Antigens and antibodies interact… • They associate: [Ag] + [Ab] 6 [AgCAb] • They dissociate: [AgCAb] 6 [Ag] + [Ab] • The velocity of the association: v1%[Ag] [Ab] • The velocity of the disssociation: v2% [AgCAb] • Proportionality can be converted to equality (by introducing a velocity constant)
Antigens and antibodies interact… Thus, v1 = k1 [Ag] [Ab] and v2 = k2 [AgCAb] At equilibrium, v1 = v2; thus, k1 [Ag] [Ab] = k2 [AgCAb] Rearranging, k1 / k2 = [AgCAb] / [Ag] [Ab] k1 / k2 = (necessarily a constant) is set equal to Ka, the association constant between antigen and antibody. Ka measures the affinity of a specific antibody for a specific antigen. (Kd, the dissociation constant is the inverse of Ka. Kd were the values seen with affinity maturation due to somatic hypermutation.)
Antigen precipitin complex…a visible, three-dimensional network or lattice that will eventually settle from solution as an “antigen-precipitin” complex. • The antibody must be bivalent; a precipitate will not form with monovalent Fab fragments. • The antigen must be either bivalent or polyvalent; that is, it must have at least two copies of the same epitope
Ag-Ab precipitins…. • Seeing a complex requires that the Ag-Ab complex exist at milligrams / milliliter
Numerous techniques make Ag’s visible • See more techniques: http://www.alzforum.org/res/com/ant/glossary.asp
Affinity versus avidity… (from the “Glossary” in your text...) “Affinity: The strength with which a ligand interacts with a binding site. It is represented quantitatively by the affinity constant Ka.” (For, “ligand” read epitope; for “binding site” read paratope.) “Avidity: The strength of antigen-antibody binding when multiple epitopes on an antigen interact with multiple binding sites of an antibody.”
Agglutination:(once again from the “Glossary”...) • “Agglutination[:] The aggregation or clumping of particles (e. g., latex beads) or cells (e. g., red blood cells).” • Hmmm, that’s a bit spare. • What happens here is that a bead or cell gets coated with an antigen of interest. Then, the cell(bead)-Ag is reacted with Ab. If the Ab is specific for the Ag, a mesh will form. This complex is more readily observed then are precipitates.
Two examples of agglutination… • ABO blood groups… • The ABO blood-group antigens are glycoproteins expressed on red blood cells... • A type O individual ... has anti-A and anti-B antibodies... • but has no antigen on the blood cell to elicit these antibodies!!! • Cross reactivity (CRM) is the basis for the presence of these blood-group antibodies, which are induced in an individual not by exposure to red blood cell antigens but by exposure to cross-reacting microbial antigens present on common intestinal bacteria. !!!!
ABO blood groups continued… • “Antibodies to the A, B, and O antigens, called isohemagglutinins, are usually of the IgM class. An individual with blood type A, for example, recognizes B-like epitopes on intestinal microorganisms and produces isohemagglutinins to the B-like epitopes. This same individual does not respond to A-like epitopes on the same intestinal microorganisms because these A-like epitopes are too similar to self and a state of self-tolerance to these epitopes should exist.” • sooooo, [conversely]your ability to resist some microorganisms is affected by lacking antibodies to microbial antigens that would react with your own blood cells; • Such is the case if you are type A, or type B, or type AB. • The missing antibodies are lost by clonal deletion because the antigens look like “self.”
The second example… • Imagine that one suspects that the Ag exists in some other solution (say, the urine of an individual.) • Then, if the Ab is reacted with the urine and there is Ag in the urine, then the Ab receptors become saturated with Ag. • When the Ab is next reacted with cell(bead) complex which is coated with the same Ag, there will be no agglutination (thus called agglutination inhibition) because the Ab will already have reacted with the Ag (in the urine.) • This protocol is used in drug testing and has been used in some pregnancy tests.
Overview: • There are techniques for suspended Ag’s that produce agglutinins • There are techniques for soluble Ag’s that produce precipitins • Modern techniques increase sensitivity:
An important refinement…. ELISA • The fundamental feature of the Enzyme Linked ImmunoSorbent Assay is to have an enzyme linked to an antibody and to have that enzyme be able to generate some visible product. • Note that because AbE has a constant region as the epitope to which it reacts, AbE can be used to detect any antibody of a particular isotype. That is to say, AbE once prepared can be used in multiple ELISA assays. • Antibodies at concentrations of 0.1 to 10 ng·ml-1can be detected.