Ch 3 Text 4

·         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

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 __

·         Immunological techniques depend on the ability to generate antibodies to a __

·         How to obtain antibodies:
o   _______________________


o   What then occurs?

·         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 __

·         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

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 

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 

· 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

Hybridoma methods lead to great things. What are some?

§  Monoclonal antibodies attached to solid supports can be used as __ to purify scarce proteins, such as interferons

·         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

Two types of ELISA. Explain them

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

Indirect ELISA is __.

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 __

·         __ 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

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 __

·         __ 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