Citizendia
Your Ad Here

Immunohistochemistry or IHC refers to the process of localizing proteins in cells of a tissue section exploiting the principle of antibodies binding specifically to antigens in biological tissues. Antibodies (also known as immunoglobulins, abbreviated Ig) are Gamma globulin Proteins that are found in Blood or other Bodily An antigen (from antibody-generating) or immunogen is a substance that prompts the generation of Antibodies and can cause an immune response Tissue is a cellular organizational level intermediate between cells and a complete organism [1] It takes its name from the roots "immuno," in reference to antibodies used in the procedure, and "histo," meaning tissue (compare to immunocytochemistry). Immunocytochemistry or ICC is a common lab practice which uses antibodies that target specific peptides or proteins antigens in the cell via specific epitopes Immunohistochemical staining is widely used in the diagnosis of abnormal cells such as those found in cancerous tumors. Specific molecular markers are characteristic of particular cellular events such as proliferation or cell death (apoptosis). IHC is also widely used in basic research to understand the distribution and localization of biomarkers and differentially expressed proteins in different parts of a biological tissue.

Visualising an antibody-antigen interaction can be accomplished in a number of ways. In the most common instance, an antibody is conjugated to an enzyme, such as peroxidase, that can catalyse a colour-producing reaction (see immunoperoxidase staining). Peroxidases ( EC number 1111x are a large family of Enzymes A majority of peroxidase protein sequences can be found in the PeroxiBase database Immunoperoxidase is a type of immunostain used in Molecular biology, medical research and clinical diagnostics Alternatively, the antibody can also be tagged to a fluorophore, such as FITC, rhodamine, Texas Red, Alexa Fluor, or DyLight Fluor(see immunofluorescence). Fluorescein isothiocyanate (FITC is a derivative of Fluorescein used in wide-ranging applications including Flow cytometry. Rhodamine (ˈroʊdəmiːn is a family of related chemical compounds Fluorone dyes Texas Red or sulforhodamine 101 acid chloride is a red Fluorescent Dye, with a molecular weight of 625 daltons used in Histology History The Alexa Fluor dyes were named after Alex Haugland son of the founders of Molecular Probes Richard and Rosaria Haugland Immunofluorescence is the labeling of antibodies or Antigens with fluorescent Dyes This technique is often used to visualize the subcellular The latter method is of great use in confocal laser scanning microscopy, which is highly sensitive and can also be used to visualise interactions between multiple proteins. Confocal laser scanning microscopy ( CLSM or LSCM) is a technique for obtaining high- resolution optical images Förster resonance energy transfer (abbreviated FRET) also known as Fluoresence resonance energy transfer or resonance energy transfer ( RET

Contents

Antibody types

The antibodies used for specific detection can be polyclonal or monoclonal. Polyclonal antibodies (or antisera are antibodies that are derived from different B cell lines Monoclonal antibodies ( mAb or moAb) are monospecific antibodies that are identical because they are produced by one type of immune cell Monoclonal antibodies are generally considered to exhibit greater specificity. Polyclonal antibodies are made by injecting animals with peptide antigens, and then after a secondary immune response is stimulated, isolating antibodies from whole serum. Thus, polyclonal antibodies are a heterogeneous mix of antibodies that recognize several epitopes. An epitope, also known as antigenic determinant, is the part of a Macromolecule that is recognized by the Immune system, specifically by antibodies

Antibodies can also be classified as primary or secondary reagents. Primary antibodies are raised against an antigen of interest and are typically unconjugated (unlabelled), while secondary antibodies are raised against primary antibodies. Hence, secondary antibodies recognize immunoglobulins of a particular species and are conjugated to either biotin or a reporter enzyme such as alkaline phosphatase or horseradish peroxidase. Vitamin H redirects here In medical slang "vitamin H" may also refer to Haloperidol. Enzymes are Biomolecules that catalyze ( ie increase the rates of Chemical reactions Almost all enzymes are Proteins Alkaline phosphatase ( ALP) ( is a Hydrolase Enzyme responsible for removing Phosphate groups from many types of molecules including The Enzyme horseradish peroxidase (HRP found in Horseradish, is used extensively in Molecular biology applications primarily for its ability to amplify Some secondary antibodies are conjugated to fluorescent agents, such as the Alexa Fluor or Dylight Fluor family, are also frequently used for detection of proteins in IHC procedures. Fluorescence is a Luminescence that is mostly found as an Protein concentration is generally measured by densitometry analysis, where the intensity of staining correlates with the amount of the protein of interest.

