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13 Cards in this Set
- Front
- Back
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Antibody |
- An antibody (Ab) or immunoglobulin(Ig) is a protein produced by B cells inresponse to a foreign molecule orinvading microorganism. - Binds to the foreign molecule therebyinactivating it or marking it fordestruction |
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Antigen |
Any substance (usually foreign) thatbinds specifically to an antibody |
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Immunogen |
A substance capable of eliciting animmune response (all immunogens areantigens) |
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Hapten |
A low molecular weight molecule thatcan be made immunogenic byconjugation to a suitable carrier |
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Epitope |
The portion of an antigen that isrecognized and bound by an antibody |
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Polyclonal Antibodies |
Definition Polyclonal antibodies are derived frommore than one antibody-producing celland are usually present within theserum Preparation - using larger animals (e.g. rabbit) - myoglobin has 3 different epitopes, so 3 different antibodies can bind to the same protein |
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Monoclonal antibodies |
Preparation - Monoclonal antibodies are generatedusing a hybridoma cell - This is an antibody-producing B-cellfused with a cancerous cell so that theantibody-generating cell becomesimmortalised - Only one specific antibody is produced |
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Detection of Antibodies |
Biotinylated antibodies Biotin conjuagted to antibodies viaamine groups (e.g lysine), 3-6 perantibody. Can be detected usingstreptavidin conjugated enzymecomplexes. Flourochrome-labelled anitbodies Provides direct detection viafluorochrome Enzyme-conjugated antibodies - Antibody is conjugated directly to areporter enzyme such as horseradishperoxidase, alkaline phosphatase - The enzyme catalyses the formation ofa coloured substrate or the release ofphotons (chemiluminescence) Proteins expressed by streptococcus Proteins expressed by Streptococcus(Protein A and Protein G) bind to the Fcregion of mammalian antibodies and canbe used in detection or purification |
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Conjugated Antibodies |
Enzymes, fluorochromes, metallic gold or radioactive metals attached to antibodies |
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Applications of antibodies |
- Detect and quantify expression offoreign protein expressed byrecombinant techniques - Detect native protein in cells ororganisms - Detect probes labelled with biotin, DIGor fluorescein - Purify fusion protein - Screen expression library |
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Western Blotting |
Transfer of PROTEINS from a polyacrylamide gel to a membrane - Using electrical current - to detect expressed proteins in cells -Add antibodies to membrane, it will recognise and bind to CDK7 (protein used for cell division) |
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Enhanced Immunogold Probes |
- Silver enhancement of immunogold - Gold is used because it has high electron density - This increases electron scatter to give high contrast dark spots (stronger signal) |
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ELISA Enzyme-linked immune-sorbent assay |
Used to: - quantify the amount of specific protein in a complex sample - quantify the level of antibody to a specific protein - requires known standards using recombinant protein to create standard curve - Absorbance quantified against standards Method - Add primary antibody - Add secondary antibody and wash off excess - Color of substrate changes depending on # of antigens in solution higher [antigen] = darker colour *measured by spectophotometry |
