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    1. 1. PRESENTED BY S .VISWANTH REDDYM.Pharmacy 1stYear(pharmacology)Gokaraju rangaraju college of pharmacy 1
    3. 3. CONTENTS ELISAIntroduction to ELISAPrinciple & procedureMaterials neededTypes of ELISAAdvantages & disadvantages of ELISAApplications RIA Introduction to RIA Principle & procedure Materials needed Advantages & disadvantages of RIA Instrumentation Applications 3
    4. 4. 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
    5. 5. Why known as ......? Enzyme Linked Immunosorbent AssayAntigen of interest is absorbed on to plastic. 1’(.surface (‘sorbentAntigen is recognised by specific antibody .2’(.(‘immunoThis antibody is recognised by second antibody .3(‘immuno’( which has enzyme attached (‘enzyme-’(.linkedSubstrate reacts with enzyme to produce product, .4. usually coloured 5
    6. 6. 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 dveloped in 1970 and became rapidly accepted 6
    7. 7. 7
    8. 8. Materials NeededTesting sampleAntibody (1st, 2nd) / AntigenPolystyrene microtiter plateBlocking bufferWashing bufferSubstrateEnzyme 8
    9. 9. 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. SYMBOL FOR ANTIGENMicroorganismsAllergens.Viruses Etc. 9
    10. 10. ANTIBODY ( Ab) Antibody: proteins produced by the immune system which help defend against antigensSYMBOL FORANTIBODY Y 10
    11. 11. Antibodies (Immunoglobulins) 11
    13. 13. 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 o2 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
    14. 14. ENZYME SUBSTRATE Initially the substrate should be colorless After degradation by the enzyme it should be strongly colored or fluorescent.ENZYME SUBSTRATE CHROMOGEN STOPPINGAlkaline p-NPP p-NPP+ 1 M NaOHPhosphatase diethandamine+Mg Cl2Horse radish H2O2 Tetramethylbenzidi 1 M H2SO4Peroxidase ne + Phosphate – Citrate bufferHorse radish H2O2 O– 1 M HClPeroxidase Phenylenediamine + HCl 14
    15. 15. Secondary antibodySubstrate e Enzym Colouredproduct Primary antibody Different antigens in sample 15
    16. 16. BasicSteps OfEnzyme-LinkedImmunosorbantAssay 16
    17. 17. TYPES OF ELISA ◦ Indirect elisa ◦ Sandwhich elisa ◦ Competetive elisa 17
    18. 18. Indirect elisa 18
    19. 19. 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 toconcentration of standard or analytical antigen 19
    20. 20. 20
    21. 21. ELISA SANDWICH FORMATYYY Antibody YYY 2nd antibody with enzyme Y YYY enzyme produces colour Y YYY YYYYAntibody/Antigen YYY 21
    22. 22. Competitive Elisa Used to determine small molecule antigens.(T3,T4,progesterone etc.) antibody coated microwell 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 (yellow) formation Substrate product concentration is inversely proportional to the concentration of standard or test antigen added 22
    23. 23. ( to detect Ab (HIV, HCV ( to detect Ag ( Tumor Markers, Hormones ( to detect Ag ( Free TestosteroneComparison between Indirect Sandwich & Competitive ELISA 23
    24. 24. Results 24
    25. 25. Importance of incubation step:-During the test performance incubation time and mentioned temperature is must required For the proper binding between antigen and antibody and also binding with conjugate and color development of substrate.Importance of Washing :- For the removal of any unbound Antibody/Antigen proper washing and taping is required other wise we get the incorrect result.So incubation & washing is much important for good results. 25
    26. 26. Elisa Plate Microtitre wells  Generally 96  wells Marked on one  side alphabetically Numerically on  the other side Comes with the  kit 26
    27. 27. 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 27
    28. 28. 28
    29. 29. ELISA PLATE READYFOR READINGMeasures the absorbance at450nm With the help ofELISA READER.Calculate the absorbancefor each sample andreference.We used Ascent Softwarefor Calculation of the result 29
    30. 30. 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. 30
    31. 31. 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. Won’t recognize any other antigen False positives/negatives possible, especially with mutated/altered antigen 31
    32. 32. LimitationsResults may not be absolute •Antibody must be available •Concentration may be unclear •False positive possible •False negative possible • 32
    33. 33. APPLICATIONS OF ELISAdetection of Mycobacterium antibodies in tuberculosisdetection of rotavirus in fecesdetection of hepatitis B markers in serumdetection of HIV antibodies in blood samplesIt has also found applications in the food industry in detecting potentialeggs food allergens, such as milk, peanuts, walnuts, almonds, and 33
