Basic Immunological

Procedures
PRECIPITATION REACTIONS
AGGLUTINATION RECTIONS
 Introduction
•combination of antigen with specific antibody
plays an important role in the laboratory in
diagnosing many different diseases.
• __IMMUNOASSAYS__ have been developed to
detect either antigen or antibody, and they vary
from easily
•These assays are based on the principles of
precipitation or agglutination
 Antigen-antibody binding

Affinity
• Initial force of attraction
that exist between single
Fab site and a single epitope
on the corresponding
antigen
Avidity
• Sum of all attractive forces
between an Ag and and Ab
• Dictates the over all stability
of the Ag-Ab complex

1 Fab + 1 Epitope Multivalent Ab=Multivalent

(Ab) (Ag) Ag
FACTORS INFLUENCING AFFINITY
• Types
• Ionic Bond
• Hydrogen bond
• Hydrophobic bond
• Vander Waals Forces
Important Terms to remember
• Precipitin: Antibody
• Precipitinogen: Soluble Antigens
• Precipitates: Insoluble complexes formed by the union
of the two
• Flocculation: Natural clumping
Major Immunoglobulins Involved
• IgG: much better precipitating Ab than IgM
• IgM: much better agglutinating Ab than IgG
• Precipitation: IgG>IgM>IgA
• IgE: Nonprecipitating
Factors Affecting Precipitation:
• pH. The pH of the medium used for testing should be
near physiologic conditions, or an optimum pH of 6.5 to
7.5.
• Temperature and Length of Incubation.
• Ideal: 37° C (98.6° F)
• 40-45ºC
• incubation time range from 15 to 60 minutes.
 PRECIPITATION
• involves combining soluble antigen with soluble
antibody to produce insoluble complexes that are
visible
• first noted in 1897 by Kraus
• Law of Mass Action: All antigen–antibody binding is
reversible and free reactants are in equilibrium with
bound reactants
 So that maximum precipitation
PRECIPITATION CURVE occurs, Ag and Ab concentation
must have an optimum ratio
Ag and Ab are equal, therefore
max precipitation occur
Prozone: More Ab (patient)
excess than Ag
remedy: Serum dilution
Post zone: Ag excess, less Ab
(Patient) may lead to false
negative
Remedy: Repeat the test after a
week to give time for antibody
production
PRECIPITATION REACTIONS
I. PRECIPITATION IN A FLUID MEDIUM
II. PRECIPITATION BY A PASSIVE IMMUNODIFFUSION
III. PRECIPITATION BY ELECTROPHOTERIC TECHNIQUES
I. PRECIPITATION IN A FLUID MEDIUM
TURBIDIMETRY
• a measure of the turbidity
or cloudiness of a solution
• It thus measures the
reduction in light intensity
due to reflection,
absorption, or scatter.
NEPHELOMETRY
• measures the light that is
scattered at a particular
angle from the incident
beam as it passes through a
suspension
• The amount of light
scattered is an index of the
solution’s concentration
 II. PRECIPITATION BY A PASSIVE
IMMUNODIFFUSION
• Ag-Ab complex may be observed in a support media
(Agarose gel)
• Passive: No electric current is used t o speed up reaction of
the Ag and Ab combination, but through DIFFUSION.
• Factors affecting Rate of Diffusion:
size of the particles the gel viscosity
the temperature amount of hydration
Radial Immunodiffusion
•Ab is uniformly distributed in support gel
and Ag is applied to a well cut into gel

1. Single Diffusion-Single Dimension (Oudin)

2. Single Diffusion-Double Dimension
(Mancini, Fahey and McKelvey)
Precipitation in gel Medium
• Single Diffusion
• Only one reactant is moving
• Double Diffusion
• Both Ag and Ab are moving through the
medium
• Single dimension
• Reaction in tubes-Ag or Ab migrate up
and down
• Double Dimension
• Petri dish-Ag or Ab diffuse radially
1. Single Diffusion- Single Dimension (Oudin)

Procedure:
1. Ab mixed in agarose
2. Antigen dilution is overlaid ( Ag must
always be greater)
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 stabilized band is
formed
 2. Single Diffusion- Double Dimensions

