CHAPTER 6: PRECIPITATION AND AGGLUTINATION REACTIONS Flashcards
(89 cards)
1
Q
Combination of (?) plays an important role in the laboratory in diagnosing many different diseases.
A
antigen with specific antibody
2
Q
have been developed to detect either antigen or antibody, and they vary from easily
A
Immunoassays
3
Q
Immunoassays are based on the principles of
A
precipitation or agglutination
4
Q
Initial force of attraction that exist between singe Fab site (paratope) and a single epitope on the corresponding antigen
A
Affinity
5
Q
The strength of attraction depends on the specificity of antibody for a particular antigen
A
Affinity
6
Q
Antibodies are capable of reacting with antigens that are structurally similar to the original antigen that induced antibody production. This is known as
A
crossreactivity
7
Q
Sum of all attractive forces between an Ag and Ab
A
Avidity
8
Q
Dictates the overall stability of the Ag-Ab complex
A
Avidity
9
Q
= decreased tendency of the complex to dissociate.
A
High Avidity
10
Q
occur between oppositely charged particles
A
Ionic bonds
11
Q
involve an attraction between polar molecules that have a slight charge separation and in which the positive charge resides on a hydrogen atom
A
Hydrogen bonds
12
Q
occur between nonpolar molecules that associate with one another and exclude molecules of water as they do so
A
Hydrophobic bonds
13
Q
occur because of the interaction between the electron clouds of oscillating dipoles
A
Van der Waals forces
14
Q
Types of Affinity
A
15
Q
Antibody
A
Precipitin
16
Q
Soluble antigens
A
Precipitinogen
17
Q
Insoluble complexes formed by the union of the two aforementioned
A
Precipitate
18
Q
Natural clumping. Fleecy, white/ coudy
A
Flocculation
19
Q
Much better precipitating Ab than IgM
A
IgG
20
Q
Much better agglutinating Ab than IgG
A
IgM
21
Q
Precipitation:
A
IgG>IgM>IgA
22
Q
Nonprecipitating
A
IgE
23
Q
Involves combining soluble antigen with soluble antibody to produce insoluble complexes that are visible
A
PRECIPITATION
24
Q
First noted in 1897 by Kraus
A
PRECIPITATION
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: All antigen-antibody binding is reversible and free reactants are in equilibrium with bound reactants.
Law of Mass Action
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Excess antibody is called the
prozone
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excess antigen concentration is called the
postzone
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Mnemonic:
ProAbPostAg
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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
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In the case of antibody excess, the (?) occurs
prozone phenomenon
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antigen combines with only one or two antibody molecules, and no cross-linkages are formed
prozone phenomenon
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This is because usually only one site on an antibody molecule is used, and many free antibody molecules remain in solution.
prozone phenomenon
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At the other side of the zone, where there is antigen excess, the (?) occurs
postzone phenomenon
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small aggregates are surrounded by excess antigen, and again no lattice network is formed
postzone phenomenon
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may lead to false negative
Prozone and postzone
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Prozone phenomenon→
Serum dilution
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Postzone phenomenon→
Repeat the test after a week to give time for antibody production.
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FACTORS AFFECTING PRECIPITATION
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The pH of the medium used for testing should be near physiologic conditions, or an optimum pH of 6.5 to 7.5
pH
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Ideal: Body temperature (37C/98.6F)
Temperature and Length of Incubation
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40-45C
Temperature and Length of Incubation
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Incubation time range from 15-60 minutes
Temperature and Length of Incubation
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FACTORS AFFECTING PRECIPITATION
Precipitation in a fluid medium
Precipitation by a passive immunodiffusion
Precipitation by electrophoretic techniques.
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Precipitation in a
Precipitation by a
Precipitation by
Precipitation in a fluid medium
Precipitation by a passive immunodiffusion
Precipitation by electrophoretic techniques.
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Precipitation in a Fluid Medium
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is a measure of the turbidity or cloudiness of a solution
Turbidimetry
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A detection device is placed in direct line with the incident light, collecting light after it has passed through the solution.
Turbidimetry
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It thus measures the reduction in light intensity due to reflection, absorption, or scatter.
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Scattering of light occurs in proportion to the size, shape, and concentration of molecules present in solution.
Turbidimetry
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It is recorded in absorbance units, a measure of the ratio of incident light to that of transmitted light.
Turbidimetry
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Measurements are made using a spectrophotometer or an automated clinical chemistry analyzer.
Turbidimetry
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measures the light that is scattered at a particular angle from the incident beam as it passes through a suspension.
Nephelometry
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The amount of light scattered is an index of the solution’s concentration.
Nephelometry
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The precipitation of antigen–antibody complexes can also be determined in a support medium such as a gel.
Precipitation by a Passive Immunodiffusion
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: No electric current is used to speed up reaction of the Ag and Ab combination, but through DIFFUSION
Passive
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Factors affecting rate of diffusion
Size of the particles
Temperature
Gel viscosity
Amount of Hydration
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Precipitation in Gel Medium
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• Only one reactant is moving
Single Diffusion
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• Either Ag or Ab is moving
Single Diffusion
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• Both Ag and Ab are moving through the medium
Double Diffusion
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• Reaction in tubes- Ag or Ab migrate up and down
Single Dimension
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• Petri dish – Ag or Ab diffuse radially
Double Dimension
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• Ab is uniformly distribute in a support gel and Ag is applied to a well cut into gel.
Radial Immunodiffusions
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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)
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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)
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Single DiffusionDouble Dimension (Macini, Fahey, and MacKelvey) Types:
A. Mancini/Endpoint Method
B. Fahey and McKelvey/Kinetic method
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Diameter= Ag Concentration
Mancini/Endpoint Method
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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
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This occurs between 24 and 72 hours.
Mancini/Endpoint Method
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The square of the diameter is then directly proportional to the concentration of the antigen.
Mancini/Endpoint Method
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Diameter= Logarithm Ag Concentration
Fahey and McKelvey/Kinetic method
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Uses measurements taken before the point of equivalence is reached.
Fahey and McKelvey/Kinetic method
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Antigen is not allowed to diffuse completely.
Fahey and McKelvey/Kinetic method
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In this case, the diameter is proportional to the log of the concentration.
Fahey and McKelvey/Kinetic method
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Readings are taken at about 18 hours.
Fahey and McKelvey/Kinetic method
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Mnemonic: “FAK ME”
FA→Kinetic
Mancini→Endpoint
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Both Ag and Ab diffuse independently through a semisolid medium in 2 dimension
Ouchterlony Double Diffusion
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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
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Possible Patterns in Ouchterlony Double Diffusion:
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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
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The arc indicates that the two antigens are identical.
A. Serological Identity: Identical Ag
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• 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
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• Two crossed lines represent two different precipitation reactions. The antigens share no identical determinants.
B. Non-Indentity: Ag are serologically distinct
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. • “Spur formation”
C. Partial Identity: Ag are not identical but do possess common determinants
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• Fusion of two lines with a spur indicates partial identity.
C. Partial Identity: Ag are not identical but do possess common determinants
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• 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
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: technique in which molecules with a net charge are separated when an electric field applied
Electrophoresis
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• Negative charged particles migrate to the
ANODE (Postive (+) Pole)
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• Positive charged particles migrate to the
CATHODE (Negative (-) pole)
