Immunolabeling

Immunolabeling - Antigen Detection of Tissue via Tagged Antigen-specific Antibody

Immunolabeling is a biochemical process that enables the detection and localization of an antigen to a particular site within a cell, tissue, or organ. Antigens are organic molecules, usually proteins, capable of binding to an antibody. These antigens can be visualized using a combination of antigen-specific antibody as well as a means of detection, called a tag, that is covalently linked to the antibody.[1] If the immunolabeling process is meant to reveal information about a cell or its substructures, the process is called immunocytochemistry.[2] Immunolabeling of larger structures is called immunohistochemistry.[3]

There are two complex steps in the manufacture of antibody for immunolabeling. The first is producing the antibody that binds specifically to the antigen of interest and the second is fusing the tag to the antibody. Since it is impractical to fuse a tag to every conceivable antigen-specific antibody, most immunolabeling processes use an indirect method of detection. This indirect method employs a primary antibody that is antigen-specific and a secondary antibody fused to a tag that specifically binds the primary antibody. This indirect approach permits mass production of secondary antibody that can be bought off the shelf.[4] Pursuant to this indirect method, the primary antibody is added to the test system. The primary antibody seeks out and binds to the target antigen. The tagged secondary antibody, designed to attach exclusively to the primary antibody, is subsequently added.

Typical tags include: a fluorescent compound, gold beads, a particular epitope tag,[5] or an enzyme that produces a colored compound. The association of the tags to the target via the antibodies provides for the identification and visualization of the antigen of interest in its native location in the tissue, such as the cell membrane, cytoplasm, or nuclear membrane. Under certain conditions the method can be adapted to provide quantitative information.[4]

Immunolabeling can be used in pharmacology, molecular biology, biochemistry and any other field where it is important to know of the precise location of an antibody-bindable molecule.[6][7][8]

  1. ^ Hyatt, A.D., & Wise, T.G. (2001). "Immunolabeling". Immunocytochemistry and in Situ Hybridization in the Biomedical Sciences. Boston, MA: Birkhauser Boston. pp. 73–107. doi:10.1007/978-1-4612-0139-7_5. ISBN 978-1-46-12-0139-7.{{cite book}}: CS1 maint: multiple names: authors list (link)
  2. ^ Nanci A, Wazen R, Nishio C, Zalzal SF (2008). "Immunocytochemistry of matrix proteins in calcified tissues: functional biochemistry on section". Eur J Histochem. 52 (4): 201–14. doi:10.4081/1218. PMID 19109094.
  3. ^ Swanson PE (September 1988). "Foundations of immunohistochemistry. A practical review". Am. J. Clin. Pathol. 90 (3): 333–9. doi:10.1093/ajcp/90.3.333. PMID 3046324.
  4. ^ a b Rapley R, Walker JM, eds. (2008). Molecular biomethods handbook (2nd ed.). Totowa, NJ: Humana Press. p. 1066. ISBN 978-1-60327-370-1.
  5. ^ Pang J, Zeng X, Xiao RP, Lakatta EG, Lin L (June 2009). "Design, generation, and testing of mammalian expression modules that tag membrane proteins". Protein Sci. 18 (6): 1261–71. doi:10.1002/pro.136. PMC 2774436. PMID 19472344.
  6. ^ Rangell LK, Keller GA (August 2000). "Application of microwave technology to the processing and immunolabeling of plastic-embedded and cryosections". Journal of Histochemistry and Cytochemistry. 48 (8): 1153–9. doi:10.1177/002215540004800812. PMID 10898808.
  7. ^ Malecki M, Hsu A, Truong L, Sanchez S (January 2002). "Molecular immunolabeling with recombinant single-chain variable fragment (scFv) antibodies designed with metal-binding domains". Proceedings of the National Academy of Sciences of the United States of America. 99 (1): 213–8. Bibcode:2002PNAS...99..213M. doi:10.1073/pnas.261567298. PMC 117541. PMID 11756693.
  8. ^ Haycock JW (September 1989). "Quantitation of tyrosine hydroxylase, protein levels: spot immunolabeling with an affinity-purified antibody". Analytical Biochemistry. 181 (2): 259–66. doi:10.1016/0003-2697(89)90240-6. PMID 2573292.