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  1. 1. (Enzymelinked ImmunoAbsorbant Assay)
  2. 2. Objectives • To understand the steps involved in performing an ELISA and how it is used as a diagnostic tool • To understand the disease-causing agent and transmission patterns of certain infectious diseases
  3. 3. Quick Recall: 1. The body possesses several lines of defense against infection by pathogenic organisms. 2. The first two defense modes are nonspecific. 1. body’s physical barriers 2. nonspecific immune system 3. Specific immune responses are tailored to the type of invading pathogen.
  4. 4. Specific Immune Response • The specific immune system is complex and involves several organs and tissues, including the thymus, spleen, lymph nodes, bone marrow, and white blood cells. • Specific immune responses are triggered by antigen molecules. Antigens include proteins and other molecules produced by pathogens. • The key players in the specific immune defense are dendritic cells, macrophages, and small white blood cells called B lymphocytes (B cells) and T lymphocytes (T cells).
  5. 5. • Phagocytic macrophages and dendritic cells break down pathogens and display antigenic fragments from the pathogens on the surface of their cell membranes. • B and T lymphocytes circulate through the body in the blood and lymph. • When T cells see displayed antigenic fragments, they stimulate specific B cells to reproduce and generate antibodies designed against the specific structure of the antigen encountered. Thus, the word antigen is derived from the term “antibody generator.”
  6. 6. • Antibodies are a group of serum proteins (also referred to as immunoglobulins) that are found in the bloodstream or bound to cell membranes. • These proteins all have the same basic Y-shaped structure, but have different antigen binding sites at their ends. • Antigen binding sites are designed to fit the shape of specific antigens. Antibodies bind to antigens like a lock and key, forming antigenantibody complexes
  7. 7. • When an antibody forms an antigen-antibody complex, generally it marks the invading organism/antigen for destruction or for clearance from the bloodstream by phagocytic cells. • This removal is designed to prevent the organism/antigen from infecting the cell. Antigenantibody complexes also stimulate additional immune responses to aid the body in clearing an infection.
  8. 8. ELISA
  9. 9. Assay for detecting specific organisms infection by • ELISA is commonly used to test blood serum for the presence of antibodies against disease-causing pathogens such as viruses and bacteria. • In this way, the assay indirectly detects infection by particular disease-causing agents.
  10. 10. • Based on the principle that antibodies produced in response to pathogens attach to their antigen targets with great specificity to form antigen-antibody complexes. • ELISAs can be used to test for infection by HIV, influenza virus, the bacterium that causes Lyme Disease, smallpox virus, SARS coronavirus, West Nile virus, and other disease agents.
  12. 12.  MATERIALS NEEDED FOR ELISA KIT  ELISA Plate  Positive control  Negative control  Dilution Buffer  Conjugate  TMB Substrate  Stop Solution
  13. 13. Indirect ELISA • Indirect ELISA is used to detect infection by testing patients’ blood for the presence or absence of ANTIBODIES against a particular pathogen. • The presence of such antibodies indicates that the individual has been infected and that their body has launched an immune response against the disease-causing agent.
  14. 14.  INDIRECT ELISA   Added Blocking buffer.  Suitable primary antibody is added.  Secondary antibody- HRPO is then added which recognizes and binds to primary antibody.   Antigen is added to plate. TMB substrate is added, is converted to detectable form. Under standard condition ,the enzyme activity measured is proportional to amount of specific antibody in the original serum.
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  16. 16.  ADVANTAGES OF INDIRECT DETECTION  Wide variety of labeled secondary antibodies are available commercially.  Versatile, since many primary antibodies can be made in one species and the Same labeled secondary antibody can be used for detection.  Immunoreactivity of the primary antibody is not affected by labeling.  Sensitivity is increased because each primary antibody contains several epitopes that can be bound by the labeled secondary antibody, allowing for signal amplification.  DISADVANTAGES OF INDIRECT DETECTION  Cross-reactivity may occur with the secondary antibody, resulting in nonspecific signal.  An extra incubation step is required in the procedure.
