Immunology and Serology

IMMUNOASSAYS
Catalan |Reyes |Servillon
Dr. Alfredo A. Hinay Jr., RMT, MSPH
 Protein Analysis
(Antibody evaluation, C omplement)
IMMUNOASSAYS
(PURPOSE) Cellular Analysis and Histocompatibility
(T-cell evaluation, Lymphokines, HLA typing)

Autoantibody Evaluation
(Anti-nuclear antibody tests)

Infectious Disease Serology

(Infectious disease Identification)
 Ch8: Precipitation
soluble antigen with soluble antibody to
produce insoluble complexes that are visible

Ch9:Agglutination
particulate antigens to form larger complexes when a
IMMUNOASSAYS specific antibody is present

(TECHNIQUES)
Ch10: Labeled Immunoassays
PRINCIPLES OF ANTIGEN-ANTIBODY
INTERACTIONS

IMMUNE COMPLEXES
• noncovalent combination of antigen with its respective specific antibody
• Agglutination visible immune complexes
• small (soluble) or large (precipitating) complexes
PRINCIPLES OF ANTIGEN-ANTIBODY
INTERACTIONS

SPECIFICITY CROSS REACTIVITY

•binding sites of Abs directed to one •occurs between bacteria that possess the
Ag are not complementary to another same cell wall polysaccharides as mammalian
dissimilar Ag erythrocytes (heterophile antibodies)
PRINCIPLES OF ANTIGEN-ANTIBODY
INTERACTIONS

AFFINITY
•initial force of attraction between a single
Fab site on an antibody molecule and a single
epitope
PRINCIPLES OF ANTIGEN-ANTIBODY
INTERACTIONS: Affinity

MOLECULAR BASIS
• Hydrophobic Bonds
• Hydrogen Bonds
• Van der Waals Forces
• Electrolastic Forces
PRINCIPLES OF ANTIGEN-ANTIBODY
INTERACTIONS: Affinity

Hydrophobic Bonds
• major bonds formed between
antigens and antibodies
• side chains interact and exclude water
molecules from the area of the
interaction.
• results in a gain in energy and forms an
energetically stable complex
PRINCIPLES OF ANTIGEN-ANTIBODY
INTERACTIONS: Affinity

Hydrogen Bonds
• Hydrogen bonding results from the
formation of hydrogen bridges
between appropriate atoms.
• Major hydrogen bonds
PRINCIPLES OF ANTIGEN-ANTIBODY
INTERACTIONS: Affinity

Van der Waals Forces

• Nonspecific attractive
forces by the interaction
between electron clouds
and hydrophobic bond
PRINCIPLES OF ANTIGEN-ANTIBODY
INTERACTIONS

AVIDITY
•functional combining strength of an
antibody with its antigen
• PROZONE excess antibody = antigen combines with only one or two antibody molecules
• ZONE OF EQUIVALENCE optimum for antigen-antibody complex formation
• POST ZONE excess antigen = every available antibody site is bound to a single antigen, and no cross-links are formed
PRECIPITATIO N AND
AGGLUTINATION

• Results of Antigen-Antibody Interaction

• Similarity: antibody crosslink antigen and
form precipitate
• Difference: nature of antigen
• Precipitation = soluble antigen
• Agglutination = insoluble antigen e.g.
RBC , bacteria, antigen fixed on beads
(HCG , bacterial toxins, etc.)
PRECIPITATION
• involves combination of soluble antigen with soluble antibody to
produce visible insoluble complexes.
• simplest methods of detecting antigen-antibody reactions

• DETECTION:
• Passive Immunodiffusion
• Electrophoresis
• Light scattering
PRECIPITATION
PASSIVE IM M UNO DIFFUSIO N

PASSIVE IMMUNODIFFUSION
• Passive diffusion method in which a concentration gradient is
established for an antigen and/or antibody
• diffusion of reactants to form Ag Ab reactions without electric
current to speed up reaction.

Rate of Diffusion 1. Size of particles 2. Temperature 3. Gel viscosity

4. Amount of hydration 5. Interactions between matrix and reactants.
 PRECIPITATION
PASSIVE IM M UNO DIFFUSIO N

PASSIVE
IMMUNODIFFUSION
• Single Immunodiffusion
• Radial Immunodiffusion
• Double Immunodiffusion
 PRECIPITATION
PASSIVE IM M UNO DIFFUSIO N

PASSIVE
IMMUNODIFFUSION
• Single Immunodiffusion
• Radial Immunodiffusion
• Double Immunodiffusion
 PRECIPITATION
PASSIVE IM M UNO DIFFUSIO N

PASSIVE
IMMUNODIFFUSION
• Single Immunodiffusion
• Radial Immunodiffusion
• Double Immunodiffusion
 PRECIPITATION
PASSIVE IM M UNO DIFFUSIO N

PASSIVE
IMMUNODIFFUSION
• Single Immunodiffusion
• Radial Immunodiffusion
• Double Immunodiffusion

Used for:
Fungal antigens: Aspergillus, Blastomyces, Coccidioides, and
Candida Extractable nuclear antigens
PRECIPITATION
ELECTROPHORESIS

• Electrophoresis separates molecules

according to differences in their electrical charge.

