ELISA, RIA, BIOLUMENSCENCE M.PHARM 1st SEM.pptx

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1. ELISA, RIA & BIOLUMINESCENCE ASSAYS Saurabh Dubey Institute of Pharmacy Bundelkhand University YouTube channel- SKD Education Study’s Point

2. CONTEN T ELISA Introduction to ELISA Principle & procedure Materials needed Types of ELISA Advantages & disadvantages of ELISA  Applicatio n RIA Introduction to RIA Principle & procedure Materials needed Advantages & disadvantages of RIA Instrumentation Applications

3. INTRODUCTION TO ELISA  ELISA, or enzyme-linked immunosorbent assay, are quantitative immunological procedures in which the Ag-Ab reaction is monitored by enzyme measurements.  The term ELISA was first used by Engvall & Perlma in 1971.  The ELISA test, or the enzyme immunoassay (EIA), was the first screening test commonly employed for HIV. It has a high sensitivity.

4. Why the term ...... Enzyme Linked Immunosorbent Assay 1. Antigen of interest is absorbed on to plastic surface. ‘sorbent’ 2. Antigen is recognized by specific antibody ‘immune’ 3. This antibody is recognized by second antibody which has enzyme attached (‘enzyme-linked’) 4. Substrate reacts with enzyme to produce product, usually colored.

5. BASIC PRINCIPLE OF ELISA  Use an enzyme to detect the binding of antigen (Ag) antibody (Ab).  The enzyme converts a colorless substrate (chromogen) to a colored product, indicating the presence of Ag : Ab binding.  An ELISA can be used to detect either the presence of Antigens or antibodies in a sample depending how the test is designed.  ELISA was developed in 1970 and became rapidly accepted

6. BASIC PROCEDURE OF ELISA

7. Materials Needed  Testing sample  Antibody (1st, 2nd) / Antigen  Polystyrene microtiter plate  Blocking buffer  Washing buffer  Substrate  Enzyme

8. ANTIGEN (Ag)  Any molecule that induces production of antibodies when introduced in the body of an animal is called antigen. OR  Any “thing”, foreign to the immune system. e.g. bacteria, viruses, (or their parts), pollen, etc.  Protein molecule  Carbohydrate molecule.  Microorganisms  Allergens.  Viruses etc. Symbol for antigen

9. ANTIBODY ( Ab) Antibody: proteins produced by the immune system which help defend against antigens. Symbol for antibody

10. Antibodies (Immunoglobulins)

11. Specimen Sample For ELISA SERUM CSF SPUTUM URINE SEMEN SUPERNATANT OF CULUTRE STOOL

12. Enzymes Used in ELISA Horseradish peroxidase (most commonly used) Alkaline Phosphatase  β- galactosidase  Lactoperoxidase Tetra Methyl benzidine  In case of peroxidase, the substrate hydrogen peroxide is converted into water and O 2 in the presence of electron donors (like diaminobenzidine or 4-chloronaphthol which themselves oxidized in the reaction).  Oxidation of diaminobenzidine produces dark brown color while that of 4-chlorornaphthol yields purple color which is the basis of ELISA.

13. ENZYME SUBSTRATE  Initially the substrate should be colorless. After degradation by the enzyme it should be strongly colored or fluorescent.

14. Basic Steps Of Enzyme- Linked Immunosorbent Assay

15. TYPES OF ELISA ◦ Indirect ELISA ◦ Sandwich ELISA ◦ Competitive ELISA

16. Indirect ELISA

17. Sandwich ELISA Antigens such as tumor markers, hormones and serum proteins may be determined. Antigen in the sample binds with the capture antibody on the microwell and becomes immobilized.  The antibody of the enzyme conjugate binds with the immobilized antigen to form a sandwich of antibody/antigen-antibody/enzyme bound to the microwell. Enzyme reaction product is directly proportional to concentration of standard or analytical antigen

18. ELISA SANDWICH FORMAT Antigens

19. Competitive ELISA  Used to determine small molecule antigens (T3,T4,progesterone etc.)  Antibody coated microwell is selected.  Serum antigen and labelled antigen added together (competition).  Antibody-antigen-enzyme complex bound is inversely related to the concentration of antigen present in the sample.  The bound enzyme conjugate reacts with the chromogenic substrate added to produce a color reaction (blue to yellow color).  Increased serum antigen results in reduced binding of the antigen-enzyme conjugate with the capture antibody producing less enzyme activity and color formation (yellow). Substrate product concentration is inversely proportional to the concentration of standard or test antigen added

20. Comparison between Indirect, Sandwich & Competitive ELISA

21. ELISA Plate Microtiter wells Generally 96 wells Marked on one side alphabetically Numerically on the other side Comes with the kit

22. Test performance  Using a clean Pipette, add 100 µL of diluted serum sample (Dilute the sera to be tested 1:100 in the sample diluents) to each well.  Incubate 1 hour at 37°C.

