Notes

1. precipitation
2. agglutination
3. complement fixation
4. labelled immunoassay

ag- ab binding

1. affinity 1st ab ag with a force fab site ab and e ag
the strength attraction depend on the specificity of ag and ab

more cross reacting antigen - more stronger bond ng ag and binding site
- can cause false +
higher affinity = high specificity

2. avidity - the addition of attraction ab and ag


law of mass action
k = ab ag / ab x ag
free reactants are equilibrium bound with reactants

the value k - depends strenght binding of ab- ag
the higher k - the larger the amount of ab -ag complec
- easily visible dectectable reaction

precipitation rx= soluble ab+ soluble ag = insoluble complexes
precitipinogen - ag
precipitin - ab
visible precipitin line - postive

precipitation curve
1. zone of equivalence - equal interaction ng ab -ag


2. prozone - ab excess
no cross linkages
antigen binds with only one or two ab
too much ab

false (-) high ab concentration - mas mataas na ab titer
false (-) resolution--> serum dilution

3. post zone - excess antigen
no lattice formation
lattice formation - cross linkages b/w ab and ag

resolution
1. repeat test - (patasin yung ab)
2. cell washing - remove excess antigen



Precipitation technique
1. nephelometry
- immunoglobulin
-complement
-c creactive protein
-other serum proteins

sensitivity 1-10 ag ab/ ml
principle:
light scattered indicates the amount of ab and ag present

turbidity - measures teh block of light
scatter light- light passes tru the soln
blocked light- light is block does pass the soln

the more light scattered - mas madaming ag/ ab present

2. radial immunodiffusion - single diffusion
- immunoglobulin
- complement


sensitivity 1-10 ag ab/ ml

antigens diffuse out into gel infused with antibody
measure -radius- indicates concentration of ag

-The wider the diameter the more concentrated the antigen


2. ouchterlony (double diffusion)
- complex antigen (fungal antigen)
2-200

both ab -ag diffuse out from the wells.
precipitin line formed- indicates relationship of antigen.

3. immunoelectrophoresis
differentiation of serum proteins
20-200

electrophoresis of the serum diffusion of ab from the wells

3 posible rx
identity - Arc ab and ag are identical
partial identity- spur formation only one antigen identical is not
non identity- no antigen are identical

4. Immunofixation electrophoresis- double diffusion
over or under production of ab

electrophoresis of the serum is followed by direct application of ab to the gel

most accurate precipitation
apply electrical - speed up the meet of ab and ag (several hours)

source- serum -to determine over- or underproduction of antibody types

Electrophoresis: migration of protein in an electrically charged field

The larger the molecular weight the slower the migration


agglutination vs. precipitation
Antigen
agglutination-particulate
precipitation - soluble

ab
agglutinin
precipitin

result
agglutination/ clumping
precipitinin line

Agglutination- visible aggregation
-produced by antibodies (agglutinins)
TWO PROCESSES
1. SENSIZATION
2. LATTICE FORMATION