Group 3 Project- Immunohistochemistry

From CellBiology

What is Immunohistochemistry?

Example of Immunohistochemistry - A ganglion in the myenteric plexus of a mouse labelled by immunohistochemical methods

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..[2]

History

Albert Coons - Courtesy of Fabian Bachrach, the Harvard Medical School Countway Library and the National Academies Press

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. [3]

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. [4]

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. [5]

1965 - Sternberger

Uranium was used to develop the first electron-opaque heavy metal technique. [6]

1966 - Graham and Karnovsky

First localised the enzyme peroxidase using cytochemical methods leading to the development of the enzyme tagging method. [7]

1967 - Nakane and Pierce

Nakane and Pierce developed the enzyme-labelled antibody technique by labelling an antibody with an enzyme.[8]

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. [9]

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. [10]

1974 - Heitzman and Richards

The Avidin-antibiotin complex (ABC) method was developed. Similar to the PAP method, it is also an unlabelled antibody method. [11]

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.[12]

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. Antibodies may be monoclonal or polyclonal.

5. A protein solution is added to prevent the antibodies binding to non-specific proteins in a process called blocking.

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

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

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

(General method adapted from IHC world [13] and "Microscopy, Immunohistochemistry, and Antigen Retrieval Methods [14])

This method varies somewhat as there are different immunohistochemistry methods including; Direct and Indirect Method, Peroxidase-Antiperoxidase (PAP) Method, Avidin-antibiotin complex (ABC) Method, Labelled StreptAvidin Biotin (LSAB) Method, Polymeric Method and Catalysed Signal Amplification (CSA) Methods.

Controls

Use of Positive and Negative controls in immunohistochemistry

Like most experiments, a control needs to be run so that the procedure can be verified and to check that the antibody being used is the correct one. The controls should be handled and processed in the same way the tissues being tested are, to ensure consistency and accurate results. A control should be set up for every set of experimental conditions.

There are two types of control in Immunohistochemistry; Positive control and Negative control.

1. Positive control is used to verify the procedure. Any negative results or steps that do not work efficiently will be eliminated or completed over again until the correct procedure and staining is established. Generally it is most appropriate to use the tissue which will be used in the experiment, as the most accurate results will assist in modifying the procedure making the experimental results the most reliable.

2. Negative staining is used to verify the specificity of the antibody being used. The antibody must be from the same species as the primary antibody and a stain must not occur when the primary antibody is removed or replaced with serum. This type of negative control is often used in IHC as it is easy.

A second type of negative staining is used, in which the staining is inhibited by a purified antigen absorbing the primary antibody. This technique is the most useful and appropriate, however isolating a purified antigen is time consuming and hard, consequently making this stain rarely used in Immunohistochemistry controls.[15]

Blocking

The process called blocking, is used to reduce or limit the amount of background staining which occurs during various Immunohistochemistry methods. Background staining is when cells that are not being stained for pick up some of the stain or fluorescence, leading to false positives. This is a common outcome when fixation is not completed quickly or adequately enough and also in the middle of large pieces of tissue. Background staining may also occur if the antibodies being used have been contaminated by other antibodies, through the use of impure antigen during immunization.

Background staining has two types, specific or non-specific.

Non-specific staining is when the antibodies bind to non-specific proteins instead of the antigens they are supposed to. The uniform stain that occurs can be limited by blocking the sites with normal serum.

Specific staining is when activity of a similar antigen within a tissue is similar to what is being added to the tissue overrides the experiment as all of the antibodies are taken up but not by the sites wanted. An example of this is during the use of the Avidin-antibiotin complex (ABC) Method. Some liver and kidney tissues have natural biotin. To stop the avidin binding with this biotin, the tissue is pretreated with unconjugated avidin and then biotin is added until saturation.

Natural fluorescence that exists within some tissues can interfere with the used of added fluorescent dyes, resulting in background staining. Generally it is best to use enzyme labeling methods with such tissues.[16]

Methods

Direct Method

Simplified drawing of Direct Method - Shows a labelled antibody reacting with an antigen

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.[17]

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.

Applications

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

Indirect Method

Simplified drawing of Indirect Method - Shows how the secondary antibody binds to the primary antibody.

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. [18]


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

Applications

The indirect method acts as a precursor to more complex methods such as the PAP method and ABC method. It is rarely used since the introduction of the more commonly used complex and sensitive successors.

