Section 1: Agglutination and Precipitation Reactions

Question 1

unlabeled immunoassays

Answer 1

-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)

Question 2

precipitation

Answer 2

-cross-linking of a soluble antigen to create an insoluble precipitate

Question 3

agglutination

Answer 3

-cross-linking of particulate antigens to form larger, also visible complexes (bacteria, cells, latex beads)

Question 4

Affinity

Answer 4

-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

Question 5

Affinity constant (Ka)

Answer 5

(Conc. of Ab-Ag complex)/ (Conc. Ab) (Conc. Ag) = (AbAg)/(Ab)*(Ag) = Ka
-the higher the affinity the more sensitive the reaction

Question 6

avidity

Answer 6

-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

Question 7

equivalence

Answer 7

-the the formation of Ag-Ab complexes the largest complexes are formed when Ag epitopes are in equivalence to Ab binding sites

Question 8

prozone

Answer 8

-when precipitation efficiency is decreased because of excess Ab which decreases cross-linking
Prozone: dilute patient serum (can try to dilute patient serum)

Question 9

Postzone

Answer 9

-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

Question 10

What is the simplest way to detect Ab-Ag interactions

Answer 10

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

Question 11

Advantage of precipitation

Answer 11

-easy and cheap

Question 12

Disadvantages of precipitation

Answer 12

-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

Question 13

Ouchterlony analysis

Answer 13

-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

Question 14

Ouchterlony (Line of identity)

Answer 14

-when identical Ags are placed in the wells, a line of identity is formed (ARC)

Question 15

Ouchterlony (Line of nonidentity)

Answer 15

-when completely different Ags are placed in the wells, two crossing lines of precipitate form, line of nonidentity

Question 16

Ouchterlony (Line of partial identity)

Answer 16

-when similar Ags are placed in the wells, a line similar to the line of identity forms with the exception of forming a spur
-the spur always points to the simpler Ag, that is the one with fewer epitopes

Question 17

Radial immunodiffusion (RID)

Answer 17

-based on a concept similar, except now there are many wells with decreasing concentrations of Ag as you move left to right across the agar media which already contains Ab
-Quantitative
-more concentrated Ag wells diffuse farther into the media
-conc. can be calculated by measuring the diameter of the precipitate line from the well and producing a standard curve with known Ag concentrations
- RID is used to measure patient IgG, IgM, and IgA levels (the Ab are being measured as Ag because an anti-human Ab is contained in the media, recognizes Fc portion)
-mobilize one antibody into the agar itself, so the agar contains one of the two factors at a constant concentration, then vary or dilute out the patient sample and see where the lines come

Question 18

Two methods to perform RID

Answer 18

1. Fahey method (kinetic method)
2. Mancini Method method (End-point method)

Question 19

Fahey method (kinetic method)

Answer 19

-difusion proceeds for 18 hours
-the diameter is proportional to the Log of the concentration
-plotted on semilog paper (x axis = diameter, y axis= concentration)

Question 20

Mancini method (end-point method)

Answer 20

-diffusion proceeds for 48-72 hours
-the square of the diameter is directly proportional to the concentration