23. Assay procedure

24. Advantages of ELISA  Reagents are relatively cheap & have a long shelf life.  ELISA is highly specific and sensitive.  No radiation hazards occur during labelling or disposal of waste.  Easy to perform and quick procedures.  Equipment can be inexpensive and widely available.  ELISA can be used to a variety of infections.

25. Disadvantages of ELISA  Measurement of enzyme activity can be more complex than measurement of activity of some type of radioisotopes.  Enzyme activity may be affected by plasma constituents.  Kits are commercially available, but not cheap.  Very specific to a particular antigen. Wouldn’t recognize any other antigen.  False positives/negatives possible, especially with mutated/altered antigen.

26. APPLICATIONS OF ELISA Detection of Mycobacterium antibodies in tuberculosis. Detection of rotavirus in feces. Detection of Hepatitis B markers in serum. Detection of HIV Antibodies in blood samples. It has also found applications in the food industry in detecting potential food allergens, such as milk, peanuts, walnuts, almonds and eggs .

27. RADIOIMMUNOASSAY  Radioimmunoassay (RIA) is a very sensitive in vitro assay technique used to measure concentrations of antigens (for example, hormone levels in the blood) by use of antibodies.  The RAS test ( RadioAllergoSorbent test) is an example of radioimmunoassay. It is used to detect the causative allergen for an allergy.  To perform a radioimmunoassay, a known quantity of an antigen is made radioactive, frequently by labeling it with gamma-radioactive isotopes of iodine attached to tyrosine.

28. Contd. This radiolabeled antigen is then mixed with a known amount of antibody for that antigen, and as a result, the antigen-antibody specifically bind to one another. Then, a sample of serum from a patient containing an unknown quantity of that same antigen is added. This causes the unlabeled (or "cold") antigen from the serum to compete with the radiolabeled antigen ("hot") for antibody binding sites . As the concentration of "cold" antigen is increased, more of it binds to the antibody, displacing the radiolabeled variant, and reducing the ratio of antibody-bound radiolabeled antigen to free radiolabeled antigen. The bound antigens are then separated from the unbound ones, and the radioactivity of the free antigen remaining in the supernatant is measured using a gamma counter.

29. Principle of Radioimmunoassay

30. Requirements for RIA 1. Preparation & characterization of the Antigen [Ligand to be analyzed] 2. Radiolabeling of the Antigen 3. Preparation of the Specific Antibody 4. Development of Assay System

31. Preparation & Radiolabeling of the Antigen  Antigens prepared by: ◦ Synthesis of the molecule ◦ Isolation from natural sources  Radiolabeling [Tagging procedure] ◦ 3 H 14 C 125 I are used as radioactive tags ◦ Antigens are tagged to 3 H 14 C 125 I ◦ Tagging should NOT affect Antigenic specificity & Antigenic activity !

32. Preparation of the Specific Antibody  Antigen injected intradermally into rabbits or guinea pigs antibody production  Antibodies recovered from the serum  Some ligands are not Antigenic ◦ Hormones, Steroids, Drugs HAPTENS ◦ Eg : Gastrin, Morphine, ◦ Haptens conjugated to albumin antigenic

33. Development of the Assay System  A crucial step is separation of unbound antigens  This is achieved by binding the antibodies to the microtiter well surface [Solid phase RIA]  Antigens bound to the fixed antibodies remain stuck to the inner surface  Decanting & washing the well removes unbound antigens  Other techniques of separation: Centrifugation

34. Assay Procedure  Add known amounts of the test sample + labelled antigen into the microtitre wells  Incubate allow the reaction to reach completion  Decant & wash contents of the well removes all unbound antigens  Radioactivity remaining in the Microtiter wells measured by a Counter [GM counter , Scintillation counter etc.]  Intensity of radioactivity is inversely correlated with the conc. of antigens in the test sample.  Sensitive to very low conc of antigens.

