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ANTIGEN RETRIEVAL
By:
1. Siti Swaprziah Bt Muhyiddin 03-200904-00273
2. Siti Nadiyah Bt Othman 03-200904-00387
3. Sunarti ...
Objectives:
 Define antigen retrieval
 Describe the principle of Proteolytic enzyme
digestion method and its procedure
...
Content:
 Definition of antigen retrieval
 Epitope
 Masking of antigen / epitope
 How to retrieve masked antigen
 Ant...
Antigen Retrieval
 Method performed to expose or retrieve
antigens which have become masked by
series of events.
 Techni...
Epitope
Masking of antigen/epitope:
 Happen when the epitope is covered and can
no longer bind to the primary antibody.
 caused ...
How to retrieve the masked
antigen?
 Pretreatment with the antigen retrieval
reagent
 Break the protein cross-links form...
Antigen Retrieval Techniques
 Antigen RetrievalTechniques basically
divided into 3 categories:
 Heat Induced Epitope Ret...
Protease-induced Epitope
Retrieval (PIER)
 Enzyme used:
 Protease
 Trypsin
 Pepsin
 Principle:
 Enzyme will breaks d...
Disadvantages of PIER
 May destroying tissue morphology
 May also destroy epitope of the antigen
 Finally leading to fa...
key factors to be considered
when performing PIER
 Concentration of enzyme is usually 0.05-0.1%
depending on type of tiss...
Heat-induced Epitope Retrieval
(HIER)
 Instruments include:
 microwave ovens
 pressure cookers
 vegetable steamers
 a...
Principle of HIER
 FirstTheory – Shi 1991
 Methylene bridges produced by formaldehyde are
cleaved and polypeptide chains...
Heat-induced antigen / epitope
retrieval mechanisms
Principle of HIER
 2nd Theory – Morgan 1997
 Calcium coordinated complex formed during
fixation prevent antibody-antigen...
Key Factors to be Considered
When Performing HIER
 Temperature of retrieval solution should be
around 95 °C.
 Incubation...
IHC images show the detection of p27 in paraffin-embedded human prostate cancer sections following
incubation of tissue fo...
Combination of HIER and
Enzyme Method
 Alternative approach to unmask antigens if other
methods did not work
 Principle:...
Antigen Retrieval Technique
 There are several techniques available, four of
them are:
 Proteolytic enzyme digestion (PI...
Proteolytic enzyme digestion -
Trypsin
Incubate sections in pre-warmed distilled water at 37ºC
Prepare 0.1% trypsin in 0.1...
Proteolytic enzyme digestion -
Protease
Incubate sections in pre-warmed distilled water at 37ºC
Prepare 0.1% protease in d...
Proteolytic enzyme digestion -
Pepsin
Incubate sections in pre-warmed distilled water at 37ºC
Prepare 0.4% pepsin in 0.01M...
Combination of trypsin digestion and
microwave antigen retrieval
Incubate section in pre-warmed distilled water at 37C
Pre...
Pressure cooker antigen retrieval
Add 1.5 liters of appropriate antigen retrieval buffer into the pressure cooker and
brin...
General steps in immunohistochemistry
Staining LSAB-peroxidase
Incubate section
slide in the oven at
56C for 30min
Bring section to
water
Trypsinise the
section...
Labeled Streptavidin Biotin
principle
 The first layer is unlabeled primary antibody.
 The second layer is biotinylated ...
References
 J.D Bancroft and A Steven (2008)Theory and
Practice of HistologicalTechniques (6th ED)
Churchill Livingstone....