Sample preparation

In the procedure, depending on the purpose and the thickness of the experimental sample, either thin (about 4-40 μm) slices are taken of the tissue of interest, or if the tissue is not very thick and is penetrable it is used whole. A micrometre ( American spelling: micrometer; symbol µm) is one millionth of a Metre, or equivalently one thousandth of a Millimetre The slicing is usually accomplished through the use of a microtome, and slices are mounted on slides. A microtome is a mechanical instrument used to cut biological specimens into transparent thin sections for microscopic examination "Free-floating IHC" uses slices that are not mounted, these slices are normally produced using a vibrating microtome. A microtome is a mechanical instrument used to cut biological specimens into transparent thin sections for microscopic examination

Direct and indirect IHC

The direct method of immunohistochemical staining uses one labelled antibody, which binds directly to the antigen being stained for.
The direct method of immunohistochemical staining uses one labelled antibody, which binds directly to the antigen being stained for.

There are two strategies used for the immunohistochemical detection of antigens in tissue, the direct method and the indirect method. In both cases, the tissue is treated to rupture the membranes, usually by using a kind of detergent such as Triton X-100. The cell membrane (also called the plasma membrane, plasmalemma, or "phospholipid bilayer" is a Selectively permeable Lipid bilayer Triton X-100 ( is a Nonionic Surfactant which has a Hydrophilic Polyethylene oxide group (on average it has 9 Some antigen also need additional step for unmasking, resulting in better detection results.

The direct method is a one-step staining method, and involves a labeled antibody (e. Staining is an auxiliary Technique used in Microscopy to enhance contrast in the microscopic image Antibodies (also known as immunoglobulins, abbreviated Ig) are Gamma globulin Proteins that are found in Blood or other Bodily g. FITC conjugated antiserum) reacting directly with the antigen in tissue sections. Fluorescein isothiocyanate (FITC is a derivative of Fluorescein used in wide-ranging applications including Flow cytometry. This short article on antiserum deals exclusively with its applications An antigen (from antibody-generating) or immunogen is a substance that prompts the generation of Antibodies and can cause an immune response This technique utilizes only one antibody and the procedure is therefore simple and rapid. Antibodies (also known as immunoglobulins, abbreviated Ig) are Gamma globulin Proteins that are found in Blood or other Bodily However, it can suffer problems with sensitivity due to little signal amplification and is in less common use than indirect methods.

The indirect method of immunohistochemical staining uses one antibody against the antigen being probed for, and a second, labelled, antibody against the first.
The indirect method of immunohistochemical staining uses one antibody against the antigen being probed for, and a second, labelled, antibody against the first.

The indirect method involves an unlabeled primary antibody (first layer) which reacts with tissue antigen, and a labeled secondary antibody (second layer) which reacts with the primary antibody. An antigen (from antibody-generating) or immunogen is a substance that prompts the generation of Antibodies and can cause an immune response A secondary antibody is an Antibody that binds to Primary antibodies or antibody fragments (The secondary antibody must be against the IgG of the animal species in which the primary antibody has been raised. Immunoglobulin G ( IgG) is a Monomeric Immunoglobulin, built of two heavy chains γ and two light chains. ) This method is more sensitive due to signal amplification through several secondary antibody reactions with different antigenic sites on the primary antibody. The second layer antibody can be labeled with a fluorescent dye or an enzyme. Enzymes are Biomolecules that catalyze ( ie increase the rates of Chemical reactions Almost all enzymes are Proteins

