CHAPTER 6.3 - PRECIPITATION 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
1 fab + 1 epitope
(Ab) (Ag)
Affinity
Sum of all attractive forces between an Ag and Ab
Avidity
Dictates the overall stability of the Ag-Ab complex
Avidity
Multivalent Ab=Multivalent Ag
Avidity
Types of Affinity
electrovalent bond (ionic charge in a chemical soln)
Ionic bonds
Hydrogen bridging
Hydrogen bonds
major bond demonstrated by an Ag and Ab
Hydrophobic bonds
combination between an ionic and a hydrophobic bond
Van der Waals forces
Antibody
Precipitin
Soluble antigens
Precipitinogen
Insoluble complexes formed by the union of the two aforementioned
Precipitate
Natural clumping
Flocculation
Major Immunoglobulins Involved
Much better precipitating Ab than IgM
IgG
Much better agglutinating Ab than IgG
IgM
Precipitation:
IgG>IgM>IgA
Nonprecipitating
IgE
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
Ideal: Temperature and Length of Incubation
Body temperature (37C/98.6F)
37C; warm reacting
IgG
40-45C; cold reacting
40-45C
Incubation time range from
15-60 minutes
involves combining soluble antigen with soluble antibody to produce insoluble complexes that are visible
first noted in 1897 by Kraus
: All antigen-antibody binding is reversible and free reactants are in equilibrium with bound reactants
Law of Mass Action
So that maximum precipitation occurs, Ag and Ab concentration must have an
optimum ratio
Ag and Ab are equal, therefore (?) occur
max precipitation
More Ab (patient) excess than Ag
prozone
remedy: Serum dilution
prozone
Ag excess, less Ab (Patient) may lead to false negative
postzone
Remedy: Repeat the test after a week to give time for antibody production
postzone
PRECIPITATION REACTIONS
PRECIPITATION IN A FLUID MEDIUM
PRECIPITATION BY A PASSIVE IMMUNODIFFUSION
PRECIPITATION BY ELECTROPHOTERIC TECHNIQUES
Precipitation in a Fluid Medium
measure of the turbidity or cloudiness of a solution
Turbidimetry
measures the reduction in light intensity due to reflection, absorption, or scatter
Turbidimetry
measures the light that is scattered at a particular angle from the incident beam
Nephelometry
amount of light scattered is an index of the solution’s concentration
Nephelometry
Ag-Ab complex may be observed in a support media (Agarose gel)
Precipitation by a Passive Immunodiffusion
Passive: No electric current is used to speed up reaction of the Ag and Ab combination, but through DIFFUSION
Precipitation by a Passive Immunodiffusion
Factors affecting rate of diffusion
o Size of the particles
o Temperature
o Gel viscosity
o 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
Types of Radial Immunodiffusions
Oudin
Single DiffusionSingle Dimension
Macini, Fahey, and MacKelvey
Single DiffusionDouble Dimension
Single DiffusionSingle Dimension (Oudin)
Procedure:
1. Ab mixed in (?)
2. Antigen dilution is overlaid ([?]must always be greater to achieve zone of equivalence)
3. (?) diffuses through the gel, containing immobilized Ab forming insoluble Ag-Ab Complexes.
4. At equivalence concentration, the Ag stops moving and a (?) is formed.
agarose
Ag
Mobile Ag
stabilized band
Single DiffusionDouble Dimension (Macini, Fahey, and MacKelvey)
Procedure:
1. Ab is mixed with liquid agar and poured into the (?)
2. Circular wells cut in (?)
3. Ag is loaded into the (?)
4. (?) expands from the well as Ag diffuses toward its equilibrium concentration
5. (?) is measured.
