Ch 3 Text 4

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  1. ·         Purifying proteins reveals __ and __, but removing it from its native context was not great. So, we use __to follow proteins within the cellà they are specific
    ·         __to specific proteins can be generated
    • function and structure
    • antibodies 
    • Antibodies
  2. o   Immunological techniques begin with the generation of __to a particular protein, which respond to __. __are specific (Due to __) and have high affinity for the antigens that elicit their synthesis
    §  __can be foreign proteins, nucleic acids, etc. and bind to antibody
    o   An antibody recognizes a specific group or cluster of amino acids on the target molecule called an __
    • antibodies 
    • antigens
    • Antibodies 
    • shape complementarity
    • Antigens 
    • antigenic determinant or epitope
  3. ·         Immunological techniques depend on the ability to generate antibodies to a __

    ·         How to obtain antibodies:
    o   _______________________

    o   What then occurs?
    • specific antigen
    • Protein injected twice—once to elicit immune response and buildup of antibodies to recognize it
    • Blood drawn and centrifuged to create serum (antiserum
  4. ·         Antibodies that are derived from multiple antibody-producing cell populations are called __
    o   Each protein is __and can be bound by more than one antibody at multiple distinct __
    • polyclonal
    • heterogeneous
    • antigenic sites
  5. ·         Monoclonal antibodies with virtually any desired specificity can be readily prepared
    o   The discovery of a way to make __ was great
    o   While mixed mixtures are bad, you also don’t want the interpretation of data to be confusing. Isolating  the clone of cells producing a single, identical antibody is hard due to short life spans
    monoclonal antibodies
  6. o   Immortal cell lines that produce monoclonal antibodies are derived from a type of cancer,__, which is a malignant disorder of antibody-producing cells
    §  In this cancer, what happens?
    · The identical cells of the myeloma secrete large amounts of __of a single kind 
    • multiple myeloma
    • a single transformed plasma cell divides uncontrollably, generating a very large number of cells of a single kind, called a clone due to the descendance from the same cell
    • immunoglobulin
  7. o   __ and __ discovered that large amounts of antibodies of nearly any desired specificity can be obtained by doing what?
    Use on rat: inject with antigen, remove spleen, which contains plasma cells fused in vitro with myeloma cells à hybrid cells called __can then be screened by a specific assay for the antigen-antibody interaction to determine which ones produced antibodies of the preferred specificity 
    • Cesar Milstein and Georges Kohler
    • fusing a short-lived antibody producing cell with an immortal myeloma cell
    • hybridoma
  8. · Cells that produce the antibody are __ and __. The procedure is repeated until a pure cell line is __, which can grow to induce __or can be frozen
    • subdivided and reassayed
    • isolated
    • myelomas
  9. Hybridoma methods lead to great things. What are some?
    prep of specific antibodies, insight into relations between antibody structure and specificity, serve as precise analytical and preparative reagents
  10. §  Monoclonal antibodies attached to solid supports can be used as __ to purify scarce proteins, such as interferons
    affinity columns
  11. ·         Proteins can be detected and quantified by using an __

    o   Antibodies can help determine the amount of protein in a biological sample. The __ makes use of an enzyme that reacts with a colorless substrate to produce a __, which covalently links to a __ that recognizes a target antigen
    §  Antigen presentàcomplex bindsà colored product
    • enzyme-linked immunosorbent assay
    • enzyme-linked immunosorbent assay (ELISA)
    • colored agent
    • specific antibody
  12. Two types of ELISA. Explain them
    §  Indirect ELISA: used to detect the presence of antibody; basis of test for HIVE infection

    • §  Sandwich ELISA: used to detect antigen rather than antibody
  13. Indirect ELISA 

    The HIV test detects the presence of antibodies that recognize __, which are adsorbed to the bottom of a well. Antibodies from the subject are then added to the coated well. If infected, antibodies bind. Finally, __ to human antibodies are allowed to react in the well, and unbound proteins are washed. Substrate is then applied. If coloredà enzyme-linked antibodies were bound to human antibodies
    • viral core protein antigens
    • enzyme-linked antibodies
  14. Indirect ELISA is __.
    quantitative: the rate of the color-formation reaction is proportional to the amount of antibody originally present
  15. Sandwich ELISA 

    · Antibody is adsorbed to bottom of well. Next, solution containing the __is added to the well and binds to the __. Finally, a second, different __to the antigen is added, which is __.
    · The rate of color formation is directly proportional to the __ and permits __
    • antigen 
    • antibody
    • antibody 
    • enzyme linked
    • amount of antigen present and permits measurement of small quantities of antigen
  16. ·         __ permits the detection of proteins in small quantities separated by gel electrophoresis
    o   First, the sample is subjected to _ on an __-> polymer sheet pressed against it to __-> __added to sheet to react with antigenà complex forms that is detected by __--> identification and quantitation of protein of interest
    • Western blotting
    • electrophoresis on an SDS-polyacrylamide gel
    • transfer resolved protein to sheet
    • antibody 
    • rinsing sheet with a second antibody (with a label) specific for the first antibody
  17. o   Alternatively, an enzyme on the second antibody generates a __
    o   Western blotting is good for finding proteins in complex mixtures and tests for hepatitis C, where it is used to detect a __ of the virus. It is also useful in __ and in __
    • colored product
    • core protein
    • monitoring protein purification and in the cloning of genes
  18. ·         __ make the visualization of proteins in the cell possible
    o   Used to examine proteins in their biological context
    o   Can be stained with __and examined by __ to reveal the location of a protein of interest
    o   Highest resolution: 2 micrometers, the wavelength of visible light
    • Fluorescent markers
    • fluorescence-labeled antibodies 
    • fluorescence microscopy
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Ch 3 Text 4
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