Peroxidase-Antiperoxidase (PAP) Method

Simplified drawing of PAP Method demonstrating how the PAP complex binds to the secondary antibody

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. [19]

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. [20]

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. [21]

Advantages Disadvantages
Sensitivity is between 100-1000 times greater than the indirect method [22] The primary antibody and the PAP complex must be from the same species [23]
Allows for the primary antibody to be more diluted
Reduces non-specific background staining

Applications

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

Proliferation, steroid receptors and clinical/pathological response in breast cancer treated with letrozole [24]

This study aims to explore the effect of treating primary breast cancers with letrozole on the proliferation, steroid receptors and other clinical aspects of the cancer. Tumour samples were taken and these were immunohistochemically tested by applying the peroxidase - antiperoxidase (PAP) method. Staining was achieved by reacting an antibody with the MIB1 (Ki-67) antigen and this allowed for the tumour cell proliferation to be measured. [25]

A modified peroxidase--antiperoxidase procedure for improved localization of tissue antigens: localization of substance P in rat spinal cord [26]

This study used a modified version of the PAP method, resulting in a greater amount of immunoreactive processes. The majority of these were found to be in the dorsal horn of the rat spinal cord. Substance P was also visualised, demonstrating the increased sensitivity of this modified PAP method could aid other studies to visualise antigens in tissues which have very small numbers of antigens.[27]

In situ immunologic characterization of cellular constituents in lymph nodes and spleens involved by Hodgkin's disease [28]

In this study, the spleens and lymph nodes of patients suffering from Hodgkin's disease were studied to determine the cell constituents. Both the PAP method and indirect immunfluorescence were utilised to find that T cells were the most common lymphocytes in these tissue samples. [29]

Avidin-Biotin Complex Method (ABC Method)

Pictorial representation of the steps involved in the 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. [30] [31]

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. Blocking of endogenous biotin and avidin is required in order to prevent non specific binding and hence resulting in non specific staining. [32] [33]


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 when a secondary antibody is used Blocking of biotin is needed to prevent the non specific binding of avidin to endogenous biotin
Does not require a large amount of primary antibody On several occasion, the complex produced can be too big to seep into the tissue
Process is very fast (within three hours) Endogenous biotin can cause non specific staining.
Once the avidin biotin complex is assembled it can be used for several days Background staining can occur, compromises the accuracy of the location of the antigen

Applications


Pathology of Natural Infections by H5N1 Highly Pathogenic Avian Influenza Virus in Mute (Cygnus olor) and Whooper (Cygnus cygnus) Swans [29]

In 2003, there was an epidemic outbreak of highly pathogenic avian influenze ( HPAI) in Southeast Asia. This outbreak was caused by the viruses of the H5N1 virus. Humans were slowly being infected by this virus and the main reason for their infection was due to direct contact with birds that were infected with the H5N1 HPAI virus. In order to understand where this particular viral influenza antigen lesions lie within the birds, the ABC method was applied to detect the antigen within the birds. The tissue of interest was sectioned off and stained. A rabbit antinucleoprotein serum was inserted into the tissue section as the primary antibody. A biotinylated goat anti-rabbit IgG1 was applied as the secondary antibody. The ABC complex was then added. Lastly, an enzyme substrate called the substrate 3-amino-9-ethylcarbazole was introduced. The overall process produced a bright red indicator at the site of antigen. It was found that the main locations for these lesions are in the pancreas, brain and liver. [34]


Vascular Endothelial Growth Factor ( VEGF ) Receptor Expression Correlates With Histologic Grade and Stage of Colorectal Cancer [30]

Colorectal carcinoma is a common malignancy and has a high fatal rate. The protein vascular endothelial growth factor ( VEGF) plays an important role in angiogenesis and vasculogenesis. The amount of VEGF detected has a direct correlation with the stage of malignancy. The ABC immunohistochemistry method was applied to expose protein VEGF within cancer patients. Clone VG1, Code M7273, LOT 00028659 were used as primary antibodies that binded to the antigen protein. The presence of VEGF protein was established by a dark bright precipitate stain. Results shows that immunohistochemistry staining using the ABC method detects the presence of VEGF and this protein has been found to be expressed in patients with colorectal carcinoma in a much higher intensity than in patients without colorectal cancer. The ABC method had contributed significantly to the search of colorectal carcinoma. [35]