petri dish
gel
wells
Ring precipitate
Diameter of the disc
Single DiffusionDouble Dimension (Macini, Fahey, and MacKelvey) Types:
A. Mancini/Endpoint Method
B. Fahey and McKelvey/Kinetic method
Diameter= Ag Concentration
Mancini/Endpoint Method
Diameter= Logarithm Ag Concentration
Fahey and McKelvey/Kinetic method
Both Ag and Ab diffuse independently through a semisolid medium in 2 dimension
Ouchterlony Double Diffusion
Ouchterlony Double Diffusion
Procedure
1. Pattern of well in cut in an (?)
2. (?) are loaded
3. Incubated until lines are (?)
agarose gel in petri dish
Reactants
precipitated
Possible Patterns in Ouchterlony Double Diffusion:
A. Serological Identity: Identical Ag
B. Non-Indentity: Ag are serologically distinct
C. Partial Identity: Ag are not identical but do possess common determinants.
: Identical Ag
A. Serological Identity
: Ag are serologically distinct
B. Non-Indentity
: Ag are not identical but do possess common determinants.
C. Partial Identity
Important in lab tests
Specificity
technique in which molecules with a net charge are separated when an electric field applied
Electrophoresis
Negative charged particles (anions) migrate to the
ANODE (Postive (+) Pole)
Positive charged particles (cations) migrate to the
CATHODE (Negative (-) pole)
FACTORS THAT INFLUENCE RATE OF PROTEIN MIGRATION
size, harder to migrate
↑
Solubility of the protein
AMOUNT OF SOLVATION
viscous, harder to migrate
↑
PH OF BUFFER
> 8
• Room temperature
TEMPERATURE
• Protein will denature once it is exposed to high temperature
TEMPERATURE
flow of ions goes toward the cathode and can impede movement of proteins toward the anode
ENDOOSMOSIS
DIFFERENT TESTS FOR ELECTROPHORESIS
Single reactant moving in one dimension
- ROCKET IMMUNOELECTROPHORESIS
Laurell Technique (1960)
- ROCKET IMMUNOELECTROPHORESIS
Radial immunodiffusion (RID) + electrophoresis
- ROCKET IMMUNOELECTROPHORESIS
End result: precipitin line that is conical in shape, resembling a rocket
- ROCKET IMMUNOELECTROPHORESIS
The height of the rocket, measured from the well to the apex, is directly in proportion to the amount of antigen in the sample.
- ROCKET IMMUNOELECTROPHORESIS
This technique has been used to quantitate immunoglobulins, using a buffer of pH 8.6
- ROCKET IMMUNOELECTROPHORESIS
- ROCKET IMMUNOELECTROPHORESIS
Procedure:
1. Antigen is pushed through antibody containing gel under influence of an applied (?)
2. When they are equivalence, precipitation will occur forming a (?)
electric field (EC)
cone/ rocket shape band
Reactant: Ag (mobile; sample) o Cut thru the gel to make wells where the Ags will be placed o Apply EC, allowing migration of Ag to the anode
Reagent: Ab (immobile; incorporated in the gel)
- ROCKET IMMUNOELECTROPHORESIS
Single reactant moving in 2 dimensions
- CROSSED IMMUNOELECTROPHORESIS
Either Ag or Ab will move (radially)
- CROSSED IMMUNOELECTROPHORESIS
Ressler’s method
- CROSSED IMMUNOELECTROPHORESIS
Either Ag or Ab will move (up or down)
- ROCKET IMMUNOELECTROPHORESIS
- CROSSED IMMUNOELECTROPHORESIS
Procedure:
1. Proteins are separated by (?)
2. Proteins are subjected to a 2nd electrophoresis where they will move through a (?) until rocket is formed (AgAb reach equivalence)
electrophoresis
Ab-containing agarose
Reactant: Ag (immobile)
Reagent: Ab (mobile)
- CROSSED IMMUNOELECTROPHORESIS
Anode: Ab – migrates toward the cathode
- COUNTER IMMUNOELECTROPHORESIS (COUNTERCURRENT ELECTROPHORESIS)
Cathode: Ag – migrates toward the anode
- COUNTER IMMUNOELECTROPHORESIS (COUNTERCURRENT ELECTROPHORESIS)
Zone of equivalence will form a precipitate
- COUNTER IMMUNOELECTROPHORESIS (COUNTERCURRENT ELECTROPHORESIS)
SPECIFICITY is important
- COUNTER IMMUNOELECTROPHORESIS (COUNTERCURRENT ELECTROPHORESIS)
Voltage Facilitated double immunodiffusions
- COUNTER IMMUNOELECTROPHORESIS (COUNTERCURRENT ELECTROPHORESIS)
DOUBLE: Double reactants moving in one dimension
- COUNTER IMMUNOELECTROPHORESIS (COUNTERCURRENT ELECTROPHORESIS)
Use: Identify bacterial, fungi or virus in fluids
- COUNTER IMMUNOELECTROPHORESIS (COUNTERCURRENT ELECTROPHORESIS)
- COUNTER IMMUNOELECTROPHORESIS (COUNTERCURRENT ELECTROPHORESIS)
Procedure: - Ag and Ab are added to separate parallel wells cut out in an (?)