In a common procedure, a biotinylated secondary antibody is coupled with streptavidin-horseradish peroxidase. Streptavidin is a 53000 dalton tetrameric Protein purified from the Bacterium Streptomyces avidinii. This is reacted with 3,3'-Diaminobenzidine (DAB) to produce a brown staining wherever primary and secondary antibodies are attached in a process known as DAB staining. 33'-Diaminobenzidine (DAB is a Chemical compound. It is usually used as the water-soluble tetrahydrochloride in permanent Immunohistochemical staining The reaction can be enhanced using nickel, producing a deep purple/gray staining. Nickel (ˈnɪkəl is a metallic Chemical element with the symbol Ni and Atomic number 28

The indirect method, aside from its greater sensitivity, also has the advantage that only a relatively small number of standard conjugated (labeled) secondary antibodies needs to be generated. For example, a labeled secondary antibody raised against rabbit IgG, which can be purchased "off the shelf," is useful with any primary antibody raised in rabbit. With the direct method, it would be necessary to make custom labeled antibodies against every antigen of interest.

Diagnostic IHC markers

IHC is an excellent detection technique and has the tremendous advantage of being able to show exactly where a given protein is located within the tissue examined. This has made it a widely-used technique in the neurosciences, enabling researchers to examine protein expression within specific brain structures. Neuroscience is a field devoted to the scientific study of the nervous system Its major disadvantage is that, unlike immunoblotting techniques where staining is checked against a molecular weight ladder, it is impossible to show in IHC that the staining corresponds with the protein of interest. The western blot (alternatively immunoblot) is an Analytical technique used to detect specific Proteins in a given sample of tissue homogenate or The molecular mass (abbreviated m of a substance, more commonly referred to as molecular weight and abbreviated as MW, is the Mass of one For this reason, primary antibodies must be well-validated in a Western Blot or similar procedure. The western blot (alternatively immunoblot) is an Analytical technique used to detect specific Proteins in a given sample of tissue homogenate or The technique is even more widely used in diagnostic surgical pathology for typing tumors (e. Surgical pathology is the most significant and time-consuming area of practice for most anatomical pathologists. g. carcinoma vs melanoma).

Directing therapy

A variety of molecular pathways are altered in cancer and some of the alterations can be targeted in cancer therapy. The cluster of differentiation (often abbreviated as CD) is a protocol used for the identification and investigation of Cell surface molecules present on Leukocytes Hodgkin's lymphoma, also known as Hodgkin's disease is a type of Lymphoma first described by Thomas Hodgkin in 1832 Alpha-fetoprotein ( AFP) is a molecule produced in the developing Embryo Endodermal sinus tumor ( EST) also known as Yolk sac tumor ( YST) is a member of the Germ cell tumor group of Cancers Hepatocellular carcinoma (HCC also called hepatoma) is a primary malignancy (cancer of the Liver. CD117, also called KIT or C-kit receptor, is a Cytokine receptor expressed on the surface of Hematopoietic stem cells as well as other cell types A gastrointestinal stromal tumor ( GIST) is a rare Tumor of the Gastrointestinal tract (1-3% of all gastrointestinal malignancies Prostate specific antigen ( PSA) is a protein produced by the cells of the Prostate gland Estrogens (US otherwise oestrogens or œstrogens) are a group of Steroid compounds named for their importance in the Estrous cycle, Progesterone is a C-21 Steroid Hormone involved in the Female Menstrual cycle, Pregnancy (supports Gestation B cells are Lymphocytes that play a large role in the humoral immune response (as opposed to the cell-mediated immune response, which is governed by Lymphoma a type of Neoplasm that originates in Lymphocytes (a type of White blood cell in the vertebrate Immune system) CD20 is a non- Glycosylated phosphoprotein expressed on the surface of all mature B-cells. T cells belong to a group of White blood cells known as Lymphocytes, and play a central role in Cell-mediated immunity. Lymphoma a type of Neoplasm that originates in Lymphocytes (a type of White blood cell in the vertebrate Immune system) Immunohistochemistry can be used to assess which tumors are likely to respond to therapy, by detecting the presence or elevated levels of the molecular target.

