Section 1: Agglutination and Precipitation Reactions Flashcards
unlabeled immunoassays
-agglutination and precipitation were the first immunoassays developed and are based on cross-linking of antibody-antigen complexes
-these reactions are not that sensitive because results are visualized by eye or using optics (turbidometry and nephelometry)
precipitation
-cross-linking of a soluble antigen to create an insoluble precipitate
agglutination
-cross-linking of particulate antigens to form larger, also visible complexes (bacteria, cells, latex beads)
Affinity
-all serological assays (labeled or unlabeled) rely on the affinity, avidity, and specificity of the Ab to the Ag
-the strength of the binding of 1 Fab region with its corresponding Ag
Affinity constant (Ka)
(Conc. of Ab-Ag complex)/ (Conc. Ab) (Conc. Ag) = (AbAg)/(Ab)*(Ag) = Ka
-the higher the affinity the more sensitive the reaction
avidity
-the number of binding sites times the affinity
-IgG with two binding sites has an avidity of 2 * (Ka)
-IgM with 10 binding sites has an avidity of 10 * (Ka)
* IgM usually has a lower affinity than IgG because the production of IgG (class switching) has occurred after somatic hypermutation events
-Steric hindrance can also play a role in lowering avidity
-the greater those numbers are the lower amount we can detect
equivalence
-the the formation of Ag-Ab complexes the largest complexes are formed when Ag epitopes are in equivalence to Ab binding sites
prozone
-when precipitation efficiency is decreased because of excess Ab which decreases cross-linking
Prozone: dilute patient serum (can try to dilute patient serum)
Postzone
-when precipitation efficiency is decreased because of excess Ag which decreases cross-linking
** both prozone and postzone will appear falsely negative
Postzone: redraw 1-2 weeks after more Ab produced
What is the simplest way to detect Ab-Ag interactions
precipitation
* this is accomplished by placing Ag and Ab in different wells of an agar plate, allow time for them to diffuse through the agar, and visualize the precipitation pattern
Advantage of precipitation
-easy and cheap
Disadvantages of precipitation
-limited sensitivity (20 ug/ml)
-cannot be amplified, either by attaching Ag to a particle or linking of a substance to the Ab to increase visualization
Ouchterlony analysis
-double diffusion gel precipitation
-qualitative
-both Ag and Ab diffuse through agar media
-as radial diffusion occurs Ag and Ab reach equivalence a precipitate is formed by cross-linking (lattice structure)
-particularly useful in identifying if unknown Ag is Identical, Partially, Identical, or Nonidentical with a known Ag
-modern uses are limited to rare fungal infection diagnosis (coccidiomycosis) and in some Ab specificity testing in autoimmune diseases
Ouchterlony (Line of identity)
-when identical Ags are placed in the wells, a line of identity is formed (ARC)
Ouchterlony (Line of nonidentity)
-when completely different Ags are placed in the wells, two crossing lines of precipitate form, line of nonidentity