- When an electric field is applied, the Ag will migrate to the (?) and Ab to the (?)
- Zone of equivalence will form a (?)
agar gel
Anode; cathode
precipitate
Cathode (+) to Anode (–)
Albumin (fastest, lightest) → a-1 globulin → a-2 globulin → b- globulin → y- globulin (immunoglobulin)
Grabar and Williams
- CLASSIC IMMUNOELETROPHORESIS
Double reactants moving in 2 dimensions
- CLASSIC IMMUNOELETROPHORESIS
Both Ag or Ab will move (radially)
- CLASSIC IMMUNOELETROPHORESIS
Use: Differentiate the Ig Class, identify abnormal proteins, myeloma proteins (BJP), Monitor purity of pharmaceutical products
- CLASSIC IMMUNOELETROPHORESIS
(+) Result: Any change in the shape of the arcs indicate abnormality
- CLASSIC IMMUNOELETROPHORESIS
- CLASSIC IMMUNOELETROPHORESIS
Procedure: - Ag is introduced in a well and an electric field is applied resulting in (?)
- Ab is introduced in a (?) parallel to the separated protein
- (?) form
separation of proteins
trough
Ag-Ab complex
Process by which particulate antigens (agglutinogen) such as cell aggregate to form larger complexes when a specific antibody (agglutinin) is present
published the first report about the ability of antibody to clump cells, based on observations of agglutination of bacterial cells by serum
1896: Gruber and Durham
This finding gave rise to the use of serology as a tool in the diagnosis of disease, and it also led to the discovery of the ABO blood groups
(Karl Landsteiner)
2 STAGES OF AGGLUTINATION
Antigen-Antibody reaction
SENSITIZATION
No agglutination yet
SENSITIZATION
Cross linking
LATTICE FORMATION
Visible agglutination
LATTICE FORMATION
Represents binding of Ag and Ab
SENSITIZATION
Stabilization of antigen–antibody complexes with the binding together of multiple antigenic determinants.
SENSITIZATION
Forms bridges between Ab and Ag
LATTICE FORMATION
FACTORS THAT AFFECT AGGLUTINATION
Buffer pH Routine:
pH 7 (close to physiological pH) 7.35 – 7.45
Affects the zoning phenomenon
Relative concentration of Ag and Ab
Abs will not detect determinants buried within the particle
Location and concentration of Antigenic determinants of the particle
More number of determinants, the higher the likelihood of cross bridging
Location and concentration of Antigenic determinants of the particle
Electrostatic interactions between particles
Non covalent interaction
in the buffer plays an important role in agglutination
Electrolyte concentration (ionic strength)
: charge bet RBC and the elctrolyte/ECF
Zeta potential
Zeta potential
in RBC
outside the body
↑
↓
reduce electrostatic charges that interfere with lattice formation
Electrolytes
Antibody isotope Best: (dealing w/ particulate Ag)
IgM
: Cold reacting (range 4-22oC)
IgM
: Warm reacting with optimum temperature at 37oC
IgG
Incubation times ranges from
15-60 minutes
No agglutinates
0
Dark, turbid, homogenous
0
Many tiny agglutinates, many free cells, may not be visible without microscope
W+
Dark, turbid
W+
Many small agglutinates, many free cells (25% are agglutinated)
1+
Turbid
1+
Clear
2+
4+
2+
Many medium sized agglutinins, moderate number of free cells
2+
(50% are agglutinated)
2+
Several large agglutinates, few free cells
3+
(75% are agglutinated)
3+
One large solid agglutination, no free
4+
(100% are agglutinated)
4+
Newer tests performed in the serolab
categorized based on the labels being used
EIA -
RIA -
FIA -
- enzymes
- radio isotopes
- flourescence