Sacroiliac Joint Biopsies in Early Sacroiliitis [31]

Sacroiliitis is an inflammatory disease in the cavity of the sacroiliac joint. Immunohistochemistry was used to find out whether inflammatory cells are present within the sacroiliac joint. The tissue section was cut and stained with haematoxylin and eosin. The ABC method was employed and different primary antibodies were used to detect different types of inflammatory cells. [36]

Labelled StreptAvidin Biotin (LSAB) Method

This method is an improved method of the ABC method and because of this it overcomes some of the disadvantages that the ABC method poses and is one of the most used techniques in immunohistochemistry today. The LSAB method replaces the avidin used in the ABC method with streptavidin. Streptavidin is similar to avidin in structure and is tetrameric. Steptavidin has a high capability in binding biotin.

simple drawing showing how the streptavidin enzyme complex binds to the secondary body.

The method works similar to the ABC method. Primary antibody is added which binds to the antigen. Secondary antibody is added and the biotinylated antibody binds to the primary antibody. Streptavidin molecule binds to enzyme molecules directly and this complex is injected into the tissue section and penetrates through to the site of antigen. Peroxidase or alkaline phosphatase is added in the next step to enable the detection of the enzyme location.

There are a few reasons why this method has its advantage over the ABC method. The advantages stems from the fact that streptavidin has more advantages over avidin. Unlike avidin, streptavidin does not contains carbohydrates which prevents the non-specific binding of lectin-like molecules which are found naturlly in the body in the kidney,brain and liver. Avidin has an isoelectric point of 10 whereas strepavidin has an isoelectric point which is close to neutral. This neutral isoelectric point is beneficial because it does not elicit the non specific electrostatic binding of the streptavidin to natural tissue cells. The stablity of the complex used in the LSAB method is alot higher than the complex used in the ABC method due to the fact that streptavidin is conjugated directly to the enzyme. This allows the complex to be stored for a much longer period of time than the ABC complex. [37]


Advantages Disadvantages [38]
High stability Time consuming
Can be stored for longer period of time than ABC complex Produces a higher background staining as opposed to the Envision System
More specific staining than ABC method


Applications

Severe Scrub Typhus Confirmed Early via Immunohistochemical Staining [32]

Scrub typhus is a disease which is caused by orientia tsutsugamushi. Immunohistochemistry was used to detect the presence of orientia tsutsugamushi within tissue specimens. The LSAB method was exploited. The primary antibody used to bind to the antigen was ICR mouse hyperimmune serum. This antibody was diluted at 1:200. Appearance of a brown stain indicted the location of the antigen. [39]


Cellular Localization of EMMPRIN Predicts Prognosis of Patients with Operable Lung Adenocarcinoma Independent from MMP-2 and MMP-9 [33]

Extracellular matrix metalloproteinase inducer ( EMMPRIN ) is a protein which regulates MMP activity. This protein is known to play a role in the metastasis of neoplasms and is able to predict the survival rate of patients. In this study, immunohistochemisty was applied to primary lung tumors to detect the location and presence of EMMPRIN in the tissue section. The method used in this study was the LSAB method. The tissue section was cut, dewaxed, rehydrated and boiled for 20 minutes in a citric acid buffer. A commercial blocking agent was used to prevent the binding of non specific antibodies. The primary antibody was added which was a monoclonal mouse antbibody called HIM6. The section was then incubated overnight. A secondary antibody was then added which was a biotinylated anti- mouse/rabbit antibody. The streptavidin conjugate was then added. Aminoethylcarbazole dissolved in dimethylformamide and an acetate buffer was added and the stained the location of EMMPRIN a red-brown colour.[40]

Polymeric Methods

Diagram showing how the polymeric conjugates binds to the primary antibody

These methods were developed in search for immunohistochemistry methods that are both reliable and does not consume too many steps. It is a two step labelling method. An example of a polymer enhanced method is the EnVision method. In this technique, the primary antibody is applied to the tissue section, proceeded by the application of polymeric conjugates. The polymer is labeled with enzymes. The polymeric conjugates are made up centrally of a dextran backbone in which secondary antibodies and perioxidase binds onto. Each dextran backbone binds approximately 100 enzyme molecules and 20 antibody molecules. This dextran backbone allows for the attachment of a huge number of enzymes onto secondary antibodies. A chromogen substrate is then added into the complex.

