Section 5: Antibody- Structure and Diversity Flashcards
(39 cards)
Antibody
-the primary function of an antibody is to recognize and bind antigen, and target that antigen to other components of the immune system
What does an antibody consist of?
4 polypeptide chains
-2 identical H chains (50kDa)
-2 identical L chains (25kDa)
-each region possesses a region of high sequence diversity (variable region, v) and more conserved sequences (constant region, c)
How was the structure of an antibody found?
-the structure was found by using various enzymes to cleave different portions of the molecule
-treatment with PAPAIN yields cuts above disulfide bond
-one fragment would crystalize but not bind antigen (Fc) fragment crystallization portion
-Fc is the variable part and Fab is the constant
-two fragments that could bind antigen; fragment antigen-binding portion (Fab)
Treatment with Pepsin yields what in the antibody structure?
-cuts below disulfide bond
-one partial Fc portion
- one fragment that consisted of two cross-linked Fab regions F(ab’)2
-allows cross-linking Fab regions, probably useful for making reagents or assay or neutralizing treatment
hinge region
-found in an antibody structure that allows for flexibility in binding antigens
-allows two Fab arms to adopt many different orientations
Isotypes
-classes of immunoglobulins defined by the differences in heavy chain C regions
1. IgG
2. IgM
3. IgD
4. IgA
5. IgE
*with their heavy chains denoted γ, μ, δ, α, ε
What are the names of the two isotopes of the light chain?
- Kappa (κ) and lambda (λ) with no functional differences found
Immunoglobulin domains
-what each antibody is made up of
- light chains: have one variable domain (VL) and one constant (CL)
-the heavy chains: have one variable (VH) and 3 constant (CH 1,2,3)
hypervariable regions (HV)
-the variations in the amino acid sequence of the V regions that determine antigen specificity are concentrated here (red) which are flanked by framework regions (yellow)
-3 HV regions are found in each V domain
-the yellow loop is constant and red is the variable region
complementary determining regions (CDRs)
-what hypervariable loops are often called
Antigen binding
-the most effective antibodies against a pathogen bind to surface molecules
-carbohydrates and proteins (glycoproteins, polysaccharides, glycolipids, peptidoglycan)
-antibodies can bind a large array of chemical structures however these are more commonly implicated in allergic or autoimmune reactions
-limiting factor to what cannot have an antibody developed is the size
antigenic determinant (epitope)
-the part of the antigen to which the antibody binds is called
-important to how well the antibody can neutralize or opsonize
systemic lupus erythematosus
-antibodies directed towards DNA are characteristic of this autoimmune disease
linear epitope
is formed from contiguous amino acids
-bind continuous amino acids in a row
discontinuous epitopes
-is formed from amino acids from different parts of the polypeptide brought together by chain folding
-bind to the structure, not the sequential amino acid
-structure can determine how well an antibody works
-pathogens can hide some epitopes in the 3D structure
affinities
-because of small differences in shape and chemical properties of the binding site, certain antibodies will bind with different strengths to antigens
-this property makes them useful reagents in detecting and quantifying a particular antigen
-Historically you could immunize an animal and harvest
its antisera, containing Ab to a specific antigen
monoclonal antibodies
-the modern method is to isolate a B-cell from the immunized animal and immortalize it by fusion with a tumor cell forming hybridoma cell lines that produce this
-there is one clone, one type of antibody, has a specificity that we can measure and a known affinity
monoclonal antibody steps
-using monoclonal antibodies to specific surface markers on cells then we add fluorescent dyes to them, to tell us more information going on
antibody diversity
-it is estimated that there are 10^8 Ab specificities that exist in each individual
-if each Ab were produced by a single gene, this would result in about 410^11 nucleotides needed to encode these genes
-there are 2.8510^9 nucleotides in the human genome, so that is not how Ab diversity is achieved
somatically recombined
-this is how Ab is encoded in a series of genes to achieve a high level of diversity
-chain variable regions are encoded by 2 or 3 gene segments
-heavy chain (3), V (variable), Diversity (D), and (J) joining
-light chain (2) has Variable (V) and Joining (J)
somatic recombination
-V region gene sequences are constructed from gene segments by the process called…
-somatic recombination of V, D, and J segments produces a functional heavy chain gene
-the various C (constant) gene segments that remain
-in the germ line organization of the human immunoglobulin heavy, the lambda chain has about 30 while kapa has up to 35
recombination signal sequences (RSS)
-each V, D, and J gene segment is flanked by these sequences
- 9-bp purple
- 7-bp orange
-these RSS are arranged in pairs that are separated by 12- and 23- bp spacers (white)
- the -12 and -23 bp change is the twisting of the DNA to be between the major and minor grooves
recombination activating genes (RAG-1 & -2)
-gene segments are joined together by this protein which recognizes the RSS
-to ensure alignment of the gene segments RAG-1 and 2 bring together specific 12 and 23 bp spacers (12/23 rule)
-not a eukaryotic gene does not have introns and exons
RNA processing
-gives rise to different isotypes and different secreted or membrane-bound forms of antibodies
-determines the types of isotopes we produce and where the antibody will localize to (secreted or transmembrane)