    34. 34. APPLICATIONS OF ELISA1- Hormones 7- Vaccine Quality Control2- Proteins 8- FOR GMO (Genetically modified organism)3- Infectious Agent ( Viral, Bacterial, 9- For Rapid TestParasitic, Fungal )4- Drug Markers 10- IgG, IgM, IgA5- Tumor Markers 11- In New Born Screening6- Serum Proteins 12- In Clinical Research 34
    35. 35. Sensitivity of various immunoassays 35
    36. 36. Equipments for performing the ELISA testPipettes Incubator ELISA reader 36
    38. 38. 38
    39. 39. ]Radioimmunoassay 39
    40. 40. INTRODUCTION Radioimmunoassay (RIA) is a very sensitive in vitro assay techniqueused to measure concentrations of antigens (for example, hormone levels intheblood) by use of antibodies. The RAST test (radioallergosorbent test) is an example ofradioimmunoassay. It is used to detect the causative allergen foran allergy To perform a radioimmunoassay, a known quantity of an antigen ismade radioactive, frequently by labeling it with gamma-radioactive isotopes of iodine attached to tyrosine. 40
    41. 41. To perform a radioimmunoassay, a known quantity of an antigen ismade radioactive, frequently by labeling it with gamma-radioactive isotopes of iodine attached to tyrosine.This radiolabeled antigen is then mixed with a known amountof antibody for that antigen, and as a result, the two specifically bind to oneanother.Then, a sample of serum from a patient containing an unknown quantity ofthat same antigen is added.This causes the unlabeled (or "cold") antigen from the serum to competewith the radiolabeled antigen ("hot") for antibody binding sites. Astheconcentration of "cold" antigen is increased, more of it binds to theantibody, displacing the radiolabeled variant, and reducing the ratio ofantibody-bound radiolabeled antigen to free radiolabeled antigen. Thebound antigens are then separated from the unbound ones, and theradioactivity of the free antigen remaining in the supernatant is measuredusing a gamma counter. 41
    42. 42. Principle of RadioimmunoassayPrinciple:Uses an immune reaction [Antigen – Antibody reaction] to estimate a ligand Ag + Ag* + Ab  AgAb + Ag*Ab + Ag + Ab* ◦ Unbound Ag* and Ag washed out ◦ Radioactivity of bound residue measured ◦ Ligand conc is inversely related to radioactivity [Ag : ligand to be measured ; Ag* radiolabelled ligand] 42
    43. 43. Advantages & Disadvantages of RIAAdvantages ◦ Highly specific: Immune reactions are specific ◦ High sensitivity : Immune reactions are sensitiveDisadvantages ◦ Radiation hazards: Uses radiolabelled reagents ◦ Requires specially trained persons ◦ Labs require special license to handle radioactive material ◦ Requires special arrangements for  Requisition, storage of radioactive material  radioactive waste disposal. 43
    44. 44. Requirements for RIA1. Preparation & characterisation of the Antigen [Ligand to be analysed]2. Radiolabelling of the Antigen3. Preparation of the Specific Antibody4. Development of Assay System 44
    45. 45. Preparation & Radiolabelling ofthe Antigen Antigens prepared by.. ◦ Synthesis of the molecule ◦ Isolation from natural sources Radiolabelling [Tagging procedure] ◦ 3 H 14 C 125 I are used as radioactive tags ◦ Antigens are tagged to 3 H 14 C 125 ◦ Tagging should NOT affect Antigenic specificity & Antigenic activity ! 45
    46. 46. 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 46
    47. 47. Development of the Assay SystemA crucial step is separation of unbound antigensThis achieved by binding the antibodies to the microtitre 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 47
    48. 48. 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 Microtitre 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 48
    49. 49. Antibodies: types oflabelling,Radio-isotopes-Enzymes -FluorescentChemi-luminescentprobesMetal tags 49
    50. 50. Radioimmunoassay (RIA) Advantages  Disadvantages ◦ Flexibility ◦ Toxicity ◦ Sensitivity ◦ Shelf life ◦ Size ◦ Disposal costs 50
    51. 51. Advantages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 51
    52. 52. limitations The main drawbacks to radioimmunoassay are the expense and hazards of preparing and handling the radioactive antigen. Both 125I or 131I emit gamma radiation that requires special counting equipment The body concentrates iodine atoms — radioactive or not — in the thyroid gland where they are incorporated in thyroxine (T4). 52
    53. 53. 53
    54. 54. INSTRUMENTATION Radiation will hit silver grains in emulsion and expose them Expose to film or emulsion Isotope will emit(radiation (usually betaIncubate tissue with radioactive ligand Autoradiography 54
    55. 55. INSTRUMENTATION 55
    56. 56. 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 13December 2012.Kuhar M, Yamamura HI (Jul 1976(. "Localization of cholinergic muscarinicreceptors 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 numberof 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 56
    57. 57. 57
    58. 58. r r ∝ [ Ag ] 58