  17. 17. Direct ELISA • assays for the presence or absence of certain ANTIGENS in patients’ blood.
  18. 18.  DIRECT ELISA 1. Apply a sample of known antigen to a surface. 2. Enzyme linked primary antibody is applied to the plate. 3. Washed, After this wash, only the antibody-antigen complexes remain attached. 4. Apply a substrate which is converted by the enzyme to elicit a chromogenic signal.  Under standard condition ,the enzyme activity measured is proportional to amount of specific antibody in the original serum.
  19. 19.  ADVANTAGES OF DIRECT DETECTION  Quick methodology since only one antibody is used.  Cross-reactivity of secondary antibody is eliminated.  DISADVANTAGES OF DIRECT DETECTION  Immunoreactivity of the primary antibody may be reduced as a result of labeling.  Labeling of every primary antibody is time-consuming and expensive.  No flexibility in choice of primary antibody label from one experiment to another.  Little signal amplification.
  20. 20.  SANDWICH ELISA 1. a. Plate is coated with suitable antibody. b. Blocking buffer is added. 2. Sample is added to plate so antigen is bounded by capture antibody. 3. A suitable biotin labeled detection antibody is added to plate. 4. Enzyme HRPO is added and binds the biotin labeled detection antibody. 5. TMB substrate is added and converted by HRPO to colored product. Under standard condition ,the enzyme activity measured is proportional to the Amount of specific antigen in the original serum.
  21. 21.  PROCEDURE OF ELISA A B C E D Wash Wash Wash Wash 3X 4X 4X 4X Wells are coated with 0.2 μg primary antibody Diluted plasma is added to coated wells, which bind to antibodies 2h Incubated overnight at 4˚C 0.1 μg of biotinylated (biotin = – ) antihuman secondary antibody 2h Add 1.2000 dilution of streptavidin conjugate to alkaline phosphatase ( E) 1h Incubated at room temperature (24˚C) Alkaline phosphatase substrate is added and developed colour is read at 405 nm wavelength to measure plasma cencentration
  23. 23. COMPETETIVE ELISA  COMPETETIVE ELISA Solid phase coated with antibody Add unknown amount of unlabeled antigen and known amount of labeled antigen Free and labeled antigen are captured Color formation by oxidation of substrate into a colored compound Under standard condition ,the enzyme activity measured is proportional to the proportion of labeled antigen in the mixture of labeled and unlabled antigen.
  24. 24.  ADVANTAGES:   Suitable for complex (crude or impure) samples, since the antigen does not require purification prior to measurement. DISADVANTAGES  Each antigen may require a different method to couple it to the enzyme.
  26. 26.  SENSITIVITY ELISAs are one of the most sensitive immunoassays available. The typical detection range for an ELISA is 0.1 to 1 fmole or 0.01 ng to 0.1 ng, with sensitivity dependent upon the particular characteristics of the antibody – antigen interaction. In addition, some substrates such as those yielding enhanced chemiluminescent or fluorescent signal, can be used to improve results. As mentioned earlier, indirect detection will produce higher levels of signal and should therefore be more sensitive. However, it can also cause higher background signal thus reducing net specific signal levels.
  27. 27.  APPLICATIONS Screening donated blood for evidence of viral contamination by     HIV-1 and HIV-2 (presence of anti-HIV antibodies) Hepatitis C (presence of antibodies) Hepatitis B (testing for both antibodies and a viral antigen) Measuring hormone levels     HCG (as a test for pregnancy) LH (determining the time of ovulation) TSH, T3 and T4 (for thyroid function) Detecting infections     Sexually-transmitted agents like HIV, syphilis and chlamydia Hepatitis B and C Toxoplasma gondii  Detecting illicit drugs.  Detecting allergens in food and house dust
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