الله بسمالرحيم الرحمن

Faculty of Allied Medical Sciences

Clinical Immunology & Serology Practice

(MLIS 201)

Prof. Dr. Ezzat M HassanProf. of ImmunologyMed Res Inst, Alex UnivE-mail: elgreatlyem@hotmail.com

Over View

Clinical Immunology & Serology Practice (Code: MLIS 201)

Teaching Objectives:1- To know the elements of serological reactions2- To define serological reactions3- To describe the basics of primary and secondary serological reactions

A N T I G E N SDefinition: 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 optimum antigenicity Low dose low- zone tolerance High dose 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

A N T I B O D I E S

Definition: Spec. glycoproteins, produced by plasma cells in response to antigenic stimulation of B lymphocytes. Immunoglobulins.

4.Ig has 2 terminal regions: Carboxyterminal - with constant amino acid sequence (constant region). Aminoterminal - with varying antibody specificity (variable region)

Structure of Immunoglobulin1.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.

IgG IgA IgM IgD IgEMolecular

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 5Activate

Complement?

Yes Alt pathway only

Yes No. No.

Classes of Immunoglobulins

Other Name

Serum Ig Secretory Ig Pentameric Ig

___ Reagenic Ig

Other Notes

can cross placenta except-major Ig in 2º Immune response

predominant Ig in secretion-with J chain-exist as monomerin serum and dimer in secretion

Predominat in 1ºimmune response-has J chain

involved in B cell activation

asso. With allergy - binds basophils and mast cells- elevated during parasitic infections

IgG IgA IgM IgD IgE

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) -emits green light***mostly usedb. Phycocyanin -emits red lightc. Texas red -emits red lightd.Tetramethyl rhodamine -emits red orange light

Techniques:1. Direct/Single Layer Immunofluorescent Assay2. 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. ALPb. Horseradish Peroxidasec. Glucose oxidased. B-galactosidase

Techniques:1. Direct2. Indirect3. Sandwich/Double Ab4. Competitive Binding5. Enzyme Inhibition

ELISA: DOUBLE ANTIBODY TECHNIQUE

Secondary tests

PRECIPITATIONAGGLUTINATIONCOMPLEMENT FIXATIONNEUTRALIZATION

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:

Compare between:

Different Classes of Immunoglobulins

40

Assignment

Write notes onA- Primary Ag-Ab reactions

اشرف – – – – مروة على فاطمة الدين عز غادة كمال شروق الحسن ابو شروق

B- Secondary Ag-Ab reactions

رمضان – – – محمد محمود فوزى محمد زغلول محمد ميلود صالح

Thanks