6 - Antibodies Flashcards
How does the structure of antibodies allow for their function?
They have two specific regions that allow linking to two objects as well as a third binding surface that can be used to bind them onto a membrane or other surface while still retaining their function.
What are IgA antibodies?
These are dimeric antibodies that line the intestines and mucosal tract as a first line of defence against pathogens.
What are IgD antibodies?
Early response antibodies, cell surface bound.
What are IgE antibodies?
Degranulation
Binding to poisons
Stimulating mast cell histamine response
What are IgG antibodies?
The primary plasma antibody, responsible for opsonisation and agglutination.
What is opsonisation?
Identification of pathogens.
What are IgM antibodies?
Early response
Expressed on B-cell surface
Can form soluble pentamers that link at their J chains
What is the chain structure of an antibody?
Two long heavy chains that combine to form two ß-sandwiches at one end and split apart at the other at a midpoint linker sequence to allow two half-length light chains to make another two ß-sandwich immunolobulin fold with either one.
How many amino acids make up the heavy and short chains respectively?
440 and 220
What is polyclonal?
An antibody sample that contains a mixture of antibodies with different variable regions.
Why can polyclonal antibodies not be used for structural analysis?
Methods such as XRD require very high purity, and the difference in the variable regions in a polyclonal mixture would prevent proper crystallisation.
What must antibodies be in order to have their structure determined?
Monoclonal, but even then the hinge region flexibility can prevent proper crystallisation.
How are monoclonal antibodies prepared from polyclonal mixtures?
By affinity chromatography using whatever the antibody is specific for.
How can monoclonal antibodies be prepared de novo?
Crossing a specific antibody producing B-cell can be crossed with a tumour cell line causing proliferation and hence large amounts of monoclonal antibody production.
How can the structure of immunoglobulins be determined despite the difficulty in crytallising even monoclonal preparations due to the flexibility of the molecule?
By cleaving them into fragments composed of their three domains.
What is used to cleave antibodies into their domain fragments?
Papain protease cleavage of the heavy chain ‘hinge’ linker sequences.
What fragments are produced by papain cleavage of anitbodies?
2 Fab fragments that are the variable domains
1 Fc fragment that was the dual heavy chain region
How many amino acids make up a ß-sandwich immunoglobulin fold?
110 residues.
Why is fragmentation useful for structural analysis?
The linker regions are the largest obstacle to whole antibody crystallisation as they allow all the regions to move with great freedom relative to one another. This allows them to have their structure analysed separately.
Describe the immunoglobulin fold.
A stable fold consisting of two ß-sandwich motifs that are packed together at a 30° angle.
How many immunoglobulin folds make up a single antibody?
12 - 14, four of them binding in pairs in each fragment region.
How are immunoglobulin domains named?
V or C depending on their constancy or variability for an antibody type, with an L or H in subscript to denote whether they are on the light or heavy chains. On the heavy chains the constant regions are notes as 1, 2, 3 and sometimes 4 starting from the variable end.
Which immunoglobulin types possess heavy chain C_H4 domains?
IgMs and IgGs, hence these are the ones that have fourteen total domains.
How are disulphide bonds used in antibodies?
Each domain is stabilised by a single disulphide bond and more are employed to join the domains at the hinge region.
What post translational modifications are present on antibodies?
The two hinge adjacent immunoglobulin domains (C_H2) in the heavy chain Fb fragment are linked by a pair of carbohydrate molecules.
How are the structure of the variable domains denoted?
The end half of the Fab fragments are known as the Fv fragments - the variable regions. These are tipped by hypervariable antigen binding sites on each chain called complementarity determining regions.
How are the antigen binding sites themselves formed?
The Fv pair of immunoglobulin domains come together to form a barrel that binds the antigen.
What is a paratrope?
The variable antigen binding tip of the Fv region.
What is an epitope?
The antigen to which a specific paratrope binds.
Which loci code for the heavy chain?
Just the Heavy Chain Locus
Which loci code for the light chains?
The lambda and kappa loci.
How was it realised that antibody genetics was employing an unusual mechanism?
Because we can produce billions of different antibodies, more than we have sufficient base pairs to code for in our entire genome.
Where are antibodies produced?
In B-cells.
How are so many antibodies produced from such little genetic material?
Each different region of the antibody is coded for by a wide variety of available versions which can be combined in any way to produce a huge number of different structures.
What is unique about antibody genetics?
The different DNA sections are spliced together at the DNA level, each B-cell becoming specific to producing a single antibody by excising and degrading the rest of the DNA.
How does splicing occur at the lambda locus?
Four regions to choose from in two pairs.
L and V regions always selected together (ie. uses L1 and V1 or L4V4)
J and C regions paired similarly.
How many LV pairs are present at the lambda locus?
30
What do the L and V sections code for?
The Light and Variable chains respectively.
How many JC pairs are present at the lamda locus?
4
What to the J and C sections code for?
The Joining and Constant regions respecitvely.
How many different combinations of light chains can be produced at the lambda locus?
30 (LV pairs) x 4 (JC pairs) = 120 combinations at the lambda locus.
What sections are coded for by the kappa locus?
LV pairs, as in the lambda locus.
J and C sections, now spliced independently from one another.
