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· 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
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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
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· 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
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· 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
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· 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
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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
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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
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· 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
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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
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§ Monoclonal antibodies attached to solid supports can be used as __ to purify scarce proteins, such as interferons
affinity columns
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· 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
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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
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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
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Indirect ELISA is __.
quantitative: the rate of the color-formation reaction is proportional to the amount of antibody originally present
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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
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· __ 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
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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
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· __ 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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