When the polymeric conjugates is added, the secondary antibodies contained within it binds to the primary antibody which has been attached to the antigen. The enzyme attached to the polymer conjugate helps detect the location of the antigen within the tissue section by staining. [41] [42]


The Immpress system is another type of polymeric method used in immunohistochemistry. Unlike the EnVision system, this system does not require a dextran backbone. A primary antibody is added and binds to the antigen of interest. Next, a polymer chain consisting of a high number of enzyme monomers are synthesized and attached to secondary antibodies. This polymer chain is added and the secondary antibody binds to the primary antibody. The advantages and disadvantages of the Immpress system is similar to the EnVision system. The one advantage the ImmPress system has over the EnVision system is that it does not use the high molecular weight backbone and therefore penetrates through the tissue more efficiently.

Advantages Disadvantages
It is a two step process which therefore is more time efficient The high molecular weight of the backbone can reduce the efficiency of penetration into the tissue ( EnVision System ).
Very little to no background staining More expensive than other methods such as the ABC method. [43]
Extremely sensitive
No non-specific binding as it does not use biotin or avidin

Applications

Pathologic and Immunohistochemical Studies of Newcastle Disease ( ND ) in Broiler Chickens Vaccinated with ND : Severe Nonpurulent Encephalitis and Necrotizing Pancreatitis [34]

Newcastle disease is a detrimental disease poultry caused by the ND virus. Immunohistochemistry staining using the polymeric method was used to detect the ND virus antigen in tissue sections. Tissues were sectioned and stained with HE. The primary antibody which was a mouse monoclonal antibody was added followed by a labeled polymer made up of amino acid monomers. The enzyme attached was peroxidase and the secondary antibody attached was a goat anti-mouse Ig. [44]


Neuroendocrine Ductal Carcinoma in Situ of the Breast: Cytological Features in 32 Cases [35]

Patients that suffer neurodendocrine tumours of the breast have neuroendocrine markers expressed within their cells. The polymeric method was employed to detect neuroendocrine markers within tissue speciemens. The primary antibody used was the polyclonal rabbit antibody.[45]

Catalysed Signal Amplification (CSA) Methods

Direct Immunofluorescent Staining of CMV infected cells - Courtesy of PerkinElmer [www.perkinelmer.com/tsa]
TSA Enhanced Direct Immunofluorescent Staining of CMV infected cells - Courtesy of PerkinElmer [www.perkinelmer.com/tsa]

The Catalysed Signal Amplification (CSA) method, also known as the Tyramide Signal Amplification (TSA) method, is a highly sensitive and powerful technique used to detect tiny amounts of proteins.[46] This method’s high sensitivity allows it to detect weak signals coming from antigens that previously could not be seen in formalin-fixed, paraffin-embedded tissue, especially in human tissue. This method is up to 100 times more sensitive that the ABC method.

The CSA method uses biotinyl tyramide or BT, which is deposited at the site of the antigen. Biotinly tyramide is a substrate of peroxidise and is activated by the enzyme horseradish peroxidase (HRP). When the biotinyl tyramide is activated it turns into a very reactive molecule which then binds with proteins close by. This binding happens very rapidly (within 10 minutes). The amplification of the signal in the process then occurs when the Tyramide is detected within the tissue.

The Catalysed Signal Amplification II method uses a relatively similar procedure to the normal CSA method, however instead of utilising biotinyl tyramide, this method uses fluorescyl-tyramide (FT). The FT precipitates on the specimen and this reaction is followed with a secondary reaction with an anti-fluorescein. It is finished with a hydrogen peroxide/chromogen and can then be viewed under a light microscope. This system, like TSA allows for detection of small amounts of antigen and the use of low affinity antibodies. As this method uses FT instead of BT, any non-specific background staining caused by reactivity with endogenous biotin from liver and kidney tissue is avoided. [47] When comparing this method to other Immunohistochemistry techniques, the CSA methods have been shown to have greater sensitivity than most other techniques

In comparison to standard immunohistochemical methods, such as labelled streptavidin biotin (LSAB) or avidin-biotin complexes (ABC), tyramide amplification methods have been reported to be many fold more sensitive. However the recent technology, in situ immuno-PCR, has been shown to be more sensitive than CSA techniques.[48]


