Group 3 Project- Immunohistochemistry

From CellBiology

What is Immunohistochemistry?

Example of Immunohistochemistry

Immunohistochemistry is a technique that is regularly used in cell biology, biological research and diagnostic pathology. It relies upon the interaction between antibodies and antigens, allowing for substances to be identified within tissue samples. An antigen is a substance that is recognised by the immune system as foreign, prompting the production of antibodies and causing an immune response within the body. Antigens are usually large, complex proteins or polysaccharide molecules. An antibody is a glycoprotein that is produced by B lymphocytes in response to the presence of an antigen. Also called immunglobulins, antibodies neutralise or destroy antigens by binding to them in a specific interaction, made possible by a 'lock and key' mechanism, where specific antibodies are only able to bind with specific antigens. [1]

Immunohistochemistry (IHC) is a technique used to locate antigens or proteins in tissue sections. It utilises this antigen-antibody interaction by labelling antibodies which will react with specific antigens. This interaction is visualised by a marker which may be a fluorescent dye, enzymes, radioactive elements or even colloidal gold.

The Process

Immunohistochemistry has a general process which differs slightly depending on which method is utilised. These are the steps involved:

1. A tissue sample is collected from an animal or the patient. It can be from almost any organ in the body.

2. The sample must be frozen or preserved quickly to prevent deterioration of the tissues. Fresh samples must be used as soon as possible. This is known as the fixation process.

3. Frozen samples are sliced to one-cell thickness and mounted.

4. Antibodies are added to the sample, which bind with the antigens present in the tissue. A protein solution is added to prevent the antibodies binding to non-specific proteins in a process called blocking.

5. The sample is then incubated and washed to remove excess primary antibodies.

6. A secondary antibody is added to the sample and similarly to previous steps, it is incubated and washed to remove any excess secondary antibodies.

7. After mounting, these antibodies are fluorescently tagged and are visualized with a microscope.

This method varies somewhat as there are different immunohistochemistry methods including direct, indirect, Peroxidase-Antiperoxidase (PAP) Method, Avidin-antibiotin complex (ABC) Method and Labeled StreptAvidin Biotin (LSAB) Method.

History

Courtesy of the Harvard Medical School Countway Library

1941 - Albert H. Coons

Coons first introduces immunofluorescence as initial attempts to label antibodies were unsuccessful as the labels were not visible enough under the microscope. Using specific antibodies, Coons labeled them with fluorescent dyes in order to localise substances in tissues. This allowed for the detection of antibodies, antigens and antigenic proteins in tissues. [2]

1942 - Albert H. Coons, Hugh Creech, Norman Jones and Ernst Berliner

Coons, Creech, Jones and Berliner succeeded in tagging antibodies. These antibodies were used to detect foreign antigens in tissues. This involved using a single antipneumococcal antibody to find pnuemococcal antigens in mice injected with large numbers of pneumococci. [3]

1959 - Singer

Singer first used an electron-dense protein in order to achieve ultrastructural localisation. The protein ferritin was used to tag an antibody. Electron microscopy could be used in immunohistochemistry as a result of this as the presence of iron in the protein makes it electron-dense. [4]

1965 - Sternberger

Uranium was used to develop the first electron-opaque heavy metal technique. [5]

1966 - Graham and Karnovsky

First localised the enzyme peroxidase using cytochemical methods leading to the development of the enzyme tagging method. [6]

1967 - Nakane and Pierce

Nakane and Pierce developed the enzyme-labelled antibody technique by labelling an antibody with an enzyme.[7]

1970 - Sternberger

Building upon the work of Graham, Karnovsky, Nakane and Pierce, Sternberger developed the peroxidase-antiperoxidase (PAP) method in an attempt to improve the enzyme-labelled method. The PAP method was an unlabelled antibody method. [8]

1971 - Faulk and Taylor

Another electron-opaque heavy metal technique was developed by Faulk and Taylor using colloidal gold. This is a popular technique and can also be called the colloidal gold technique. [9]

1974 - Heitzman and Richards

The Avidin-antibiotin complex (ABC) method was developed. Similar to the PAP method, it is also an unlabelled antibody method. [10]

1990's - Antigen Retrieval

It was discovered that the retrieval of nonreactive antigens in formalin-fixed, paraffin-embedded tissues was possible by heating sections in buffer solutions. This increased the detection of antigens and sensitivity of methods.[11]

Methods

Direct Method

Simplified drawing of Direct Method

Direct fluorescent methods is the oldest and simplest method. It utilises one labelled primary antibody which reacts directly with an antigen within a tissue sample. A sample is prepared and is exposed to a primary antibody. The antibodies react with the antigens resulting in an antigen-antibody interaction. The sample is washed to remove excess antibodies and is mounted and visualised under a microscope. This method is rarely used since the introduction of more complicated modern techniques.[12]

Advantages Disadvantages
Procedure is short and quick. Procedure is insensitive.
It can be used for quick diagnostic testing. Only one tagged antibody binds with each antigen. If antigen concentration is low then the the concentration of tagged antibodies is low and may not be enough for detection under the microscope.

