl ph º C End Product intensified High signal/low noise

35 CIHRT Exhibit P-1764 Page 35 SECTION QUALITY

36 CIHRT Exhibit P-1764 Page 36

37 CIHRT Exhibit P-1764 Page 37

38 CIHRT Exhibit P-1764 Page 38

39 CIHRT Exhibit P-1764 Page 39

40 CIHRT Exhibit P-1764 Page 40 BACKGROUND STAINING ENDOGENOUS PEROXIDASE ACTIVITY ENDOGENOUS AVIDIN-BINDING ACTIVITY

41 CIHRT Exhibit P-1764 Page 41 Section without H2O2

42 CIHRT Exhibit P-1764 Page 42 Section with H2O2 applied

43 CIHRT Exhibit P-1764 Page 43 ENDOGENOUS AVIDIN- BINDING ACTIVITY Biotin is a vitamin and coenzyme. Biotin binds avidin or streptavidin specifically. Non-specific staining resembles a diffuse,cytoplasmic pattern. Eliminate with avidin-biotin blocking.

44 CIHRT Exhibit P-1764 Page 44 Section without Avidin Biotin Block

45 CIHRT Exhibit P-1764 Page 45 Section with Avidin/Biotin Block

46 CIHRT Exhibit P-1764 Page 46 CHROMOGENS IMMUNOFLUORESCENCE IMMUNOENZYMATIC

47 CIHRT Exhibit P-1764 Page 47 IMMUNOFLUORESCENCE FITC TEXAS RED DAPI

48 CIHRT Exhibit P-1764 Page 48

49 CIHRT Exhibit P-1764 Page 49 Triple Fluorescent Staining DAPInuclear FITC- TUNEL Texas RedvWF 400x Rat lung

50 CIHRT Exhibit P-1764 Page 50 IMMUNOENZYMATIC STAINING Allows the visualization of cell components Enzyme-substrate reactions convert colourless chromogens into coloured end products which precipitates at the site of the antigen that is localized. Reaction is insoluble upon oxidation.

51 CIHRT Exhibit P-1764 Page 51 ENZYMATIC MARKERS DAB Brown end product which is highly insoluble in alcohol and other organic solvents. NOVA Red Red end product which is insoluble in alcohol and other organic solvents.

52 CIHRT Exhibit P-1764 Page 52 HMB45 DAB NovaRED

53 CIHRT Exhibit P-1764 Page 53 MART-1 DAB NovaRED

54 CIHRT Exhibit P-1764 Page 54 SPECIMENS Formalin fixed, paraffin embedded pretreatments Frozen specimens acetone fixed (preserves immunoreactive sites) Blood Smears acetone fixed Cytospins alcohol fixed (preserves chromatin & nuclear changes)

55 CIHRT Exhibit P-1764 Page 55 Factors affecting the Quality of Immunostaining Antibody titre Antibody Dilution Incubation Time Incubation Temperature Pretreatments Buffers, ph

56 CIHRT Exhibit P-1764 Page 56 Ag Loss Upon Storage

57 CIHRT Exhibit P-1764 Page 57 Freshly Cut Section