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Immunoassay - RIA & ELISA

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This is the powerpoint containing Radioimmunoassay and Enzyme-linked immunosorbent assay (ELISA). Don't forget to watch the YouTube video inside.

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Immunoassay - RIA & ELISA

  1. 1. RIA & ELISA 1
  2. 2. Objectives Principle of immunoassay Types of immunoassays Principle of radioimmunoassay & ELISA Various types of ELISA Steps of ELISA Urine pregnancy test by lateral flow immunoassay 2
  3. 3. Immunoassay Immunoassay is an analytical technique used for the quantification of an analyte based on the antigen-antibody reaction. 3
  4. 4. Labels in immunoassay The advantage of immunoassay is that either the antigen or antibody can be labelled. This label can be an enzyme, radioactive compound, chemiluminescent or fluorescent. The labelled component of an immunoassay is sometimes called the tracer. 4
  5. 5. Types of immunoassay Radioimmunoassay (RIA) Enzyme-linked immunosorbent assays (ELISA) Fluoroimmunoassay (FIA) Chemiluminescence immunoassay (CLIA) 5
  6. 6. Constituents of labelled immunoassay Specific antibody/antibodies (labelled) Antigen (known/unknown) A method to separate the unbound components from bound immune complex A method for detection of the label Standards 6
  7. 7. Radioimmunoassay Dr. Rosalyn Yalow and Dr. Solomon Berson Physiology or Medicine 1977 7
  8. 8. RIA-Principle Competitive binding assay “Radio labelled antigen (Ag*) competes with the unlabelled antigens for the binding site of the fixed amount of antibody (Ab).” 8
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  31. 31. RIA- Principle: Ab + Ag* ⇌ Ab-Ag* + Ag ⇌ Ab-Ag The proportion of Ab-Ag* and Ab-Ag at equilibrium will be equal to the original proportion of Ag* to Ag 31
  32. 32. Disadvantages of RIA Very high cost of equipments and reagents. Half life of I125 is 60 days only. Hazards of radioactivity. Lengthy procedure. 35
  33. 33. Enzyme-Linked Immunosorbent Assay ELISA is the simplified and modified version of RIA. Instead of the radiolabelled antibodies, enzyme- linked antibodies are used in ELISA. It eliminates all the hazards associated with radioactivity and the equipment needed for measuring radioactivity. 36
  34. 34. Types of ELISA 1. Direct ELISA 2. Indirect ELISA 3. Sandwich ELISA 4. Competitive ELISA 37
  35. 35. Direct ELISA 38
  36. 36. Direct ELISA Advantages: Quick because only one antibody and fewer steps are used. 39
  37. 37. Direct ELISA Disadvantages: Immunoreactivity of the antibody might be affected by labeling. Labelling primary antibodies for each specific ELISA system is time-consuming and expensive. Less signal amplification. 40
  38. 38. Indirect ELISA
  39. 39. Indirect ELISA - Advantages Versatile - any primary antibodies can be made in one species and the same labelled secondary antibody can be used for detection. Maximum immunoreactivity of the primary antibody is retained. Sensitivity is increased because of signal amplification. 42
  40. 40. Signal-Amplification in ELISA Enzyme labelled secondary antibodies are responsible for the signal amplification. Each primary antibody can be bound by more than one secondary antibody. Similarly, each enzyme molecule will process multiple substrates into the product. This leads amplification at each step, responsible for high sensitivity of the ELISA. 43
  41. 41. Sandwich ELISA 44
  42. 42. Sandwich ELISA 45
  43. 43. Sandwich ELISA The antigen is sandwiched between two primary antibodies recognizing two different epitopes of the antigen. The first antibody is coated on the plates. The second antibody, which is added after the antigen addition, is linked to the enzyme. It is an Ag detection test. The first antibody is known as ‘capture’ antibody and the second antibody is known as ‘detection’ antibody. 46
  44. 44. Sandwich ELISA It is important to note that the antibodies used should be able to recognise the different epitopes of the same antigen. So, haptens can’t be detected using sandwich ELISA. 47
  45. 45. Competitive ELISA Competitive ELISA is similar to RIA. Both unknown antigen (sample) and the known antigen (standard) compete with each other for a fixed amount of antibody. Ag is detected. Normally used for hapten detection. 48
  46. 46. Competitive ELISA 49
  47. 47. Comparison of the 4 types of ELISA Type of ELISA Well coated with Detects in serum Example Direct Antigen Antigen Beta hCG Indirect Antigen Antibody Anti HBV antibody Sandwich Antibody Antigen HIV: p24 antigen Competitive Antigen Antigen Hapten detection 50
  48. 48. Identify the type of ELISA: 51
  49. 49. Which ELISA will you use? Histamine Complement C3 Insulin Interleukins Leukotrienes Hepatitis B surface antigen Dengue IgM Antibodies 52 - Competitive - Competitive - Sandwich - Sandwich - Competitive - Indirect - Sandwich
  50. 50. Steps of ELISA 1. Coating 2. Blocking 3. Addition of Antibodies 4. Washing 5. Detection 53
  51. 51. Coating 54
  52. 52. Blocking Blocking is done after coating and it ensures no empty spaces are left on the plate surface. Blocking buffers are used to prevent non-specific binding of proteins to the plate. Optimal blocking buffer maximizes the signal to noise ratio during estimation. 55
  53. 53. Blocking agents 1% Bovine serum albumin (BSA) Serum Non-fat dry milk (NFM) Casein or Gelatin in PBS 56
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  59. 59. Detection The final stage in all ELISA systems is a detection step. This involves the introduction of an enzyme- substrate system. The enzyme converts the substrate to a detectable product. 62
  60. 60. Commonly used enzymes Horseradish peroxidase Alkaline phosphatase 63
  61. 61. Horse-radish peroxidase 64
  62. 62. Chromogenic Substrate 65
  63. 63. Alkaline phosphatase 66
  64. 64. ALP chromogenic substrate 1. Nitrophenyl phosphate (NPP) 2. p-nitrophenyl pyrophosphate (PNPP) 3. Bromo chloro indolyl phosphate (BCIP) & Nitro blue tetrazolium (NBT) 4. Fluorescein diphosphate 67
  65. 65. Various types of detection 68
  66. 66. 69 URINE PREGNANCY TEST
  67. 67. Point-of-care-testing 70
  68. 68. Human chorionic gonadotrophin hCG is synthesized in the syncytiotrophoblast cells of the placenta. Minute amounts are also made in the pituitary. A single gene located on chromosome 6 encodes the α subunit. Chromosome 19 contains genes for beta subunit. 71
  69. 69. Alpha subunit is shared 72 Alpha subunit is shared by glycoprotein hormones (TSH, LH [luteinizing hormone], FSH, and hCG).
  70. 70. hCG receptor 73
  71. 71. hCG and pregnancy Synthesis of hCGβ peaks at about 8 to 10 weeks of gestation. 74 First-morning specimens are preferred for qualitative urine pregnancy tests because they are concentrated and contain abundant hCG.
  72. 72. 75 Lateral flow Immunoassay
  73. 73. 76 Lateral flow Immunoassay
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  77. 77. 80 Interpret:
  78. 78. 81 Thank You!

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