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Immunoassay

Chemiluminescence immunoassay

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Immunoassay

  1. 1. An immunoassay is a test that uses antibody and antigen complexes as a means of generating a measurable result
  2. 2. * It is the light emission that is produced in certain chemical oxidation reactions. [A] + [B] → [◊] → [Products] + light
  3. 3. *About 1000 B.C., a Chinese emperor possessed a magic paint on which the image of an ox appeared at sunset. This is the first known case of a man-made substance capable of storing daylight for later recovery. *Robert Boyle first estabilished the significance of oxygen in bioluminescence *luminous fungus and luminous bacteria was first reported by Heller in 1843
  4. 4. *In 1877, Radziszewski reported for the first time on the CL exhibited by the synthetic organic compound lophine (2,4,5-triphenylimidazole)
  5. 5. *The term “chemiluminescence” was first coined by Eilhardt Weideman in 1888. *Albrecht, who described the intense luminescence associated with the alkaline oxidation of luminol *Seitz and Neary reported in the same year on the advantages of CL and BL for chemical analysis
  6. 6. *Chemiluminescence (CL)defined as the emission of electromagnetic radiation produced by a chemical reaction. *reactions generally yield one of the reaction products in an electronic excited state producing light on falling to the ground state *the emission intensity is a function of the concentration of the chemical species involved in the CL reaction
  7. 7. *In general, a chemiluminescent reaction can be generated by two basic mechanisms: *Direct Reaction *Indirect reaction
  8. 8. *In a direct reaction, two reagents, usually a substrate and an oxidant in the presence of some cofactors, react to form a product or intermediate, sometimes in the presence of a catalyst. Then some fraction of the product or intermediate will be formed in an electronically excited state, which can subsequently relax to the ground state with emission of a photon. The catalyst, enzyme or metal ions, reduces the activation energy and provides an adequate environment for producing high CL efficiency out of the process.
  9. 9. *indirect or sensitized CL is based on a process of transfer of energy of the excited specie to a fluorophore. This process makes it possible for those molecules that are unable to be directly involved in CL reactions to transfer their excess of energy to a fluorophore that in turn is excited, releasing to its ground state with photon emission
  10. 10. *Luminol *Isoluminol *Acridium ester *derivatives of lucigenin
  11. 11. *Hydrogen peroxide *Oxygen *Iodide *Hypochlorite *Permangnate
  12. 12. *Transition meta ion *Heme compounds *ferricyanide
  13. 13. *reaction must be exothermic to produce sufficient energy to form the electronically excited state. *reaction pathway must be favorable to channel the energy for the formation of an electronically excited state
  14. 14. *direct CL; *molecular dissociation *chemical reaction with other species *Intramolecular energy transfer *intermolecular energy transfer *Isomerization *physical quenching.
  15. 15. *chemical structure of the CL precursor *nature and concentration of other substrates affecting the CL pathway *selected catalyst *presence of metal ions, especially transition metals involved in the processing of the oxidant *Temperature *pH and ionic strength *presence of energy transfer acceptors
  16. 16. *It is the emission of light caused by a chemical reaction, typically an oxidation reaction, producing an excited molecule that decays back to its original ground state. * A large number of molecules are capable of chemiluminescence, but some of the most common substances used are luminol, acridinium esters, ruthenium derivatives, and nitrophenyl oxalates. *When these substances are oxidized, typically using hydrogen peroxide and an enzyme for a catalyst, intermediates are produced that are of a higher energy state. *These intermediates spontaneously return to their original state, giving off energy in the form of light. *The emitted light is measured by using luminometer.
  17. 17. *In a CL reaction, A and B react to form a product C, some fraction of which is present in an electronically excited state, C∗, which subsequently relaxes to the ground state emitting a photon: A + B → C∗ → C + hν
  18. 18. *The fraction of molecules emitting a photon on return to the ground state is the quantum yield (φcl). It is the product of three ratios: φcl = φc × φe × φf Where, Φc:fraction of reacting molecules giving an excitable molecule, Φe:fraction of such molecules in an electronically excited state, Φf:fraction of these excited molecules that return to the ground state by emitting a photon,
  19. 19. *Heterogeneous immunoassay *Homogeneous immunoassay
  20. 20. In heterogeneous assays, competitive and sandwich formats are the ones most often used. Smaller analytes such as therapeutic drugs and steroid hormones are measured using competitive assays, while the sandwich format is used for larger analytes such as protein and hormones.
  21. 21. *Two different types of nonseparation chemiluminescent immunoassays have been described. One is based on the enhancement of light emission when a labeled antigen binds to a specific antibody. *The other exploits an energy transfer between chemiluminescently labeled and a fluorophore-labeled assay components
  22. 22. *Chemiluminescent assays have an excellent sensitivity, comparable to EIA and RIA *The reagents are stable and relatively nontoxic. *The sensitivity of some assays has been reported to be in the range of attomoles (10-18 mol) to zeptomoles (10-21 mol). *Because very little reagent is used, they are also quite inexpensive to perform. *The relatively high speed of detection also means a faster turnaround time. *Detection systems basically consist of photomultiplier tubes, which are simple and relatively inexpensive.
  23. 23. *false results may be obtained if there is lack of precision in injection of the hydrogen peroxide or if some biological materials such as urine or plasma cause quenching of the light emission.
  24. 24. *Determination of bioanalyte in clinical specimens *Chemiluminescence detection in Liquid Chromatography *Chemiluminescence detection in capillary electrophoresis *Bioanalytical applications of chemiluminescent imaging *Chemiluminescence in DNA analysis *Determination of drugs using direct chemiluminescence
  25. 25. •Chemiluminescence in analytical chemistry by Ana M Gracia Campana, •Immunoassay by Eleftherios P. Diamandis, Theodore K. Christopoulos •Tietz Text Book of Clinical Chemistry and Molecular Diagnosis

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Chemiluminescence immunoassay

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