The bonds involved are non-covalent, so reactions are reversible and antibody and antigen are not altered by interaction
4 types of non-covalent forces involved:
(1) van der Waals
– interaction distance important; too close-repel; too far-no attraction; non-specific
(2) hydrophobic
– more energetically favorable for non-polar/non-polar than mixed non-polar/polar
(3)
electrostatic – between fixed charges of opposite sign
more discriminating than above forces (charge basis) but less than hydrogen
(4)
hydrogen bonds – most specific in terms of geometric requirements and atom type
Involves sharing of a hydrogen atom by 2 electronegative atoms (i.e. O and N)
There is an almost unlimited potential to generate antibodies reactive with selected ligands
Classification of antigens by epitope
:
unideterminant univalent (hapten) – one antigen
unideterminant multivalent (polysaccharide) – repeating sequence of antigen
multideterminant univalent (monomeric protein) – multiple separate antigens with no sequence
Multideterminant multivalent (virus) – repeating sequence of antigen
Classification by antigen-antibody interaction
monovalent – antibody binds antigen with one arm
monogamous bivalent – antibody binds same antigen with both arms (in different places)
more tightly bound than monovalent
non-monogamous bivalent – antibody binds one antigen with one arm and separate distinct antigen with the other
allows for X-linking of soluble or suspended molecules, or agglutination
antigen-antibody affinity is a measure of binding strength
Valence
: # of antibody molecules that can bind to a molecule of the antigen at saturation
Almost always < # of epitopes due to steric hindrance of competing antibodies at nearby sites
Two types of affinity:
(1) Intrinsic Affinity
– the interaction between one antibody binding site (peritope, P) and one antigenic determinant (epitope, E) ; IA = equilibrium association constant for P + Eunivalent
Û PE
= Ka = [PE]/[P][E] therefore Ka = 1/[Ag] at 50% occupancy of P
(2) Functional Affinity (avidity)
– the overall ability of an antibody to bind an antigen
FA = Ka = !K(n) != factorial K = Ka specific epitope n= valence of antigen
Note: For bivalent antigen K1
¹K2 due to steric factors etc.
However: Ý valence Þ Ka(functional affinity) > Ka(intrinsic affinity) by as much as 10 4 or 106
antibodies are flexible, so they form various geometric shapes via antigen binding (multivalent)
complex formation: antigen or antibody excess Þ small complexes, less crosslinking
Zone of Equivalence
Þ increased crosslinking characteristic of a specific antigen-antibody interaction
agar gel diffusion of antigen and antibody form characteristic precipitate band at equivalence
Medical Applications of Antibodies
In vivo
Passive immunotherapy: antibodies injected to protect an individual
Prevention of immunization: maternal fetal incompatibility–transfer antibody to mother (Rh-) so she won’t attack Rh+ child
Elimination of toxins: digitalis overdose cleared faster with antibodies
Localization of molecules of interest through radionucleotide imaging (cancer therapy?)
In vitro
Direct binding using radioisotope to measure concentration of substance in body fluid or on cell surface
Indirect binding using enzyme to localize molecules within cells or tissues
Competative binding using fluorescent molecule to assess structural relatedness of different molecules
Monoclonal Antibodies
Antigen produced by hybridoma formed from fusion of B lymphocyte from mouse spleen (antigen specific) with non-Ig-producing mouse myeloma cell
Screening for desired antibody will produce homogenous antibodies that have exact same V domain and antigen reactivity
Genetically engineered Antibody
Fab fragments or single chain Fv fragments can be expressed as soluble molecules in periplasm of E. coli.
Expression of Fab’s or Fv’s as fusion proteins with bacteriophage coat proteins (phage display) permit large numberss of different VH-VL pairs (called combinatorial library) to be screened for reactivities of interest.
Engineered antibodies can be made with any combination of domains, even across species