TYPES
• Immunoelectrophoresis
• Rocket Immunoelectrophoresis
• Countercurrent Immunoelectrophoresis
PRECIPITATION
IMMUNOELECTROPHORESIS

redAlert! Typically, the source

of the antigens is serum

These lines or arcs can be compared in shape,

intensity, and location to that of a normal
serum control to detect abnormalities.
 PRECIPITATION
IMMUNOELECTROPHORESIS
Uses
• Immunodeficiencies can be detected by this procedure,
if no precipitin band is formed for a particular Ig.
• Deficiencies in complement can also be detected.
• Overproduction of serum proteins.
• Identification of monoclonal protein Free kappa and lambda
light chains May be used to identify urine proteins.
• Testing normal & abnormal proteins in serum/urine.
• Purity of Ag.
PRECIPITATION

COUNTERCURRENT
ELECTROPHORESIS
 PRECIPITATION
ELECTROPHORESIS

ROCKET IMMUNO-
ELECTROPHORESIS
(LAURELL TECHNIQUE)
 PRECIPITATION
ELECTROPHORESIS

ROCKET IMMUNO-
ELECTROPHORESIS
(LAURELL TECHNIQUE)

Screening tool for differentiation of more than 30 serum proteins.

• Major classes of immunoglobulins
Standard set-up

Used for the detection of:

• Myelomas
• Malignant lymphomas
• Other lymphoproliferative disorder.
PRECIPITATION
LIG HT SC ATTERING

TURBIDIMETRY
• measure of the turbidity or cloudiness of a solution
• measures the reduction in light intensity due to
reflection, absorption, or scatter

NEPHELOMETRY
• measure light scatter at angles ranging from 10
degrees to about 90 degrees

• more sensitive than turbidimetry

• End-point
• Kinetic
Factors affecting Agglutination:
1. Ionic strength 2. pH
3. Temperature 4. Enhancement medium
4. Nature of antibody Goal: form a VISIBLE stable lattice formation
 TYPES OF AGGLUTINATION

AGGLUTINATION 1. Direct Agglutination

2. Indirect/ Passive Agglutination
3. Reverse Passive Agglutination
4. Agglutination Inhibition
5. Co-agglutination
DIRECT AGGLUTINATION
• combination of an insoluble particulate antigen
with its soluble antibody
• In this case, the A g-A b reaction
forms an agglutination, which is
directly visible.
• The particle antigen may be a bacterium, Parasite,
or RBC
e.g. Serotyping of E. coli, Salmonella using a
specific antiserum, Serodiagnosis of
Toxoplasmosis, Determination of blood groups
INDIRECT AGGLUTINATION (PASSIVE)
• An agglutination reaction that employs particles
that are coated with antigens not normally
found in the cell surfaces
• Converting a precipitating
test to an agglutinating
test
• Chemically link soluble antigen to inert particles
• Particle carriers include:
• Red blood cells
• Polystyrene latex
• Bentonite
• charcoal

Detection of :
• -Rheumatoid factor
• -Antinuclear antibody in LE
• -A b to g roup A streptococcus ag
• -A b to Trichinella spiralis
REVERSE PASSIVE AGGLUTINATION
• antigen binds to soluble antibody coated on
carrier particles and result in agglutination
detects antigens
• Example detecting cholera toxin
 HEMAGGLUTINATION INHIBITION

AGGLUTINATION INHIBITION
• Agglutination inhibition reactions are based on
competition between particulate and soluble
antigens for limited antibody-combining sites
• The lack of agglutination is an indicator of a
positive reaction
• The technique is called hemagglutination
inhibition if the particle in the reaction is a RBC

Example: detecting antibodies to influenza

COAGGLUTINATION
• The name given to systems using bacteria as
the inert particles to which antibody is
attached
• Staphylococcus aureus is most frequently
used, because it has a protein on its outer
surface, called protein A which naturally
adsorbs the FC portion of antibody molecules
• The Fab region is free to interact with antigens
present in the applied specimens
AGGLUTINATION
ANTIBO DY Q UANTITATIO N
 Immunology and Serology

IMMUNOASSAYS
Catalan |Reyes |Servillon
Dr. Alfredo A. Hinay Jr., RMT, MSPH