Ligand binding: antibodies Flashcards
What is the difference between the innate and adaptive immune system?
Innate is non-specific and just looks for general non-human features that would indicate a pathogen. The adaptive immune system is specific to particular pathogens
What are the two subdivisions of the adaptive immune system?
Cellular and humoral
What does the cellular immune system do?
Detects and destroys infected cells through T cell receptors
What does the humoral immune system do?
Recognizes and attacks pathogens in body fluids by using antibodies
What protein family are antibodies part of?
Immunoglobulins
What are the two polypeptide chains in antibodies? What’s the difference between them?
Heavy and light. Heavy chains are longer
What are the two domains on one of the chains in an antibody?
Variable and constant
How many variable and constant domains are on a heavy chain?
3 constant, 1 variable
How many variable and constant domains are on a light chain?
1 constant, 1 variable
Which domains make up the antigen binding site on an antibody?
The variable domains of the heavy and light chains
How many disulfide bonds are in an antibody?
4
Where are the disulfide bonds in an antibody?
2 closer to the top near the antigen binding site on both sides, between the heavy and light chains. 2 in the middle hinge region between the two heavy chains
What are the two fragments of an antibody?
Fab (antigen binding) and Fc (crystallizable)
How do we break apart the two fragments in an antibody?
Papain
What is an antigen?
Molecule that binds selectively to an antibody
What is the epitope?
The particular site on the antigen that is recognized by the antibody
What are the two types of epitopes?
Linear and conformational
What is a linear epitope?
The specific amino acid sequence is recognized, so binding will still occur if the antigen is unfolded
What is a conformational epitope?
The 3D structure is recognized, so binding does not occur if the antigen is unfolded
How do macrophages recognize pathogens targeted for destruction by antibodies?
Have an Fc receptor that will bind to that region of the antibody and cause the macrophage to engulf the pathogen
What is structural motif is present in the constant domains?
Beta sandwich (two antiparallel beta sheets)
What structural feature is present in the antigen binding sites of antibodies?
Hypervariable loops
How do antibodies improve the complementarity of antigen binding?
Induced fit for shape, charge, hydrophobicity and H bonding
What are 4 applications for antibodies?
Western blot, ELISA, immunofluorescence, co-immunoprecipitation
What is an ELISA?
Enzyme-linked immunosorbent assay. Provides a quantitative measurement of the amount of antibody of antigen
What are the two types of ELISA?
Indirect and sandwich
What does indirect ELISA detect?
The presence of the antibody
How do you do indirect ELISA?
Coat the well with the antigens, then add the fluid you’re testing. Add secondary antibodies with an enzyme attached then add the substrate for that enzyme. If there were antibodies in the fluid, we would get a colour
What does sandwich ELISA detect?
The presence of the antigen
How do you do sandwich ELISA?
Coat the well with antibodies, then add the fluid you’re testing. Add the secondary antibodies with the linked enzyme that is also specific to the antigen, then add the enzyme substrate and look for a colour change
What do we use immunofluorescence for?
Detection of proteins in living cells
What does co-immunoprecipitation detect?
Protein-protein interactions
How do you do a co-immunoprecipitation experiment?
Add antibodies attached to resin beads for one of the binding partners, then remove the supernatant and run the pellet on a gel. Do a western blot and probe for the other binding partner