Chemical inhibitors

Tumor biology allows for a number of potential intracellular targets. Many tumors are hormone dependent. The presence of hormone receptors can be used to determine if a tumor is potentially responsive to antihormonal therapy. One of the first therapies was the antiestrogen, tamoxifen, used to treat breast cancer. Tamoxifen is an orally active Selective estrogen receptor modulator (SERM that is used in the treatment of Breast cancer and is currently the world's largest Such hormone receptors can be detected by immunohistochemistry. [3] Imatinib, an intracellualar tyrosine kinase inhibitor, was developed to treat chronic myelogenous leukemia, a disease characterized by the formation of a specific abnormal tyrosine kinase. Imatinib is a drug used to treat certain types of Cancer. It is currently marketed by Novartis as Gleevec ( USA) or Glivec A tyrosine kinase is an Enzyme that can transfer a Phosphate group from ATP to a Tyrosine residue in a Protein. Chronic myelogenous (or myeloid leukemia ( CML) is a form of Leukemia characterized by the increased and unregulated growth of predominantly Myeloid cells Imitanib has proven effective in tumors, that express other tyrosine kinases, most notably KIT. Most gastrointestinal stromal tumors express KIT, which can be detected by immunohistochemistry. A gastrointestinal stromal tumor ( GIST) is a rare Tumor of the Gastrointestinal tract (1-3% of all gastrointestinal malignancies [4]

Monoclonal antibodies

Main article: Monoclonal antibody therapy

Many proteins shown to be highly upregulated in pathological states by immunohistochemistry are potential targets for therapies utilising monoclonal antibodies. Monoclonal antibody therapy is the use of Monoclonal antibodies (or mAb to specifically target cells Monoclonal antibodies ( mAb or moAb) are monospecific antibodies that are identical because they are produced by one type of immune cell Monoclonal antibodies, due to their size, are utilized against cell surface targets. Among the overexpressed targets, the members of the epidermal growth factor receptor (EGFR) family, transmembrane proteins with an extracellular receptor domain regulating an intracellular tyrosine kinase, [5] Of these, HER2/neu (also known as Erb-B2) was the first to be developed. The epidermal growth factor receptor (EGFR ErbB-1 HER1 in humans is the cell-surface receptor for members of the Epidermal growth factor family (EGF-family HER2/neu (also known as ErbB-2) stands for "Human Epidermal growth factor Receptor 2" and is a protein giving higher aggressiveness in Breast cancers It is a The molecule is highly expressed in a variety of cancer cell types, most notably breast cancer. As such, antibodies against HER2/neu have been FDA approved for clinical treatment of cancer under the drug name Herceptin. There are commercially available immunohistochemical tests, Dako HercepTest and Ventana Pathway. Ventana Medical Systems Inc is a global leader in tissue diagnostics that develops manufactures and markets medical diagnostic instrument and reagent systems that automate tissue preparation [6] Similarly, EGFR (HER-1) is overexpressed is a variety of cancers including head and neck and colon. Immunohistochemistry is used to determined patients who may benefit from therapeutic antibodies such as Erbitux (cetuximab). Cetuximab (IMC-C225 -- marketed under the name Erbitux) is a chimeric Monoclonal antibody, an Epidermal growth factor receptor (EGFR inhibitor [7] Commercial systems to detect EGFR by immunohistochemistry include the Dako pharmDx.

