Luminoscence immunoassay

Explanation of Luminoscence immunoassay are described in this slide

1. LUMINOSCENCE IMMUNOASSAY SUBMITTED BY- SK AZIZ UDDIN SUBMITTED TO- DR.RIKESHWAR PRASAD DEWANGAN COURSE-M.PHARM BRANCH-PHARMACEUTICAL ANALYSIS COLLEGE NAME- JAMIA HAMDARD YEAR-2020-2022

2. CONTENTS INTRODUCTION (LUMINESCENCE) TYPES OF LUMINESCENCE. PRINCIPLE( CHEMILUMINESCENCE) PRINCIPLE OF CLIA TYPES OF CLIA APPLICATION OF CLIA ADVANTAGES & DISADVANTAGES OF CLIA REFERENCES

3. INTRODUCTION Luminescence refers to the release of energy from an electronically excited molecule in the form of visible light. When the excited molecule returns to the ground state it releases a photon of light. The catch-all title of luminescence covers a range of detection systems, including chemiluminescence, phosphorescence, bioluminescence and fluorescence. Incandescence, which is light emitted by a substance as a result of heating.

4. LUMINESCENCE Cold light that can be emitted at lower temperature. Source kicks an electron of an atom out of its lowest energy ‘ground’ state higher energy ‘excited’ state. Finally electron returns the energy in the form of light so it can fall back to its ‘ground’ state.

5. TYPES LUMINESCENCE Excitation event process Chemicals Luminol Isoluminol acridinium ester Chemiluminescen-ce Biochemical Luciferin aequorin Bioluminescence Electromagnetic Ruthenium Tris (bipyridly) chelate Electroluminescence Photons Inorganic phosphors Photoluminescence

6. CHEMILUMINESCENCE Emission of light with limited emission of heat , as the result of a chemical reaction. [A]+[B] →[◊]→[products]+ light Where ;- [A], [B] ; reactants [◊] is excited intermediate Commonly used catalysts in this reaction are Alkaline phosphate(ALP) label is 1,2 dioxane Horse radish peroxidase(HRP) label is luminol. Metal ions complexes (copper and iron phthalocyanine complex) It has sensitivity- 10 -15 (femtograms)

7. CONTINUE;- For example if [A] is luminol and [B] is hydrogen peroxide in the presence of a suitable catalyst we have: Luminol+ H 2 O 2 → 3-APA[◊]→3-APA + light Where 3-APA is 3- amino phthalate 3-APA[◊] is the excited state producing light as it decays to lower energy level.

8. CONTINUE;-

9. Application of chemiluminescence Chemiluminescence immunoassay DNA hybridization detection Western blotting Forensic science Food analysis

10. CHEMILUMINESENCE IMMUNOASSAY(CLIA) Principle-: CLIA utilize chemical probe which could generate light emission through chemical reaction to label the antibody. It is an assay that combine chemiluminescence technique with immunochemical reactions. Similar with other labelled immunoassays(RIA, FIA, ELISA).etc Provides a sensitive , high throughput alternative to conventional colorimetric methodologies. Uses chemiluminescent substrate, hydrogen peroxide , enhancers Stopping reagent is not required. Incubation period is small

11. Types of chemiluminescent assay. Direct chemiluminescent assay In this method luminophore markers used are acridinium and ruthenium esters. This kind of chemical with special structure can transfer to an excited state through chemical reaction. Exposure of an acridinium ester label to an alkaline hydrogen peroxide solution triggers a flash of light.  A subsequent development has been the acridinium sulfonamide ester labels. It is also triggered by alkaline hydrogen peroxide to emit a flash of light. The light emission mechanism of acridinium ester is shown in Figure 

12. Direct chemiluminescent assay

13. Indirect chemiluminescent assay Enzymatic markers used indirect methods are alkaline phosphates with adamantly 1,2 dioxetane aryl phosphate(AMPPD) and horse radish peroxidase(HRP) each has its own luminescent substrates. Luminol is a very common chemiluminescent substrate used for detection of HRP. HRP catalyzes the decomposition of luminol in the presence of peroxide to produce an excited state intermediate.  Flashes of visible light (maximum at 425nm) is emitted on decay of the singlet intermediate.

14. Indirect chemiluminescent assay  AMPPD is a derivative of 1, 2-dioxetane substrates. It has a similar mechanism of chemiluminescence. On enzymatic cleavage of the phosphate group, this compound becomes destabilized and decomposes via an intermediate anion, AMPD, which is moderately stable. The wavelength of maximum light emission is 470nm.

15. Sandwich chemiluminescent immunoassay Sandwich CLIA is a kind of detection method combined double antibody sandwich method with chemiluminescence detection method. It regularly includes lots of steps, which are shown below. At first, the microtiter plate has been pre-coated with an antibody specific to analyte. Then, standards or samples are added to the appropriate microtiter plate wells, the analyte in standards and samples will bound to the immobilized Ab. Next, biotin-conjugated antibody is added and binds to the analyte absorbed on the plate. The complex of two antibodies and analytes in the wells act as a “sandwich” structure. After the unbound biotin-conjugated antibody is washed away, avidin-conjugated Horseradish Peroxidase (HRP) is added to each micro plate well, after incubation, luminol is added into the micro wells, and then relative luminosity values (RLU) will be scanned by photon counter reader . 

16. USES Used to estimate analytes which have extremely low conc. In the blood such as hormones, serological markers and drugs. Hormones ; insulin, thyroxin, estradiol,testosterone,progesterone,prolactin, LH, FSH etc. Vitamin; vit B 12 Tumor markers: bone morphogenic protein-2, carcino embryonic antigen(CEA), alpha fetoprotein(AFP) Also COVID-19 marker, biochemical marker is also detected Human beta chorionic gonadotropin C- reactive protein. Tumor necrosis factor To detect ATP specific enzymes.(Luciferase,creatin kinase) Used for determination of co-enzyme like NADH,NADPH.

17. ADVANTAGES OF CLIA It is a analytical methods reside in the wide dynamic range. It has high signal intensity, absence of interfering emissions(i.e. High specificity) Rapid acquisition of the analytical signal. High stability of reagents and their conjugates. Low consumption of reagents. Random access, reduced incubation time.

18. DISADVANTAGES OF CLIA Light leaks from assay reagent & reaction vessels Ultra sensitive- stringent controls on purity of reagents High intensity light emission leads to pulse pile up in photomultiplier tubes leads to underestimation. Limited Ag detection High costs Limited tests panel

19. REFERENCES Internet browsing (GOOGLE) http://www.cloud-clone.com/topic/201511200859289789.htm#:~:text=Sandwich%20CLIA%20is%20a%20kind,method%20with%20chemiluminescence%20detecion%20method.&text=The%20complex%20of%20two%20antibodies,as%20a%20%E2%80%9Csandwich%E2%80%9D%20structure . https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5483212/ You tube video Google Slide share Immunoassay book.

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