Since the introduction of the more accurate and sensitive indirect method, the direct method is rarely used.


Indirect Method

Simplified drawing of Indirect Method

The indirect method is used far more commonly than the direct method. It involves using both primary and secondary antibodies. Similarly to the direct method, the sample is exposed to primary antibodies and the antigens in the sample react with the antibodies resulting in an antigen-antibody interaction. The sample is washed to remove the excess antibodies and is then exposed to the labelled secondary antibodies which are directed against the primary antibodies causing them to bind together. The sample is mounted and can be visualised through a microscope.

The secondary antibodies may be labelled with various substances. If they are labelled with fluorescent dyes such as Texas Red, rhodamine or FITC the method becomes known as Indirect Immunofluorescence Method. Alternatively, they may be labelled with enzymes such as peroxidase or glucose oxidase. This is known as Indirect Immunoenzyme Method. [13]


Advantages Disadvantages
Only the secondary antibodies, which tend to be cheaper, need to be labelled, thus preventing wastage of primary antibodies. Secondary antibodies must be from a different animal species
Sensitivity is much greater than that of direct method Procedure is laborious


The indirect method acts as a precursor to more complex methods such as the PAP method and ABC method. It is not as commonly used as its more complex successors.


Peroxidase-Antiperoxidase (PAP) Method

Simplified drawing of PAP Method

The PAP method was pioneered by Sternberger in the 1970's and is a development of the indirect technique. Similar to the indirect method, the PAP method exposes an unlabelled primary antibody to the antigens in the sample. Following this, the sample is rinsed to remove any excess primary antibodies, allowing for a secondary antibody to be introduced to the sample. The primary antibodies react with the antigens in an antigen-antibody reaction, binding them together. The secondary antibodies react with the primary antibodies. [14]

Following this step and unique to the PAP method is the introduction of the PAP molecule. Horseradish peroxidase acts as an antigen when injected into an animal and when combined with immunoglobins, creates a stable antigen-antibody complex, known as the PAP complex. As this combination does not damage enzyme activity, the PAP complex is a versatile tool for the detection of binding sites of anti-antibodies. [15]

The PAP complex acts as an antigen and reacts with the secondary antibodies, making up the third layer. The sample is then visualised under a microscope. This multiple layer method is most commonly used in diagnostic laboratories working with formalin-fixed, paraffin-embedded section. [16]


Advantages Disadvantages
Sensitivity is between 100-1000 times greater than the indirect method [17] The primary antibody and the PAP complex must be from the same species [18]
Allows for the primary antibody to be more diluted
Reduces non-specific background staining


PAP method is used in both diagnostics and research due to its high specificity and sensitivity. Recent uses include:

  • Proliferation, steroid receptors and clinical/pathological response in breast cancer treated with letrozole [19]
  • c-kit Expression in small cell carcinoma of the urinary bladder: prognostic and therapeutic implications [20]


Avidin-Biotin Complex Method (ABC Method)

The ABC method is an indirect method of immunohistochemistry.

Firstly, the tissue of interest is sectioned and is incubated with a primary antiserum that targets the antigen we would like to locate in the tissue section. An antiserum is a serum containing antibodies such as agglutinins and antitoxins. This antiserum is known as the primary antibody and is injected into the tissue to target the antigen and causes an antibody-antigen reaction. After the primary antibody is inserted. A secondary labeled antibody is added, namely a biotinylated antibody. The secondary antibody reacts with the first antibody and launches a large mass of biotin into the area in which the antigen is situated. The secondary antibody is reacts in an opposite manner to the primary antibody. The secondary antibody does not have the intention in reacting with the antigen.

The addition of the avidin biotin enzyme complex causes binding to the secondary antibody. The avidin biotin enzyme complex consists of avidin, biotin and enzymes. Avidin has a high attraction for biotin with four uniting sites in each molecule. Enzymes readily bind to biotin and it is because of those two properties that enables the formation of the avidin biotin complex. The last step in the formation of the complex before it is ready to use is by combining it with a solution. The avidin biotin complex is inserted into the tissue and the biotinylated secondary body that is already attached to the antigen binds to any free biotin sites on the avidin molecule.