Advantages Disadvantages
Most sensitive of the IHC methods Complex staining proceedure
Reduction of use of reagents Time consuming
If FT is used, background staining from biotin is eliminated Endogenous biotin can cause non specific staining when BT is used.
Results are hard to reproduce


The use of automated immunostaining assays in labs, has recently sped up the CSA procedure and thus increasing the reproducibility of results in pathology labs. This new technology has allowed this sensitive technique to be applied in clinical and diagnostic settings rather than just as a research technique. [49]

Applications

Catalyzed Signal Amplification for Cyclin D1 Detection in Mantle Cell Lymphoma

This study was on Mantle cell lymphoma which only has a median survival rate of 3-5 years. CSA was used as a potential new tchnology to identify the different types of lymphoma and at an early time so that survival rates of cancer and in particular Mantle cell lymphoma can increase. [50]

Locked nucleic acid in situ hybridization analysis of miR-21 expression during colorectal cancer development

This study’s purpose was to understand better the function in carcinogenesis of microRNA miR-21. A biotin-free tyramide signal amplification system was used in order to minimise the over fluorescence of the proteins that already contain biotin. [51]

Proof of principle: an HIV p24 microsphere immunoassay with potential application to HIV clinical diagnosis

This study’s aim was to see whether the HIV-1 p24 antigen could be measured and what the conditions were that would be needed to process plasma samples. Using CSA increased the fluorescence levels, making the results clearer and more useful. [52]


Limitations

There are various limitations associated with immunohistochemistry:

  • There are some tumours for which there are no antibodies available which can be accepted to be specific to that type of tumour. This means that there is no specific test for this tumour and it requires a multimodal approach in order to reach a diagnosis. An example of this is malignant mesothelioma which is a malignant epithelial neoplasm which originates from the serosal surfaces of body cavities. [53]
  • The specificity of antibodies needs to be tested using controls to avoid false-positive results which are often a result of non-specific binding. [54]
  • There is a limited amount of antibodies that can sourced from a single animal and specificity is reduced when the protein used for immunisation and the contaminants are co-purified. This is overcome by using monoclonal antibodies, though monoclonal antibodies are more expensive. [55]


Sensitivity

A visual representation of the Sensitivity of Immunohistochemistry methods from least sensitive to most sensitive

Immunohistochemistry has numerous methods that can be used in the lab, with some having substantial advantages over others. One such property, sensitivity is often viewed as a limitation as many methods will not produce the required results. The sensitivity of a technique is important as it dictates a method’s use and also the results that will be obtained. A range of sensitivities can be seen within the methods and generally the more sensitive the method, the better the results that will be obtained.

The spectrum of sensitivity in the methods can be seen clearly in the diagram on the right with the most sensitive at the top of the curve and the least at the bottom.

The direct method was not included in this diagram as this technique is the least sensitive and is rarely used. Indirect methods such as the PAP, ABC, LSAB, polymeric methods are more sensitive and therefore more favoured.[56]

Useful Links

The Journal of Histochemistry and Cytochemistry [36]

The Histochemical Society [37]

Applied Immunohistochemistry and Molecular Morphology [38]

Glossary

ABC: Avidin-Biotin Complex

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.

Antiserum: A serum containing antibodies.

Blocking: A process used to limit or reduce the amount of background staining.

BT: Biotinyl Tyramide; a substrate of peroxidase.

Control: A standard which variables can be compared against.

CSA: Catalysed Signal Amplification

FT: Fluorescyl Tyramide

Glycoprotein: Proteins that contain oligosaccharide chains attached to polypeptide chains.

Immunocytochemistry: Another name for immunohistochemistry.

Immunofluorescence: Labelling of antibodies with fluorescent dyes

Immunoglobins: Another name for antibodies

LSAB: Labeled StreptAvidin Biotin

PAP: Peroxidase-Antiperoxidase

Pneumococci: Bacteria of the genus pneumococcus which causes diseases such as pneumonia.

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.

StreptAvidin: Derived from streptococcus avidini, it is an uncharged molecule used in the LSAB method.

TSA: Tyramide Signal Amplification. This is another name for Catalysed Signal Amplification (CSA).

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2010 Projects

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