Antigen retrieval (ANATOMY)
Antigen retrieval (ANATOMY)
Antigen retrieval (ANATOMY)
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Antigen retrieval (ANATOMY)

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Antigen retrieval (ANATOMY)

  1. 1. ANTIGEN RETRIEVAL By: 1. Siti Swaprziah Bt Muhyiddin 03-200904-00273 2. Siti Nadiyah Bt Othman 03-200904-00387 3. Sunarti Bt Mansur 03-200904-00199 4. Syloveria Binti 03-200904-00201 5. Nurhanisa Bt Gulamu 03-200904-00130 6. Suierina Lawansing 03-200904-00226 7. Randy B. Gubud 03-200904-00264 DMLT 0904 Group 3 MLTA 3812 ANATOMY PATHOLOGY 4 PRESENTING:-
  2. 2. Objectives:  Define antigen retrieval  Describe the principle of Proteolytic enzyme digestion method and its procedure  Describe the principle of Heat induced epitope retrieval method and its procedure  State the staining procedure  LSAB-peroxidase
  3. 3. Content:  Definition of antigen retrieval  Epitope  Masking of antigen / epitope  How to retrieve masked antigen  Antigen retrieval technique categories  PIER  HIER  Combination of HIER & Enzyme  Procedure of PIER, HIER+Enzyme and HIER  LSAB staining
  4. 4. Antigen Retrieval  Method performed to expose or retrieve antigens which have become masked by series of events.  Technique in which the masking of an epitope is reversed and epitope-antibody binding is restored.
  5. 5. Epitope
  6. 6. Masking of antigen/epitope:  Happen when the epitope is covered and can no longer bind to the primary antibody.  caused by:  Fixation can alter protein biochemistry  cross-linking of amino acids within the epitope  Cross-linking unrelated peptides at an epitope  Altering the conformation of an epitope  Altering the electrostatic charge of the antigen
  7. 7. How to retrieve the masked antigen?  Pretreatment with the antigen retrieval reagent  Break the protein cross-links formed by formalin fixation  Thus uncover hidden antigenic sites
  8. 8. Antigen Retrieval Techniques  Antigen RetrievalTechniques basically divided into 3 categories:  Heat Induced Epitope Retrieval (HIER)  Proteolytic Induced Epitope Retrieval (PIER)  Combination of Heat Mediated and Proteolytic Enzyme Method
  9. 9. Protease-induced Epitope Retrieval (PIER)  Enzyme used:  Protease  Trypsin  Pepsin  Principle:  Enzyme will breaks down formalin cross-linking and hence the antigenic sites for antibodies binding are uncovered
  10. 10. Disadvantages of PIER  May destroying tissue morphology  May also destroy epitope of the antigen  Finally leading to false negative results
  11. 11. key factors to be considered when performing PIER  Concentration of enzyme is usually 0.05-0.1% depending on type of tissue and fixation.  Incubation time could be 5-30 minutes and 10-15 minutes is commonly used.  Incubation temperature is usually at 37 °C.
  12. 12. Heat-induced Epitope Retrieval (HIER)  Instruments include:  microwave ovens  pressure cookers  vegetable steamers  autoclaves  water baths
  13. 13. Principle of HIER  FirstTheory – Shi 1991  Methylene bridges produced by formaldehyde are cleaved and polypeptide chains extended to expose their hydrophobic regions.  During cooling process, the polypeptide chains rapidly refold.  Hydrophobic attractive force and electrostatic repulsion force based on positively or negatively charged polypeptides may balance to prevent intertwining of polypeptide chains and expose antigenic determinants for antigen-antibody interaction
  14. 14. Heat-induced antigen / epitope retrieval mechanisms
  15. 15. Principle of HIER  2nd Theory – Morgan 1997  Calcium coordinated complex formed during fixation prevent antibody-antigen reaction.  Hydroxy-methyl groups and other oxygen-rich group (carboxyl or phosphoryl groups) interact with calcium ion  Produce large coordinate complex that mask epitope.  High temperature weakens or breaks the calcium coordinated bonds.
  16. 16. Key Factors to be Considered When Performing HIER  Temperature of retrieval solution should be around 95 °C.  Incubation time should be at least 10 minutes and it is usually around 20 minutes.  pH value of retrieval solution is depending on which solution you are using.
  17. 17. IHC images show the detection of p27 in paraffin-embedded human prostate cancer sections following incubation of tissue for 10 minutes at 95 °C in the specified antigen retrieval solution. Compared to no HIER treatment, p27 detection was enhanced following incubation in neutral (pH 7.0) and basic (pH 9.5) but not acidic (pH 5.0) antigen retrieval solution. P27 was detected using anti-human/mouse/rat p27 antibody.
  18. 18. Combination of HIER and Enzyme Method  Alternative approach to unmask antigens if other methods did not work  Principle:  Heat pretreatment increases the sensitivity of sections to subsequent proteolytic enzyme digestion.  Proteolytic enzyme digestion increases the sensitivity of sections to microwaves antigen retrieval.  Therefore, proteolytic enzyme digestion can be carried out before or after heating.