References

  1. ^ Ramos-Vara, JA (2005). "Techical Aspects of Immunohistochemistry". Vet Pathol 42: 405-426.  
  2. ^ Leader M, et al. An analysis of the sensitivity and specificity of the cytokeratin marker CAM 5. 2 for epithelial tumours. Results of a study of 203 sarcomas, 50 carcinomas and 28 malignant melanomas. Histopathology. 1986;10:1315"{{{title}}}" .  
  3. ^ Jørgensen, Jan Trøst; Kirsten Vang Nielsen, Bent Ejlertsen (April 2007). "Pharmacodiagnostics and targeted therapies - a rational approach for individualizing medical anticancer therapy in breast cancer.". The Oncologist 12 (4): 397-405. United States: AlphaMed Press. doi:10.1634/theoncologist.12-4-397. A digital object identifier ( DOI) is a permanent identifier given to an Electronic document. ISSN 1083-7159. An International Standard Serial Number ( ISSN) is a unique eight-digit number used to identify a print or electronic Periodical publication. PMID 17470682.  
  4. ^ Gold, Jason S. ; Ronald P. DeMatteo (August 2006). "Combined surgical and molecular therapy: the gastrointestinal stromal tumor model.". Annals of Surgery 244 (2): 176-184. United States: Lippincott Williams & Wilkins. doi:10.1097/01.sla.0000218080.94145.cf.. A digital object identifier ( DOI) is a permanent identifier given to an Electronic document. ISSN 0003-4932. An International Standard Serial Number ( ISSN) is a unique eight-digit number used to identify a print or electronic Periodical publication. PMID 16858179.  
  5. ^ Harari, P M (December 2004). "Epidermal growth factor receptor inhibition strategies in oncology". Endocrine-Related Cancer 11 (4): 689-708. England: Society for Endocrinology. doi:10.1677/erc.1.00600. A digital object identifier ( DOI) is a permanent identifier given to an Electronic document. ISSN 1351-0088. An International Standard Serial Number ( ISSN) is a unique eight-digit number used to identify a print or electronic Periodical publication. PMID 15613446.  
  6. ^ Press, Michael F. ; Guido Sauter, Leslie Bernstein, Ivonne E. Villalobos, MartinaMirlacher, Jian-Yuan Zhou, RoobaWardeh, Yong-Tian Li, Roberta Guzman, Yanling Ma, Jane Sullivan-Halley, Angela Santiago, Jinha M. Park, Alessandro Riva, Dennis J. Slamon (September 15, 2005). Events 668 - Eastern Roman Emperor Constans II is assassinated in his bath at Syracuse Italy. Year 2005 ( MMV) was a Common year starting on Saturday (link displays full calendar of the Gregorian calendar. "Diagnostic evaluation of HER-2 as a molecular target: an assessment of accuracy and reproducibility of laboratory testing in large, prospective, randomized clinical trials.". Clinical Cancer Research 2005 15;11(18): (18): 6598-6607. United States: American Association for Cancer Research. . doi:10.1158/1078-0432.CCR-05-0636. A digital object identifier ( DOI) is a permanent identifier given to an Electronic document. ISSN 1078-0432. An International Standard Serial Number ( ISSN) is a unique eight-digit number used to identify a print or electronic Periodical publication. PMID 16166438.  
  7. ^ Bibeau, Frédéric; Florence Boissière-Michot, Jean-Christophe Sabourin, Sophie Gourgou-Bourgade, Michèle Radal, Frédérique Penault-Llorca, Philippe Rochaix, Laurent Arnould, Marie-Pierre Bralet, David Azria, Marc Ychou (July 25, 2006). Events 285 - Diocletian appoints Maximian as Caesar, co-ruler Year 2006 ( MMVI) was a Common year starting on Sunday of the Gregorian calendar. "Assessment of epidermal growth factor receptor (EGFR) expression in primary colorectal carcinomas and their related metastases on tissue sections and tissue microarray". Virchows Archiv 449 (3): 281–287. Germany: Springer International. doi:10.1007/s00428-006-0247-9. A digital object identifier ( DOI) is a permanent identifier given to an Electronic document. ISSN 0945-6317. An International Standard Serial Number ( ISSN) is a unique eight-digit number used to identify a print or electronic Periodical publication. PMID 16865406.  

External links

Medical Subject Headings ( MeSH) is a huge Controlled vocabulary (or metadata system for the purpose of indexing journal articles and books

Dictionary

immunohistochemistry

-noun

  1. The analytical process of finding proteins in cells of a tissue microtome section exploiting the principle of antibodies binding specifically to antigens in biological tissues.
© 2009 citizendia.org; parts available under the terms of GNU Free Documentation License, from http://en.wikipedia.org
 Dapyx Software network: MP3 Explorer | Ebook Manager | Zenithic