35. Antibodies: types of labelling Radio-isotopes Enzymes - Fluorescent Chemi -luminescent probes Metal tags

36. Radioimmunoassay (RIA)  Advantages ◦ Flexibility ◦ Sensitivity ◦ Size  Disadvantages ◦ Toxicity ◦ Shelf life ◦ Disposal costs ◦ Radiation hazards: Uses radiolabeled reagents ◦ Requires specially trained persons ◦ Labs require special license to handle radioactive material ◦ Requires special arrangements for  Requisition, storage and handling of radioactive material  radioactive waste disposal.  Radioimmunoassay is widely-used because of its great sensitivity.  Using antibodies of high affinity, it is possible to detect a few picograms (10 −12 g) of hormone in the tube.  The greater the specificity of the antiserum, the greater the specificity of the assay

37. Bioluminescence Assay Bioluminescence occurs when an EXOTHERMIC CHEMICAL reaction releases energy to generate electromagnetic radiation which gives off light.

38. Types: Chemiluminescence - Reactions with synthetic compounds (i.e. H 2 O 2 etc.) Bioluminescent reactions – from a living organism Electrochemiluminescent reactions – use electric current

39. Enzymes: luciferases Firefly luciferase Renilla luciferase (sea pansy) Aequorin (jellyfish- Aequorea victoria ) Substrates (photon-emitting): luciferins Enzyme catalyzed reactions

40. Firefly Bioluminescence Light Intensity ~ Chemical Concentration [ Chemical ] of interest may be ATP, luciferin, or luciferase (hold all others constant) Very large linear range Most commonly luciferase used in the development of High-Throughput Screening (HTS) Assays

41. Contd. Yellow - green light λ max = 560 nm Firefly

42. Signal detection

43. How can this reaction be used in HTS

44. HTS: Luciferase Concentration Goal: investigate intracellular events by monitoring gene transcription. May include internal control (dual-luciferase assay) Simple and efficient (HTS) Commonly used for GPCR and nuclear receptor assays

45. Examples of bioluminescence assay ATP Concentration monitoring Enzyme must be consistent!  Often use “ stabilized ” luciferase enzymes for HTS Used in: Cytotoxicity screens  ATP concentrations ~ cell viability Kinase activity screens  all kinases consume ATP in phosphorylation rxn Real-time detection of ATPase activity 4 ~ 100X more sensitive and significantly faster than some dye assays used to look at cell metabolism

46. Luciferase Enzymatic Activity monitoring assays: Sensitive & broad detection range Protein- Protein interaction assays BRET – Bioluminescence resonance energy transfer PCA – Protein fragment complementation assay Real-time bioluminescence to analyze inhibitors of polymerases (DNA & RNA).

47. BL/CL recombinant whole-cell biosensors: Genetically engineered cells to create a luminescent signal ~ to a specific analyte (analyte should be regulating gene expression) Used for monitoring: Stress, oxidants, metals, xenobiotics, receptor activating molecules, etc.

48. Conclusions Chemiluminescence and Bioluminescence are common, extremely versatile, and useful analytical tools. HTS methods are becoming increasingly more dependent upon this “background-free” technique. The ATP Detection Assay could have huge implications in pharmacology as it evolves for different types of detection.

49. REFERENCES  Engvall E, Perlman P ,1971, "Enzyme-linked immunosorbent assay (ELISA). Quantitative assay of immunoglobulin G". Immunochemistry 8 (9): 871–4. Gofflot ; El (2004).  Journal of Immunoassay and Immunochemistry 25 (3): 241–58. Retrieved 13 December 2012.  Kuhar M, Yamamura HI (Jul 1976(. "Localization of cholinergic muscarinic receptors in rat brain by light microscopic radioautography". Brain Res. 110 (2): 229–43.  E. Rutherford and H. Geiger (1908) "An electrical method of counting the number of α particles from radioactive substances," Proceedings of the Royal Society (London), Series A, vol. 81, no. 546,  A Handbook of Radioactivity Measurements Procedures, 2nd edition: (Report No. 58), National Council on Radiation Protection and Measurements (NCRP), 1985 ISBN 0-913392-71-5,pages 30-31  WWW.GOOGLE.COM/IMAGES WWW.SLIDESHARE.NET