IMMUNOLOGY AND SEROLOGY

LECTURE / WEEK NO.12 / MACARUBBO L.

GENERAL PRINCIPLES OF ANTIGEN ANTIBODY REACTIONS REVIEW OF ANTIGEN

I. Review of Antibody, Antigen  Antigens are substances that when introduced into the
II. Epitope and Paratope body stimulates the production of an antibody.
III. Factors that Affect Antigen Antibody Reactions  Antigens stand for non-self molecules and cells. These
IV. 3 Types of Antigen Antibody Interactions can be foreign proteins, viruses, environmental
V. Agglutination pollutants, bacteria and parasites, foreign transplanted
VI. Precipitation tissues or cancerous cells.
 Antigens contain a region called epitope which is an
REVIEW OF ANTIBODY antigenic determinant. It is the part of an antigen that is
recognized by the immune system, specifically by
 Antibodies, also called Immunoglobulins (Ig), are
antibodies, B cells or T cells. For example, the epitope is
specialized soluble proteins produced by B cells and
the specific piece of the antigen to which an antibody
plasma cells that interacts with antigen.
binds.
o B cells differentiate into plasma cells that
produce antibody molecules closely modelled
after the receptors of the precursor B cell.
o Each antibody is designed to bind to a specific
surface binding site or epitope on the antigen.
 Antibodies are Y shaped Immunoglobulins. Comprised of
2 heavy and 2 light chains.
o There are 5 different types: IgA (dimer), IgD, IgE,
IgG, and IgM (pentamer). Each with a specific
immune response role. Antibodies contain
EPITOPE AND PARATOPE
variable regions which recognize and bind
antigen via lock and key.
 Antibodies contain an antigen-binding site called a
Paratope. The paratope is the part of the antibody which
recognizes and binds to an antigen. It is a small region at
the tip of the antibody's antigen-binding fragment and
contains parts of the antibody's heavy and light chains.

 Pathogens possess highly specific antigenic determinants

(epitopes)
 Antibody paratopes (modified to fit epitopes) are
complementary to specific antigenic determinants.
 Antibody will bind to antigens epitope using its paratope.

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 IMMUNOLOGY AND SEROLOGY
LECTURE / WEEK NO.12 / MACARUBBO L.

FACTORS THAT AFFECT ANTIGEN ANTIBODY REACTIONS

 Affinity is the strength of binding between a single

antigenic determinant and an individual antibody
combining site. Initial force that exists between a single
Fab site of antibody and single epitope of corresponding
antigen. Strength of 1 Antigen and 1 antibody.
 Avidity is the overall strength of binding between
multivalent antigens and antibodies. Sum of all attractive  ANTIGEN – describing the number of epitope
forces between an antigen and an antibody. It measures  Monovolent – 1 epitope
overall stability of antigen antibody complex.  Bivalent – 2 epitope
 Valency is the number of antigenic determinants that an  Multivalent – 3 or more epitopes
individual antibody molecule can bind. Simply, it is the
number of antigen binding sites an antibody has.  Tertiary
 The pentameric IgM has lower affinity than IgG. However, o It is purely in vivo (performed within the body)
IgM has higher avidity than IgG due to its higher valency, and is performed for research purpose.
which enables it to bind effectively to the antigen. o Examples are phagocytosis, opsonization,
chemotaxis, immune adherence and cellular
degradation.

3 TYPES OF ANTIGEN ANTIBODY INTERACTIONS

 Primary
o It is a combination of a single Antigen and
Antibody.
o It is non-covalent and does not show a visible
reaction.
o It is automated and the most sensitive type.
o Examples are IFA (Immunofluorescence), RIA
(Radioimmunoassay), ELISA (Enzyme linked
immunosorbent assay)
 Secondary
o It involves sensitization and lattice formation.
o The antigen involved here is multivalent.
o It shows a visible reaction.
o Examples are precipitation, agglutination, and
complement fixation.

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 IMMUNOLOGY AND SEROLOGY
LECTURE / WEEK NO.12 / MACARUBBO L.

AGGLUTINATION TESTS THAT UTILIZE DIRECT OR PASSIVE AGGLUTINATION

 Agglutination refers to making antigens clump together Direct Agglutination Passive Agglutination
with their specific antibodies. 1. Blood Group 1. Syphilis
2. Mycoplasma 2. Rubella
 It is the most sensitive method and the simplest to pneumoniae (Cold 3. Rheumatoid Arthritis
perform. Agglutinin) Latex Test
 The most sensitive carrier is the RBC. 3. Heterophile Antibodies 4. Hepatitis B and C
(Epstein Barr Virus) 5. Human Chorionic
 IgMs are better antibodies in agglutination.
4. Weil Felix (Rickettsia) Gonadotropin (HcG)
TYPES OF AGGLUTINATION BASED METHODS 5. Phadebact (Gonococci) 6. C-Reactive Protein (CRP)
6. Streptococcus MG 7. Drugs of Abuse
1. Flocculation Method – Uses fine particles of antigen to 7. Viral Hemagglutination
detect antibodies in serum in which they remain as
suspension. PRECIPITATION
2. Latex Agglutination  Precipitation occurs when insoluble antigens interact
3. Direct Bacterial Agglutination – Uses dyed bacterial cell to with their antibody at the right temperature and pH.
detect antibodies in patients.  This results into the creation of a precipitate.
4. Hemagglutination  It is normally done in semi-solid medium such as agar.
5. Agglutination Inhibition  Soluble Antigen + Soluble Antibody = Insoluble
6. Coagglutination – antibodies are reacting to two or more Complexes.
proteins/ antigens.
 Precipitation is affected by temperature and
TWO STAGES OF AGGLUTINATION concentration of antibodies.
 At 40-45C, there is rapid precipitation, and the preferred
1. Sensitization (Ag-Ab Reaction) – No visible reaction yet
pH is 7.0.
but binding already occurs
 Prozone is antibody excess
2. Lattice Formation (Cross Linking) – Visible agglutination is
 Zone of Equivalence is where maximum precipitation
now seen
occurs
 Postzone is antigen excess.

MAJOR CATEGORIES OF AGGLUTINATION

Category of Agglutination Unknown (Serum) Known (Reagent)

Direct Agglutination Antigen (RBCs) Antibody
Antibody (serum) Antigen
Indirect/Passive Antibody Antigen with carrier
Reverse Passive Antigen Antibody with carrier

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 IMMUNOLOGY AND SEROLOGY
LECTURE / WEEK NO.12 / MACARUBBO L.

APPLICATIONS OF PRECIPITATION TECHNIQUE

Technique Sensitivity (ug/mL) Application

Nephelometry 1-10 Immunoglobulin, Complement,
CRP
Radial Immunodiffusion 10-50 Immunoglobulin, Complement
Ouchterlony 20-200 Complex Antigen (Candida,
Blastomyces, and Aspergillus)
Rocket Electrophoresis 2 Immunoglobulin, Complement,
AFP
Immunoelectrophoresis 20-200 Serum Proteins
Immunofixation Variable HIV, Lyme Disease, Syphilis,
Serum Proteins
*When the number is lower meaning it is sensitive.

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