CHAPTER 6: PRECIPITATION AND AGGLUTINATION REACTIONS Flashcards
Combination of (?) plays an important role in the laboratory in diagnosing many different diseases.
antigen with specific antibody
have been developed to detect either antigen or antibody, and they vary from easily
Immunoassays
Immunoassays are based on the principles of
precipitation or agglutination
Initial force of attraction that exist between singe Fab site (paratope) and a single epitope on the corresponding antigen
Affinity
The strength of attraction depends on the specificity of antibody for a particular antigen
Affinity
Antibodies are capable of reacting with antigens that are structurally similar to the original antigen that induced antibody production. This is known as
crossreactivity
Sum of all attractive forces between an Ag and Ab
Avidity
Dictates the overall stability of the Ag-Ab complex
Avidity
= decreased tendency of the complex to dissociate.
High Avidity
occur between oppositely charged particles
Ionic bonds
involve an attraction between polar molecules that have a slight charge separation and in which the positive charge resides on a hydrogen atom
Hydrogen bonds
occur between nonpolar molecules that associate with one another and exclude molecules of water as they do so
Hydrophobic bonds
occur because of the interaction between the electron clouds of oscillating dipoles
Van der Waals forces
Types of Affinity
Antibody
Precipitin
Soluble antigens
Precipitinogen
Insoluble complexes formed by the union of the two aforementioned
Precipitate
Natural clumping. Fleecy, white/ coudy
Flocculation
Much better precipitating Ab than IgM
IgG
Much better agglutinating Ab than IgG
IgM
Precipitation:
IgG>IgM>IgA
Nonprecipitating
IgE
Involves combining soluble antigen with soluble antibody to produce insoluble complexes that are visible
PRECIPITATION
First noted in 1897 by Kraus
PRECIPITATION
: All antigen-antibody binding is reversible and free reactants are in equilibrium with bound reactants.
Law of Mass Action
Excess antibody is called the
prozone
excess antigen concentration is called the
postzone
Mnemonic:
ProAbPostAg
Zone of equivalence: Area wherein maximum precipitation will occur because Ag and Ab concentration must have an
optimum ratio or Ag and Ab are equal
In the case of antibody excess, the (?) occurs
prozone phenomenon
antigen combines with only one or two antibody molecules, and no cross-linkages are formed
prozone phenomenon
This is because usually only one site on an antibody molecule is used, and many free antibody molecules remain in solution.
prozone phenomenon
At the other side of the zone, where there is antigen excess, the (?) occurs
postzone phenomenon
small aggregates are surrounded by excess antigen, and again no lattice network is formed
postzone phenomenon
may lead to false negative
Prozone and postzone
Prozone phenomenon→
Serum dilution
Postzone phenomenon→
Repeat the test after a week to give time for antibody production.
FACTORS AFFECTING PRECIPITATION
The pH of the medium used for testing should be near physiologic conditions, or an optimum pH of 6.5 to 7.5
pH
Ideal: Body temperature (37C/98.6F)
Temperature and Length of Incubation
40-45C
Temperature and Length of Incubation
Incubation time range from 15-60 minutes
Temperature and Length of Incubation
FACTORS AFFECTING PRECIPITATION
Precipitation in a fluid medium
Precipitation by a passive immunodiffusion
Precipitation by electrophoretic techniques.
Precipitation in a
Precipitation by a
Precipitation by
Precipitation in a fluid medium
Precipitation by a passive immunodiffusion
Precipitation by electrophoretic techniques.
Precipitation in a Fluid Medium
is a measure of the turbidity or cloudiness of a solution
Turbidimetry
A detection device is placed in direct line with the incident light, collecting light after it has passed through the solution.
Turbidimetry
It thus measures the reduction in light intensity due to reflection, absorption, or scatter.
Scattering of light occurs in proportion to the size, shape, and concentration of molecules present in solution.
Turbidimetry
It is recorded in absorbance units, a measure of the ratio of incident light to that of transmitted light.
Turbidimetry
Measurements are made using a spectrophotometer or an automated clinical chemistry analyzer.
Turbidimetry
measures the light that is scattered at a particular angle from the incident beam as it passes through a suspension.
Nephelometry
The amount of light scattered is an index of the solution’s concentration.