The last step of the procedure is the addition of an enzyme substrate to the tissue section. This acts as a marker for site of the antibody-antigen reaction. The ABC method increases the influx of enzymes which increases the efficiency in detecting the antigen.


Advantages Disadvantages
Increases efficiency in detecting the antigen Blocking of biotin is needed
Does not require a large amount of primary antibody
Process is very fast (within three hours)
Once the avidin biotin complex is assembled it can be used for several days

Labeled StreptAvidin Biotin (LSAB) Method

Glossary

Antibody Glycoproteins that are produced by B lymphocytes in response to the presence of an antigen.

Antigen A complex protein or polysaccharide that is identified as a foreign substance within the body.

Immunocytochemistry Another name for immunohistochemistry.

Primary Antibody The antibody that reacts with the antigen in the sample; is generally unlabelled except when used in direct method.

Second Antibody The antibody which combines with the primary antibody; may or may not be labelled.

References

  1. Hayat M. A., Microscopy, Immunohistochemistry, and Antigen Retrieval Methods: For light and Electron Microscopy, Kluwer Academic/Plenum Publishers, New York, 2002, pp 31-33
  2. Mao, Su-Yau, Javois, Lorette C., Kent, Ute M. Overview of Antibody Use in Immunocytochemistry From: Methods in Molecular Bology, Vol. 115: Immunocytochemical Methods and Protocols, Edited by L. C. Javois, Humana Press Inc, Totowa, New Jersey
  3. http://www.nap.edu/readingroom.php?book=biomems&page=acoons.html
  4. Bozzola,John J.,Russell,Lonnie Dee,Electron microscopy: principles and techniques for biologists,1999 [1]
  5. History of Immunohistochemistry [2]
  6. Bozzola,John J.,Russell,Lonnie Dee,Electron microscopy: principles and techniques for biologists, 1999 [3]
  7. Bozzola,John J.,Russell,Lonnie Dee,Electron microscopy: principles and techniques for biologists, 1999 [4]
  8. History of Immunohistochemistry [5]
  9. History of Immunohistochemistry [6]
  10. History of Immunohistochemistry [7]
  11. Ramos-Vara JA, Segalés J, Duran CO, et al. Diagnosing infectious porcine diseases using immunohistochemistry. Swine Health Prod. 1999;7(2):85–91.
  12. Introduction to Immunohistochemistry [8]
  13. Introduction to Immunohistochemistry [9]
  14. Introduction to Immunohistochemistry [10]
  15. Kiernan, J. A., Histological & Histochemical Methods: Theory and Practice, The Bath Press, Somerset, Third Edition, 1999
  16. Ramos-Vara JA, Segalés J, Duran CO, et al. Diagnosing infectious porcine diseases using immunohistochemistry. Swine Health and Production. 1999;7(2):85–91[11]
  17. Ramos-Vara JA, Segalés J, Duran CO, et al. Diagnosing infectious porcine diseases using immunohistochemistry. Swine Health and Production. 1999;7(2):85–91[12]
  18. Bratthauer, Gary L., The Peroxidase-Antiperoxidase (PAP) Method and other All-Immunologic Detection Methods, From: Methods in Molecular Bology, Vol. 115: Immunocytochemical Methods and Protocols, Edited by L. C. Javois, Humana Press Inc, Totowa, New Jersey
  19. W. R. Miller, S. White, J. M. Dixon, J. Murray, L. Renshaw, T. J. Anderson,Proliferation, steroid receptors and clinical/pathological response in breast cancer treated with letrozole, British Journal of Cancer (2006) 94, 1051–1056.doi:10.1038/sj.bjc.6603001 www.bjcancer.com. Published online 14 March 2006 [13]
  20. Chong-Xian Pan, Ximing J. Yang, Antonio Lopez-Beltran, Gregory T. MacLennan, John N. Eble, Michael O. Koch, Timothy D. Jones, Haiqun Lin, Kelly Nigro, Veronica Papavero, Maria Tretiakova,Liang Cheng,c-kit Expression in small cell carcinoma of the urinary bladder: prognostic and therapeutic implications, Modern Pathology (2005) 18, 320–323, advance online publication, 15 October 2004; doi:10.1038/modpathol.3800318. [14]

2010 Projects

Fluorescent-PCR | RNA Interference | Immunohistochemistry | Cell Culture | Electron Microsopy | Confocal Microscopy | Monoclonal Antibodies | Microarray | Fluorescent Proteins | Somatic Cell Nuclear Transfer