Download antigens

‫بسم هللا الرحمن الرحيم‬
Faculty of Allied Medical
Sciences
Clinical Immunology & Serology
Practice
(MLIS 201)
Over View
Prof. Dr. Ezzat M Hassan
Prof. of Immunology
Med Res Inst, Alex Univ
E-mail: [email protected]
Clinical Immunology & Serology Practice
(Code: MLIS 201)
Teaching Objectives:
1- To know the elements of serological reactions
2- To define serological reactions
3- To describe the basics of primary and secondary
serological reactions
ANTIGENS
Definition: Any molecular structure that when
introduced is capable of Antibody production.
Parts of Antigen
Carrier portion - responsible for the molecular weight
of antigen
Epitope or Determinant - determines specificity of
antigen
Two Properties of Antigens
1. IMMUNOGENICITY
- inherent ability of a substance to induce the
specific immune response resulting in the
formation of immune lymphocytes or antibodies
Factors influencing Immunogenicity (Summary)
1-Foreigness :
Foreign substances are immunogenic
2- Molecular size:
High molecular weight increase immunogenicity
3- Chemical structure complexity:
High complexity increase immunogenicity
4- Route of administration:
Parenteral routes are more immunogenic to oral route
SC>IP>IV>Intragastric
Factors influencing Immunogenicity
(Summary) (cont.)
5- Degradability of the immunogen
6-Genotype of the recipient
7- immunogen dose:
Appropriate dose
Low dose
High dose
optimum antigenicity
low- zone tolerance
high-zone tolerance
8- Adjuvant:
Substance when injected with an immunogen
enhance immunogenicity
2. ANTIGENICITY/SPECIFICITY
- the ability to react specifically with the antibody
or cell that caused it to be produced
ANTIBODIES
Definition: Spec. glycoproteins, produced by plasma cells in
response to antigenic stimulation of B lymphocytes.
Immunoglobulins.
Structure of Immunoglobulin
1.Four (4) polypeptide chains:
2 identical LIGHT chains and 2
identical HEAVY chains.
2.Both light and heavy chains are
held together by DISULFIDE
BONDS.
3.Heavy chains are interconnected
by DISULFIDE bonds in
the HINGE region.
4.Ig has 2 terminal regions:
Carboxyterminal - with constant
amino acid sequence
(constant region).
Aminoterminal - with varying
antibody specificity (variable
region)
Classes of Immunoglobulins
IgG
IgA
IgM
IgD
IgE
Molecular
Weight
Daltons
150 T
160-400 T
900 T
180 T
190 T
Halflife
21-23
days
5-6 days
5 days
2-3 days
2-3 days
Subclasses
4
2
2
-
-
Domains
4
4
5
4
5
Activate
Complement?
Yes
Alt pathway only
Yes
No.
No.
IgG
Other
Name
Serum Ig
Other can cross
Notes placenta
except
-major Ig
in 2º
Immune
response
IgA
Secretory Ig
predominant
Ig in
secretion
-with J chain
-exist as
monomerin
serum and
dimer in
secretion
IgM
Pentameric
Ig
IgD
___
IgE
Reagenic
Ig
Predominat involved
asso.
in 1ºimmune in B cell
With
response
activation allergy
- binds
-has J chain
basophils
and mast
cells
elevated
during
parasitic
infections
Complement
System
COMPLEMENT SYSTEM
- A set of serum proteins that play a role in cytolytic
destruction of cellular antigen by specific antibody.
- reaction is nonspecific to the target cell
- destroyed at 56ºC for 30 minutes
- the in vitro study of antigen-antibody reaction
- laboratory study of the activities of the components of
blood serum that contributes to immunity
Immunologic Reactions:
Primary - combination of Ag-Ab; non-visible
reaction
Secondary - demonstrable Ag-Ab rxn (e.g.
precipitation, agglutination)
PRIMARY IMMUNOLOGIC TESTS.
IMMUNOASSAYS
Ligand - any substance that will complex to another substance;
(the substance to be measured.
A. FLUORESCENCE IMMUNOASSAY
Fluorescent Probes used:
a. FITC (Fluorescein isothiocyanate)
b. Phycocyanin
c. Texas red
d.Tetramethyl rhodamine
-emits green light***mostly used
-emits red light
-emits red light
-emits red orange light
Techniques:
1. Direct/Single Layer Immunofluorescent Assay
2. Indirect/Double Layer Immunofluorescent Assay
positive FA test for rabies
(Image: Centers for Disease Control)
negative FA test for rabies
(Image: Centers for Disease Control)
C. ENZYME IMMUNOASSAY
colorimetric reaction
Enzymes Used:
a.
b.
c.
d.
ALP
Horseradish Peroxidase
Glucose oxidase
B-galactosidase
Techniques:
1. Direct
2. Indirect
3. Sandwich/Double Ab
4. Competitive Binding
5. Enzyme Inhibition
ELISA: DOUBLE ANTIBODY TECHNIQUE
Secondary tests
PRECIPITATION
AGGLUTINATION
COMPLEMENT
FIXATION
NEUTRALIZATION
I.PRECIPITATION RXNS
- Antigens involved are soluble antigens.
Types of Precipitation reaction:
1. Single diffusion, Single Dimension
Px serum w/ soluble Ag
(+) rxn - formation of pptn line
Gel/Agar impregnated w/ known Ab
2. (RADIAL IMMUNODIFFUSION)
- uses a plate containing agar with known antibody
- Px serum is placed on the wells
- Diameter of the pptn line is directly proportional to the
concentration of the target antigen
5. Immunoelectrophoresis (IEP)
- useful procedure for the ID of monoclonal proteins
(Bence Jones Protein)
- utilizes both double diffusion and electrophoresis
a. Ags migrate under an electric current (-) Ag
(+)
b. Rgt. Ab Added. Diffusion through gel (-)
(+)
Antibody
c. (+) rxn - Pptn arcs formed
(-)
(+)
STEP 1.
STEP 2.
II. AGGLUTINATION
REACTIONS
-
Ags involved are particulate antigens.
Types of Agglutination Rxn
1.Direct Agglutination
(e.g. Blood Typing)
2. Antiglobulin Technique
(CoombsTest)/Indirect Agglutination
- anti-human IgG is added to bridge the gap between the cells
- to demonstrate incomplete antibodies
3.Passive Agglutination
- A soluble Ag is artificially attached to a particulate carrier
(e.g. cells, latex, bentonite, celloidin, or charcoal
6. Hemagglutination
- Agglutination of rbc due to antibody, viruses, bacteria, or other
biologic substance.
- It is not the antigen of RBC but the artificially attached Ag
(after undergoing tx) that are made to react with the Ab
- ex. TPHA
Study Questions:
40
 Compare between:
Different Classes of Immunoglobulins
‫‪Assignment‬‬
‫‪Write notes on‬‬
‫‪A- Primary Ag-Ab reactions‬‬
‫شروق ابو الحسن – شروق كمال – غادة عز الدين – فاطمة على – مروة اشرف‬
‫‪B- Secondary Ag-Ab reactions‬‬
‫صالح ميلود – محمد زغلول – محمد فوزى – محمود محمد رمضان‬
Thanks