Procedure: Types:
1. Ab is mixed with liquid agar A. Mancini/ Endpoint
and poured into the petri dish Diameter= Ag conc
2. Circular wells cute in gel
3. Ag is loaded into the wells B. Fahey and McKelvey/
4. Ring ppt expands from the Kinetic
well as Ag diffuses toward its D=Log Ag conc
equilibrium concentration
5. Diameter of the disc is
measured
 3. Ouchterlony Double Diffusion
• Both Ag and Ab diffuse
independently through a
semisolid medium in 2
dimension
Procedure:
1. Pattern of wells in cut in an
agarose gel in a petri dish
2. Reactants are loaded
3. Incubated until lines are
precipitated
• Possible Patterns:
A. Serological Identity: identical
Ag
B. Non identity: Ag are
serologically distinct
C. Partial Identity: Ag are not
identical but do possess
common determinants
*spur formation
III. PRECIPITATION BY ELECTROPHOTERIC
TECHNIQUES

•Electrophoresis: Technique in which molecules

with a net charge are separated when an electric
field is applied
•Negative charged particles migrate to the ANODE
(+ Pole)
•Positive charged particles migrate to the CATHODE
(- Pole)
Factors that Influence Rate of Protein
Migration
1. Size and shape of protein
2. Amount of solvation
3. Viscosity of the buffer
4. pH of Buffer: >8
5. Temperature: Room tempt
6. Endo-osmosis
-flow of ions goes toward the cathode and can impede
movement of proteins toward the anode
1. Rocket Immunoelectrophoresis
(Laurell Technique)
• Single reactant moving in one dimension
Procedure:
1. Ag is pushed through Ab containing gel under influence of an
applied electric field
2. When they are at equivalence, precipitation will occur forming a
cone/ rocket shape band
ROCKET IMMUNOELECTROPHORESIS
2. Crossed Immunoelectrophoresis
(Ressler’s Method)
• Single reactant moving in 2 dimensions
Procedure:
1. Protein are separated by electrophoresis
2. Protein are subjected to a 2nd electrophoresis where they will move
through a Ab-containing agarose gel until a rocket is formed (Ag-Ab
reach equivalence)
3. Counter Immunoelectropheresis
(Countercurrent electrophoresis)
• Procedure:
Or Voltage Facilitated double
1. Ag and Ab are added to immunodiffusions
separate parallel wells cut out
in an agar gel
2. when an electric field is Double reactants moving in one
Double dimension
applied the Ag will migrate to
the Anode and Ab to the
cathode
Use: Identify Bacterial, fungi, or virus
3. Zone of equivalence will form Use in fluids
a precipitate
COUNTERCURRENT IMMUNOELCECTROPHORESIS
4. Classic Immunoelectrophoresis
(Grabar and Williams)
• Procedure:
1. Ag is introduced in a well and an
electric field is applied resulting
in separation of proteins
2. Ab is introduced in an trough
parallel to the separated protein
3. Ag-Ab complex form
• Double reactants moving in 2
dimensions
• Use: Differentiate the Ig Class,
Identify Abnormal proteins,
myeloma proteins, Monitor
purity of pharmaceutical
products
Classic Immunoelectrophoresis
(Grabar and Williams)
AGGLUTINATION REACTIONS
Agglutination

• Process by which particulate antigens such as cell aggregate

to form larger complexes when a specific antibody is present
•Agglutination occurs in 2 stages:
1. Sensitization: Antigen-Antibody reaction

2. Lattice formation: Cross linking

Gruber and Durham

•ability of antibody to clump cells,

based on observations of
agglutination of bacterial cells by
serum
 Factors that Affect Agglutination
1. Buffer pH
2. Relative conc. Of Ag and Ab
3. Location and concentration
of Antigenic determinants of
the particle
4. Electrostatic interactions
between particles
5. Electrolyte concentration
Routine: pH 7 (close to physiological pH)
Affects the zoning phenomenon
• Abs will not detect determinants buried within the
particle
• More number of determinants the higher the likelihood
of cross bridging
Non covalent interaction
• Electrolyte conc (Ionic strength) in the buffer plays an
important role in aggln
• Electrolytes reduce electrostatic charges that interfere
with lattice formation
 Factors that Affect Agglutination
6. Antibody isotype Best: IgM
7. Temperature IgM: Cold reacting (range: 4-
22oC)
IgG: Warm reacting with
optimum tempt at 37oC
8. Centrifugation

