Immunology Lab 2 Flashcards
precipitation
the reaction of a specific antibody with a SOLUBLE antigen to form an insoluble lattice Ag-Ab complex
agglutination
the reaction of a specific antibody with a PARTICULATE antigen to form a visible clump
particulate antigen
antigens that will not mix into solution; attached to beads
3 Requirements for Optimal Ag-Ab Reaction
- Ab should be at least bivalent (have two or more binding sites)
- Ag should be multivalent (have multiple epitopes)
- Ratio of Ag-Ab should be in the equivalence zone
equivalence zone
the region where the maximum amount of precipitation occurs
IgG is ___valent
bivalent (2)
IgM is ___valent
multi (10)
What are the three zones?
- Antibody excess (this is what’s bound to the bottom in vitro)
- Equivalence
- Antigen excess
For in vivo reactions, what zone will result in disease?
antigen excess - overwhelms the #/ability of Ab to bind so the macrophages in the body cannot fight it off
Agar Double Diffusion
place dilutions of serum (Ab) in wells surrounding a center well of Ag; 24-48 hours look for a precipitin line where the two meet to determine titer
What two factors can affect location and shape of the precipitin line?
- Molecular Weight (line closer/curved to that of higher MW)
- Concentration of the Ab (larger dilutions pull the line closer/curved towards the Ab/outer well; doesn’t travel as far because there isn’t as much of it)
What can you use agar double diffusion for clinically?
titers of bacterial, viral, and fungal infections (including Blastomyces and Histoplasma)
How are titers reported?
as the inverse of the dilution (1:4 –> titer = 4)
Hemoagglutination
agglutination of RBCs
Clinical Application of Hemoagglutination?
blood typing
Why can an antibody not be univalent (rare anyway) in an agglutination reaction?
no clumping could occur! A singular Ab would not be able to bind multiple antigens on cells together
Rheumatoid Factor
IgM anti-IgG autoantibodies (meaning an IgM that contains Fc regions specific for canine IgG)
Rheumatoid Arthritis
chronic crippling disease [in dogs] involving inflammatory infiltrate in the joint(s) that can eventually lead to its destruction
How accurate is diagnosing rheumatoid arthritis (RA) in dogs with a latex agglutination reaction?
~70% of dogs that have RA will be positive with this test (the rest of the diagnosis comes from clinical symptoms)
Is rheumatoid factor found in diseases besides RA?
yes - lupus erythematosus
Rheumatoid Arthritis Agglutination Test
serum put in well (the flat black plate) and mixed with a suspension of latex beads coated in IgG; clumps will form in positive patients
Is an Ag-Ab reaction permanent?
no - it is reversible (noncovalent) binding and the reactants are not chemically altered
affinity
combined strength of bonding between a single Ag binding site (Fab) on the Ab and a single epitope on the Ag
most bonds over the shortest distance
Do we want low or high affinity Abs?
High!
