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13 Cards in this Set

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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

Antigen

Any substance (usually foreign) thatbinds specifically to an antibody

Immunogen

A substance capable of eliciting animmune response (all immunogens areantigens)

Hapten

A low molecular weight molecule thatcan be made immunogenic byconjugation to a suitable carrier

Epitope

The portion of an antigen that isrecognized and bound by an antibody

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

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

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

Conjugated Antibodies

Enzymes, fluorochromes, metallic gold or radioactive metals attached to antibodies

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

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)



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)

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