9. Time of incubation of coated Incubation times ranges from 15-

particles with patient’s serum 60 minutes
 Agglutination Reading:
Grade Description
Cells Supernatant
No agglutinates Dark, Turbid, Homogenous
0
Many tiny agglutinates, Many free cells, May not Dark, Turbid
W+ be visible without microscope

Many small agglutinates, Many free cells (25% are Turbid

1+ agglutinated)

Many medium sized agglutinates, Moderate Clear

2+ number of free cells (50% are agglutinated)

Several large agglutinates, Few free cells (75% are Clear

3+ agglutinated)

One large solid agglutinate, No free (100% are Clear

4+ agglutinated)
 Major Categories of Agglutination
Reactions
1. Direct/ Active Agglutination 5. Quantitative Assays
• Direct Immune • SPIA/ Sol Particle Immunoassay
• Direct Non immune (Viral HA, • DIA/ Disperse Dye
Viral HAI) Immunoassay
2. Indirect/Passive • IMPACT/ Immunoassay by
Particle Counting
3. Reverse Passive
4. Antiglobulin Test
• Direct Coombs
• Indirect Coombs
1. Direct Agglutination (Active)
Direct Immune
Reaction is due to an Ag-AB
reaction where in the Ag is
inherent native to the cell
Eg: ABO blood grouping
(hemagglutination), Widal test
Direct Non Immune
Aggregation of indicator red
blood cells are NOT due to
Ag-Ab reactions
Eg: Viral Hemagglutination test
2. Viral Hemagglutination

• Virus can stick to agglutinate RBCs in a process

• Rubella virus, dengue virus, influenza virus, mumps virus
• Viral receptor: Peplomers
3. Viral Hemagglutination Inhibition (HAI)

• Competitive binding Assay

• Procedure:
1. Patient serum incubated with viral particles (Commercially
Available)
2. Viral Particles will bind to the Fab region of Anti-viral Abs
3. Indicator RBCs added to reaction mixture
4. Positive result: Inhibition or Absence of Agglutination
(Presence of Ab)
Negative result: Agglutination
4. Indirect/ Passive Agglutination

• Reactions where Ag has been affixed or absorbed to a

carrier/inert particle
E.g ASTO
• Different passive carriers:
• Human RBCs
• Clay (Bentonite)
• Latex particles
• Colloidal gold
• Charcoal particles
5. Reverse Passive Agglutination

• Antibody is bound to the carrier

• Fluid is detected for the presence of Ag
• Eg: CRP, Reverse Agglutination test for Candida and
Neisseria
6. Latex Particle Agglutination Inhibition

• Patient sample (Ag) incubated with Ab in test kit

• Complex will form if the patient sample contains
the corresponding Ag and the Fab sites are no
longer available for the Ag-coated latex particles
• Eg: HCG / pregnancy test, Screening test for Drugs
Coaglutination
• System using bacteria as the inert particles to which
antibody is attached
• Staphylococcus aureus
• The active sites face outward and are capable of
reacting with specific antigen
7. Antiglobulin test
Direct Anti-globulin test
• Detects IgG Ab bound to Ag
on Red cells (in-vivo)
• Purpose:
• HDN investigation
• HTR investigation
• AIHA
• Drug induced HA
• Eg: Direct Coomb’s test
Indirect Anti-globulin test
• Detects presence of Abs in
the serum that is still to be
attached to an analyte
• Crossmatching
• Ab determination
• Ab identification
• RBC Ag phenotyping
• Indirect Coomb’s test
 Quantitative Agglutination Reaction

IMMUNOASSAY INERT PARTICLE

SPIA/ Sol Particle Gold-inorganic colloidal

Immunoassay particle

DIA/ Disperse Dye Dye-organic colloidal

Immunoassay particle

IMPACT/ Immunoassay Latex particle Best

by Particle Counting
End of the chapter ☺