ANTIGEN RETRIEVAL

By:1. Siti Swaprziah Bt Muhyiddin 03-200904-002732. Siti Nadiyah Bt Othman 03-200904-003873. Sunarti Bt Mansur 03-200904-001994. Syloveria Binti 03-200904-002015. Nurhanisa Bt Gulamu 03-200904-001306. Suierina Lawansing 03-200904-002267. Randy B. Gubud 03-200904-00264

DMLT 0904 Group 3MLTA 3812ANATOMY PATHOLOGY 4

PRESENTING:-

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

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

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.

Epitope

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

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

Antigen Retrieval Techniques

Antigen Retrieval Techniques basically divided into 3 categories:

Heat Induced Epitope Retrieval (HIER) Proteolytic Induced Epitope Retrieval

(PIER) Combination of Heat Mediated and

Proteolytic Enzyme Method

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

Disadvantages of PIER

May destroying tissue morphology May also destroy epitope of the

antigen Finally leading to false negative

results

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.

Heat-induced Epitope Retrieval (HIER)

Instruments include: microwave ovens pressure cookers vegetable steamers autoclaves water baths

Principle of HIER First Theory – 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

Heat-induced antigen / epitope retrieval mechanisms

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.

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.

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.

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.

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)

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

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

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

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

Pressure cooker antigen retrievalAdd 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

General steps in immunohistochemistry

Staining LSAB-peroxidaseIncubate 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% alcoholDCM

Result: Grey formation

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.

End of PresentationThank You!!

References

J.D Bancroft and A Steven (2008) Theory and Practice of Histological Techniques (6th ED) Churchill Livingstone.

J.Ochei and A Kolhatkar (2000) Medical Laboratory Science, Theory and Practice (4th ED). The Tata McGraw Hill Company.