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Chemiluminescence Immunoassay (CLIA) Technique

Chemiluminescence Immunoassay (CLIA) using Microplate luminometers provides a sensitive, high throughput, and economical way to quantitatively measure antigen in cell lysates, plasma, urine, saliva, tissue and culture media samples.
Chemiluminescence Immunoassay does not require long incubations and the addition of stopping reagents, as is the case in conventional colorimetric assays such as Enzyme-linked ImmunoSorbent Assays (ELISA).
Among various enzyme assays that employ light-emitting reactions, one of the most successful assays is the enhanced chemiluminescent immunoassay involving a horseradish peroxidase (HRP) labeled antibody or antigen and a mixture of chemiluminescent substrate, hydrogen peroxide, and enhancers.

In recent years, CLIA has become very popular in clinical chemistry and environmental analysis, due to its high sensitivity, wide dynamic range and complete automation. With the development and application of recombinant Ab (rAb) technology, markers and related techniques, solid-phase materials and improvements in automation, integration and miniaturization, CLIA has acquired an entirely new appearance.

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Chemiluminescence Immunoassay (CLIA) Technique

  1. 1. ChemiluminescenceChemiluminescence Immunoassay (CLIA) TechniqueImmunoassay (CLIA) Technique TapeshwarYadav (Lecturer) BMLT, DNHE, M.Sc. Medical Biochemistry
  2. 2. Background: Immunoassay (IA) as an analytical technique is widely applied in clinical chemistry, bioanalysis, toxicological analysis, pharmaceutical analysis, and environmental analysis, due to its high sensitivity, high selectivity, rapid detection and possible analysis of difficult matrices without extensive pre-treatment. Radioimmunoassay (RIA) has been developed since Yalow and Berson introduced it in 1959 by using 125 I as a label. Although RIA methods are reliable and accurate, they suffer from the problems associated with radioisotopes, which restrict their use to specialized laboratories. They also suffer from the drawback of the short half-life using 125 I as a label
  3. 3. Contd… Enzyme-linked immunosorbent assays (ELISAs) are among the most extensively used types of IA and are safer and easier than the RIA. ELISA could be based on colorimetric, fluorescence or Chemiluminescence (CL) detection. However, the sensitivity of conventional colorimetric detection is relatively low. IA has been applied to develop assays with very high sensitivity detection, which strongly depends on the affinity of specific antibodies (Abs) and on the sensitivity of the detection method.
  4. 4. Contd… Among assay methods, Chemiluminescence (CL) detection represents a versatile, ultrasensitive tool with a wide range of applications in biotechnology. It also gives a sensitive, rapid alternative to radioactivity as a detection principle in IA for the determination of molecules (e.g., proteins, hormones, drugs, nucleic acids and environmental pollutants). CL is now commonly used for IA in the form of a CL label or as a CL detection reaction for an enzyme or a nanoparticle (NP) label.  In recent years, CLIA has become very popular in clinical chemistry and environmental analysis, due to its high sensitivity, wide dynamic range and complete automation. With the development and application of recombinant Ab (rAb) technology, markers and related techniques, solid-phase materials and improvements in automation, integration and miniaturization, CLIA has acquired an entirely new appearance.
  5. 5. LUMINESCENCELUMINESCENCE “ Cold light” that can be emitted at lower temperature Source kicks an electron of an atom out of its lowest energy “ground” state into a higher energy “excited” state  Finally electron returns the energy in the form of light so it can fall back to its “ground” state
  6. 6. TYPES LUMINESCENCETYPES LUMINESCENCE Excitation event process Chemicals Luminol Isoluminol acridinium ester Chemiluminescence Biochemical Luciferin aequorin Bioluminescence Electromagnetic Ruthenium Tris (bipyridly) chelate Electroluminescence Photons inorganic phosphors Photoluminescence
  7. 7. CHEMILUMINESCENCE Emission of light with limited emission of heat (luminescence), as the result of a chemical reaction. [A] + [B] [◊] [→ → Products] + light [A], [B]: reactants [◊]: excited intermediate
  8. 8. For example, if [A] is luminol and [B] is hydrogen peroxide in the presence of a suitable catalyst we have: luminol + H2O2 →3-APA[◊] →3-APA + light Where: 3-APA is 3-aminophthalate 3-APA[◊] is the excited state producing light as it decays to a lower energy level.
  9. 9. CHEMILUMINISCENCE Luminol and peroxidase before adding H2O2 Chemiluminiscence after addition H2O2
  10. 10. Application of Chemiluminiscence  Chemiluminesence immunoassay  DNA hybridization detection  Western blotting  Forensic science  Food analysis
  11. 11. ChemiluminescenceChemiluminescence Immunoassay (CLIA) TechniqueImmunoassay (CLIA) Technique
  12. 12. Introduction: Chemiluminescence Immunoassay (CLIA) using Microplate luminometers provides a sensitive, high throughput, and economical way to quantitatively measure antigen in cell lysates, plasma, urine, saliva, tissue and culture media samples. Chemiluminescence Immunoassay does not require long incubations and the addition of stopping reagents, as is the case in conventional colorimetric assays such as Enzyme-linked ImmunoSorbent Assays (ELISA). Among various enzyme assays that employ light-emitting reactions, one of the most successful assays is the enhanced chemiluminescent immunoassay involving a horseradish peroxidase (HRP) labeled antibody or antigen and a mixture of chemiluminescent substrate, hydrogen peroxide, and enhancers.