  19. 19. Antigen Retrieval Technique  There are several techniques available, four of them are:  Proteolytic enzyme digestion (PIER)  Trypsin  Pepsin  protease  Microwave enzyme digestion (HIER+Enzyme)  Microwave and trypsin antigen retrieval (HIER+Enzyme)  Pressure cooker antigen retrieval (HIER)
  20. 20. Proteolytic enzyme digestion - Trypsin Incubate sections in pre-warmed distilled water at 37ºC Prepare 0.1% trypsin in 0.1% calcium chloride in distilled water (37C). Adjust pH to 7.8 using 0.1M sodium hydroxide solution. Incubate the sections in the trypsin solution for 10 minutes at 37C Wash section in cold running tap water to prevent further digestion Proceed with immunostaining method of choice
  21. 21. Proteolytic enzyme digestion - Protease Incubate sections in pre-warmed distilled water at 37ºC Prepare 0.1% protease in distilled water (37C). Adjust pH to 7.8 using 0.1M sodium hydroxide solution. Incubate the sections in the protease solution for 6 minutes at 37C Wash section in cold running tap water to prevent further digestion Proceed with immunostaining method of choice
  22. 22. Proteolytic enzyme digestion - Pepsin Incubate sections in pre-warmed distilled water at 37ºC Prepare 0.4% pepsin in 0.01M hydrochloric acid (pH2.0) at 37C. Incubate the sections in the trypsin solution for 15-60 minutes at 37C Wash section in cold running tap water to prevent further digestion Proceed with immunostaining method of choice
  23. 23. Combination of trypsin digestion and microwave antigen retrieval Incubate section in pre-warmed distilled water at 37C Prepare 0.1% trypsin in 0.1 calcium chloride in distilled water at 37C. Adjust pH to 7.8 using sodium hydroxide Incubate the sections in the trypsin solution for 3o seconds at 37C Wash sections in cold running tap water to prevent further digestion Using a plastic staining rack, place the sections in 600ml of 0.01M citrate buffer pH 6.0. use a microwaveable plastic container Irradiate on high power (800W) for 15minutes Carefully remove the container from the microwave and flood with cold water Proceed with the immunostaining method of choice
  24. 24. Pressure cooker antigen retrieval Add 1.5 liters of appropriate antigen retrieval buffer into the pressure cooker and bring to boil (without lid) When the antigen retrieval buffer is boiling, carefully place the slides racks into the hot solution and seal the lid Allow the pressure cooker to reach full pressure (15psi).Then incubate for two minutes.Timing start only when full pressure is reached. Transfer the pressure cooker to a sink and run cold water over the lid until all of the pressure is released. Flood the pressure cooker with cold water. Do not remove the slides until cool Proceed with the immunostaining method of choice
  25. 25. General steps in immunohistochemistry
  26. 26. Staining LSAB-peroxidase Incubate section slide in the oven at 56C for 30min Bring section to water Trypsinise the section at 37C for 30minutes (AG retrieval) Undergo proteination based on the type of reagent Wash with distilled water Put into 3% hydrogen peroxide Wash with distilled water Rinse with buffer solution Incubate with primary antibody 30min Insert antibody link 15min Wash with buffer solution Insert streptavidin for 15min Wash with buffer solution Rinse with distilled water Put substrate until developing brown colour and leave 10min Wash with distilled water Stain with H&E Rinse with water Rinse with 70% alcohol DCM Result: Grey formation
  27. 27. Labeled Streptavidin Biotin principle  The first layer is unlabeled primary antibody.  The second layer is biotinylated secondary antibody.  The third layer is Enzyme-Streptavidin conjugates to replace the complex of avidin- biotin peroxidase.  The enzyme is then visualized by application of the substrate chromogen solutions to produce different colorimetric end products.
  28. 28. References  J.D Bancroft and A Steven (2008)Theory and Practice of HistologicalTechniques (6th ED) Churchill Livingstone.  J.Ochei and A Kolhatkar (2000) Medical Laboratory Science,Theory and Practice (4th ED).TheTata McGraw Hill Company.

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