SEROLOGY

SEROLOGY
Subdiscipline of immunology
Concerns with the development and control of
the most simple, most sensitive, and specific
test
The demonstration of Ag and Ab reactions in
vitro

Immunologic specificity is not absolute

 INTERMOLECULAR FORCES
Multiple, reversible
Non-covalent bonds

1. Hydrogen bond
2. Electrostatic/Ionic bonds
3. Van der Waals
4. Hydrophobic bonds
 LAW OF MASS ACTION

The free reactants are in equilibrium with

bound reactants

association dissociation
Ag + Ab Ag-Ab complex
 Strength of attractive forces

Affinity : binding of a single antigenic

determinant on 1 binding site of the Ab
: reaction is not visible

Avidity : multivalent Ag reacting to Ab

: repeating determinants
 Interaction happens in:
PRIMARY: formation of immune complex
: uses labelled immunoassays
SECONDARY: immediate effect of formation of
immune complexes
: detection of Ag-ab complexes
TERTIARY: biologic consequence of the Ag binding
to Ab

Visible reactions are seen if the conc. of Ag is at

optimal proportion to conc. of Ab
 FACTORS INFLUENCING BINDING OF
Ag and Ab
Ag-Ab ratio
Effective binding strength: distance between the
Ag and Ab
Physical conditions
1. pH: 5.5-8.5; optimal is neutral pH
2. Temperature of medium
3. Nature of Ag
4. Incubation time: 30 mins to 1 hour
5. Ionic strength
6. Steric hindrance: mutual blocking
 AGGLUTINATION
Secondary reaction
Ag is insoluble/particulate
Formation of agglutinates/aggregates
Most sensitive
2 mechanisms:
1. Sensitization
2. Lattice formation
 TYPES OF AGGLUTINATION

Direct
Indirect/Passive
Reverse passive
Agglutination -inhibition
 COAGGLUTINATION
Term given to systems using bacteria as the
inert particles to which antibody is attached.
Staphylococcus aureus is most frequently used
With surface protein called protein A which
naturally adsorbs the Fc portion of Ab
The active sites face outward and are capable
of reacting with specific antigen.
Exhibits greater stability than latex particles
and are more refractory to changes in ionic
strength.
 Antiglobulin-mediated
agglutination

Direct Antiglobulin Test

Indirect Antiglobulin Test

 PRECIPITATION
Involves soluble Ag
2 phases: INITIAL
FORMATION of LATTICE WORK
Phase change reaction: as more complexes are
incorporated, the size of the lattice work
becomes bigger, therefore it becomes
insoluble and precipitated
Precipitin curve: shows change in ratio of the
amount of Ag to the amount of Ab
 PRECIPITATION
Procedures:

BETA : Ramon procedure

: Ab conc (varying), Ag conc. (fixed)
ALPHA: Dean Webb procedure
: Ag conc (varying), Ab (fixed)
 TYPES OF PRECIPITATION REACTIONS

Qualitative

Quantitative
 FORMS OF PRECIPITATION REACTION

Liquid medium

Semi-solid medium/ Gel medium

 LIQUID MEDIUM
Test tube Method: turbidity

Slide Method: use microscope

Capillary method: dense granulation in the

interphase

Ring test: precipitin ring formation

 GEL MEDIUM
Uses agarose gel

Passive diffusion techniques

A. Single diffusion
B. Double diffusion
 OUDIN
Single radial immunodiffusion technique

Mancini : end point method (24-72 h)

: the square of the diameter is
directly proportional to the conc. of Ag
Fahey and McKelvey: kinetic method (18 h)
: the diameter is proportional to the
log of the concentration of Ag
 OUCHTERLONY
IMMUNODIFFUSION TECHNIQUE

Line of Identity

Line of Partial Identity

Line of Non-identity
 ELECTROPHORETIC TECHNIQUES

Diffusion can be combined with

electrophoresis to speed up or sharpen the
results
 ROCKET
IMMUNOELECTROPHORESIS
One-dimension electroimmunodiffusion
Was developed by Laurell
Ab is distributed in the gel, and Ag is placed in
wells cut in the gel
The end result is a precipitin line that is
conical in shape, resembling a rocket
The height of the rocket, measured from the
well to the apex, is directly proportional to the
amount of Ag
 IMMUNOELECTROPHORESIS
Double-diffusion technique that incorporates
current to enhance results.
Serum: source of Ag
Trough: place the antiserum
Double diffusion occurs at right angles
Precipitin line/arc develops
 IMMUNOFIXATION
ELECTROPHORESIS
First described by Alper and Johnson
Similar to immunoelectrophoresis except that
antiserum is applied directly on the gels
surface rather than placed in a trough.
Agarose or cellulose acetate can be used
Eg: Western, Southern, and Northern blotting
COUNTERIMMUNOELECTROPHORESIS

Double immunodiffusion assay with voltage

applied w/ pH = 8.6 to the medium
Apply Ag to one side , Ab to the other
End result: precipitin line formation
 SOURCES OF ERROR IN
ELECTROPHORESIS
Applying the current in the wrong direction. This may
cause :
-samples run off the gel
-samples not separated
Incorrect pH of the buffer and incorrect
electrophoresis time: hinder proper separation
Too concentrated Ag or ab: no visible reaction occurs
Not too strong current is applied: separation may be
incomplete
Too strong current: heat is generated, causes
denaturation of protein
 IMMUNOCHROMATOGRAPHY

Uses nitrocellulose membrane : supported by

plastic
With sample well and reaction well
Reaction well: for test and control
 FLOCCULATION

Midway reaction between agglutination and

precipitation
Involves noncellular particulate Ags
Eg: RPR, VDRL slide test
 COMPLEMENT FIXATION
Ag reacts w/ Ab , followed by uptake of C
2 systems
Bacteriolytic system
: interaction of Ag, Ab, C
: source of C- guinea pigs serum

Hemolytic system
: sensitized RBCs (RBCs coated with
hemolysin
 NEUTRALIZATION
Ag reacts with Ab
Loss of reactivity of the Ag
Ag is nullified

Toxin: neutralization of toxic effects

Viral: neutralization of infectivity of the virus
 NEUTRALIZATION
Eg: ASO Tube method/ASO titer test

-prepare 12 tubes
-dilute the px serum
-Tube 11: RED CELL CONTROL (clear)
-Tube 12: STREP O CONTROL (w/hemolysis)
 NEUTRALIZATION

In vivo neutralization
Control: toxin
Test: toxin + serum
3 weeks observation
Control: sick; Test: healthy
 NEUTRALIZATION

In vivo neutralization
Control: Virus
Test: Anti-viral (immunize) + virus
Control: infection; Test: healthy
 NEUTRALIZATION
In vitro
a. Chick embryo- Pock reduction Test
Control: Viral suspension
Test: Viral suspension + px serum (Ab)

- count the no. of pocks

Control: w/ pocks
Test: w/o pocks
 NEUTRALIZATION
In vitro
b. Tissue culture cells: monolayer of cells
-infect with virus
-observe CPE
-infected cell: formation of
plaques

-plaque reduction test