Nephelometry
The precipitation of antigen–antibody complexes can also be determined in a support medium such as a gel.
Precipitation by a Passive Immunodiffusion
: No electric current is used to speed up reaction of the Ag and Ab combination, but through DIFFUSION
Passive
Factors affecting rate of diffusion
Size of the particles
Temperature
Gel viscosity
Amount of Hydration
Precipitation in Gel Medium
• Only one reactant is moving
Single Diffusion
• Either Ag or Ab is moving
Single Diffusion
• Both Ag and Ab are moving through the medium
Double Diffusion
• Reaction in tubes- Ag or Ab migrate up and down
Single Dimension
• Petri dish – Ag or Ab diffuse radially
Double Dimension
• Ab is uniformly distribute in a support gel and Ag is applied to a well cut into gel.
Radial Immunodiffusions
Procedure:
1. Ab mixed in agarose
2. Antigen dilution is overlaid (Ag must always be greater to achieve zone of equivalence)
3. Mobile Ag diffuses through the gel, containing immobilized Ab forming insoluble Ag-Ab Complexes.
4. At equivalence concentration, the Ag stops moving and a satbilized band is formed.
Single DiffusionSingle Dimension (Oudin)
Procedure:
1. Ab is mixed with liquid agar and poured into the petri dish
2. Circular wells cut in gel
3. Ag is loaded into the wells
4. Ring precipitate expands from the well as Ag diffuses toward its equilibrium concentration
5. Diameter of the disc is measured.
Single DiffusionDouble Dimension (Macini, Fahey, and MacKelvey)
Single DiffusionDouble Dimension (Macini, Fahey, and MacKelvey) Types:
A. Mancini/Endpoint Method
B. Fahey and McKelvey/Kinetic method
Diameter= Ag Concentration
Mancini/Endpoint Method
In this technique, antigen is allowed to diffuse to completion, and when equivalence is reached, there is no further change in the ring diameter.
Mancini/Endpoint Method
This occurs between 24 and 72 hours.
Mancini/Endpoint Method
The square of the diameter is then directly proportional to the concentration of the antigen.
Mancini/Endpoint Method
Diameter= Logarithm Ag Concentration
Fahey and McKelvey/Kinetic method
Uses measurements taken before the point of equivalence is reached.
Fahey and McKelvey/Kinetic method
Antigen is not allowed to diffuse completely.
Fahey and McKelvey/Kinetic method
In this case, the diameter is proportional to the log of the concentration.
Fahey and McKelvey/Kinetic method
Readings are taken at about 18 hours.
Fahey and McKelvey/Kinetic method
Mnemonic: “FAK ME”
FA→Kinetic
Mancini→Endpoint
Both Ag and Ab diffuse independently through a semisolid medium in 2 dimension
Ouchterlony Double Diffusion
Procedure
1. Pattern of well in cute in an agarose gel in petri dish
2. Reactants are loaded
3. Incubated until lines are precipitated
Ouchterlony Double Diffusion
Possible Patterns in Ouchterlony Double Diffusion:
Fusion of the lines at their junction to form an arc represents serological identity or the presence of a common epitope
A. Serological Identity: Identical Ag
The arc indicates that the two antigens are identical.
A. Serological Identity: Identical Ag
• Pattern of crossed lines demonstrates two separate reactions and indicates that the compared antigens share no common epitopes
B. Non-Indentity: Ag are serologically distinct
• Two crossed lines represent two different precipitation reactions. The antigens share no identical determinants.
B. Non-Indentity: Ag are serologically distinct
. • “Spur formation”
C. Partial Identity: Ag are not identical but do possess common determinants
• Fusion of two lines with a spur indicates partial identity.
C. Partial Identity: Ag are not identical but do possess common determinants
• The two antigens share a common epitope, but some antibody molecules are not captured by antigen and travel through the initial precipitin line to combine with additional epitopes found in the more complex antigen
C. Partial Identity: Ag are not identical but do possess common determinants
: technique in which molecules with a net charge are separated when an electric field applied
Electrophoresis
• Negative charged particles migrate to the
ANODE (Postive (+) Pole)
• Positive charged particles migrate to the
CATHODE (Negative (-) pole)