Opposite charge attracts, negative charge repels
Ionic bonds
most of Ags are proteins (hydrophobic/waterfearing)
Hydrophobic bonds
Result: Precipitate (smaller complexes)
Precipitin
counterpart of precipitin
Precipitinogen
Precipitin + precipitinogen =
precipitates
Same w/ precipitation
Consistency: smoother
Result: Fleecy mass
Flocculation
Monomer: precipitates
IgG
Pentamer: agglutinates
IgM
Precipitation : best
IgG
lowest pH
physiologic conditions
optimum pH
6.5 to 7.5
Blood pH:(ideal)
7.35 – 7.45
IgG: warm-reacting
37oC or 98.6oF
IgM: cold-reacting
40-45oC
Much complicated procedure
PRECIPITATION
Reversible esp. in Ag-Ab binding
PRECIPITATION
Shows optimal reaction
Zone of equivalence
Ag and Ab are equal
Zone of equivalence
Leading to lattice formation (result: precipitates/agglutinates)
Zone of equivalence
may lead to false negative
Prozone phenomenon
Postzone phenomenon
Amount of light scattered =
Soln’s/analyte conc (Ag or Ab)
Same w/ agar used in culture media w/ the exception of additional nutrients or inhibitors to be selective
passive immunodiffusion
Ex. In chromatography – addition of soln to allow movement of reactant
DIFFUSION
slower
↑ Size of the particles - slower
↓ Temperature - slower
↑ Gel viscosity - slower
↓ Amount of Hydration - slower
Direction of movement
Single Dimension
Single diffusion: Ag (moves)
Mancini/Endpoint Method
Antigen is allowed to diffuse to completion
Mancini/Endpoint Method
↑ Ag diffusion = ↑ Ag conc
Mancini/Endpoint Method
occurs between 24 and 72 hours
Mancini/Endpoint Method
Measures diameter of ring before Ag diffuses
Fahey and McKelvey/Kinetic method
Antigen is not allowed to diffuse completely
Fahey and McKelvey/Kinetic method
Mnemonic:
“FAK ME”
FA→Kinetic Mancini→Endpoint
Ouchterlony Double Diffusion Types:
Single diffusion: Ag (moves)
Mancini/Endpoint Method
Antigen is allowed to diffuse to completion
Mancini/Endpoint Method
↑ Ag diffusion = ↑ Ag conc
Mancini/Endpoint Method
occurs between 24 and 72 hours
Mancini/Endpoint Method
Measures diameter of ring before Ag diffuses
Fahey and McKelvey/Kinetic method
Antigen is not allowed to diffuse completely
Fahey and McKelvey/Kinetic method
Performed using petri dish
Double Diffusion
Used to compare 2 Ags that are the same and capable of reacting w/ the
same Ab
Double Diffusion
Fusion of the lines at their junction to form a smooth arc represents serological identity or the presence of a common epitope
Serological Identity: Identical Ag
The arc indicates that the two antigens are identical.
Serological Identity: Identical Ag
Pattern of crossed lines demonstrates two separate reactions
Non-Indentity: Ag are serologically distinct
indicates that the compared antigens share no common epitopes
Non-Indentity: Ag are serologically distinct
Two crossed lines represent two different precipitation reactions
Non-Indentity: Ag are serologically distinct
The antigens share no identical determinants.
Non-Indentity: Ag are serologically distinct
“Spur formation”
Partial Identity: Ag are not identical but do possess common determinants.
Fusion of two lines with a spur indicates partial identity.
Partial Identity: Ag are not identical but do possess common determinants.
The two antigens share a common epitope
Partial Identity: Ag are not identical but do possess common determinants.
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
Partial Identity: Ag are not identical but do possess common determinants.