  13. 13. Contd… CLIA are designed to detect glow-based chemiluminescent reactions. This provide a broader dynamic assay range, superior low-end sensitivity, and a faster protocol than the conventional Enzyme- linked immunosorbent assays (ELISA). It covers Thyroid function markers, Gonadal hormones, Tumor markers, Diabete marker, Cardiac marker and other markers. They can be used to replace conventional colorimetric ELISAs that have been widely used in many research and diagnostic applications.
  14. 14. CHEMILUMINESCENCE IMMUNOASSAY (CLIA) Provides a sensitive, high throughput alternative to conventional colorimetric methodologies. Principle: -same as ELISA -uses chemiluminescent substrate, hydrogen peroxide, enhancers -stopping reagent is not required -Incubation period is small
  15. 15. Principle: In the presence of complimentary antigen and antibody, the paratope of the antibody binds to the epitope of the antigen to form an Ag-Ab or an immune complex.  Estimating the levels of such immune complex by use of labeled antibodies form the basis of CLIA. It involves use of stationary solid particles coated either with the Ag or Ab of interest. Post incubation, which ensures intact immune complexes are formed, substrate is added. This results in generation of light, the intensity of which is directly proportional to the amount of labeled complexes present and which indirectly aids in quantification of the analyte of interest. The intensity of light is measured in terms of Relative Light Units (RLU).
  16. 16. Monoclonal antibody coated well Test specimen (serum) HRP labelled antibody conjugate Test antigen: sandwich between solid phase Ab and enzyme labelled Ab
  17. 17. Incubate for 1 hr at 37° C Remove unbound enzyme labeled Ab Chemiluminescence reagent added Read relative light unit with luminometer
  18. 18. Principle:
  19. 19.  Labels: Reagents required for reactions that produce CL may be coupled to Abs or antigens (Ags) and used as labels for IA. Since the first report on CL labels in 1976 This category involves labels that are consumed in the CL analytical reaction (e.g., luminol derivatives, acridinium esters and NPs). Luminol is the best known and one of the most efficient CL reagents. It is coupled to ligands via reactions involving the amino group.  However, the resulting conjugates have lower CL efficiencies than the parent compounds. Labels derived from isoluminol have been more successful.    Solid-phase materials: Commonly used solid-phase are 96-well microtitrationplates prepared with polystyrene. For the purposes of IA, the microplates are pre-coated with capturing protein like Ab to allow analyte immobilization.
  20. 20. Instrumentation:
  21. 21. • USES Hormones: insulin, thyroxin, estradiol, testosterone, progesterone Vitamin: vit B12 Tumor markers: bone morphogenic protein-2, carcino embryonic antigen (CEA), alpha fetoprotein (AFP) Human beta chorionic gonadotropin C-reactive protein Tumor necrosis factor
  22. 22. DNA hybridization detection Southern blotting Involves DNA separation, transfer and hybridization Hybridization - Process of forming a double-stranded DNA molecule between a single-stranded DNA probe and a single- stranded target DNA
  23. 23. 3.The restriction fragments present in the gel are denatured with alkali and transferred onto a nitrocellulose filter or nylon membrane by blotting.  This procedure preserves the distribution of the fragments in the gel, creating a replica of the gel on the filter.
  24. 24. 4. The filter is incubated under hybridization conditions with a specific HRP labelled DNA probe. The probe hybridizes to the complementary DNA restriction fragment.  Detection reagent containing H2O2 & luminol is added onto the membrane
  25. 25. USES Identifying DNA in crime case Paternal Dispute Classify DNAs of various organism
  26. 26. Steps in southern blotting 1.The DNA to be analyzed, such as the total DNA of an organism, is digested to completion with a restriction enzyme. 2.The complex mixture of fragments is subjected to gel electrophoresis to separate the fragments according to size.
  27. 27. Western blotting Western blotting (or protein immunoblotting) is a technique widely used to detect specific proteins in samples of tissue homogenate, cell lysates, cell culture supernatants or body fluids.
  28. 28. Western blotting
  29. 29. Forensic science Chemiluminescence is used by criminalists to detect traces of blood at crime scene Solution: luminol powder (C8H7O3N3), hydrogen peroxide, and a hydroxide (eg. KOH) sprayed where blood might found Tiny amount of iron from Hb in blood serves as catalyst for the chemiluminescence reaction, luminol to glow
  30. 30. A trail of blood made visible with the use of the reagent luminol.
  31. 31. Food analysis Organophosphorous most popular pesticide Most commonly used organophosphorous: QUINALPHOS (O,O diethyl-o-quinoxalinly phosphorothioate)
  32. 32. Food analysis Quinalphos +H2O2 peroxophosphonate Peroxophosphonate+ luminol 3aminophthalate anion* 3-aminophthalate* 3- aminophthalate + observed emission
  33. 33. Limitations •Light leaks from assay reagent & reaction vessels •Ultra sensitive – stringent controls on purity of reagents •High intensity light emission leads to pulse pileup in photomultiplier tubes leads to underestimation

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