immunological reactions and immune response ppt

IMMUNOLOGICAL REACTIONS AND IMMUNE RESPONSE

Dr Vijay Marakala MDvkunder637@gmail.com

Edward Jenner(1749–1822) Often termed the founder of immunology

Louis Pasteur(1822–1895) French. Father of immunology

Immunological reactions [ ANTIGEN-ANTIBODY Reactions]

• Any substance which introduced parenterally into the body, stimulates the production of an antibody with which it reacts specifically and in an observable manner

ANTIGEN

1. Induction of an immune response

2. Specific reaction with antibodies

Antigen

1. Induction of an immune response

2. Specific reaction with antibodies

Antigen

Complete antigen Hapten

Antigen

Epitope or Antigenic determinant:

The smallest unit of antigenicity is known as epitope

Smallest area 4-5 amino acids or monosaccharide residuesSpecific chemical structure, charge and configurationAntibodies are specific for epitopes

Antibodies or Immunoglobulins

Immunoglobulin Provides Structural and Chemical concept

Antibody Is a biological and functional concept

All antibodies are Immunoglobulins, but all Ig may not antibodies

Antigen-Antibody Reaction

• Ag& Ab by definition combines with each other specifically& in an observable manner

The reaction serve several purposes

In vivo In vitro

Immunity , Autoimmune diseases Serological reactions

Strength of Antigen-AntibodyInteractions

The reactions b/w Ag-Ab occur in 3 stages

Primary Secondary Tertiary

General features of Ag-Ab reaction

The reaction is specific

Entire molecule react and not fragment

There is no denaturation of Ag or Ab

Combination occurs at the surface

Combination is firm but reversible

Both Ag and Ab participate in the reaction

Ag-Ab can combine in varying proportions

Measurement of Ag and Ab

• The Ab titer of a serum is the highest dilution

of the serum that shows an observable

reaction with the Ag in the particular test

In terms of MASS(mg nitrogen ) or as units or titer

Sensitivity

• Highly sensitive – False negative reactions are absent or minimal

Specificity

• Highly specific – False positive reactions are absent or minimal

Measurement of Ag and Ab

Precipitation reaction

Agglutination

Complement fixation test

Neutralisation test

Opsonisation

Immunoflurorescence

Radioimmunoassay

Enzyme Immunoassays

Chemiluminescence immunoassay

Immunoelectroblot Techniques

Immunochromatographic tests

Immunoelectronmicroscopic tests

Serological Reactions

Precipitation occurs

Electrolyte

Antibody Soluble antigen

Precipitation reaction

• Flocculation • Medium • The amount of precipitation influenced by

relative proportion of Ag and Ab• Mechanism of precipitation - Prozone - Zone of equivalence - Postzone

Precipitation reaction

Prozone—Ab Excess

Prozone: Too much Antibody relative to Antigen

LATTICE HYPOTHESIS

Precipitation reactions in fluids

Precipitation reaction

Application:

Both qualitative and quantitative tests

Very sensitive in detecting antigens

Forensic application

Precipitation reaction

TYPES

Ring test

Slide test

Tube test

Immunodiffusion

Electroimmunodiffusion

Immunodiffusion

1. Single diffusion in one direction

2. Double diffusion in one direction

3. Single diffusion in two dimension

4. Double diffusion in two dimension

5. Immunoelectrophoresis

Radial Immunodiffusion (Mancini)

• Interpretation– Diameter of ring is

proportional to the concentration

• Quantitative– Ig levels– Screaning sera for Abs

to influenza virus among others

• Method– Ab in gel– Ag in a well

Ag Concentration

Dia

met

er2

AgAgAgAg

Ab in gel

Double diffusion in 2 dimensions

IMMUNOELECTROPHORESIS

• First do electrophoretic separation of a mixture of soluble antigens in a gel medium.

• Individual antigens are detected using an antiserum containing antibodies for all antigens in the mixture diffused at a right angle to the direction of electrophoresis.

Antigen applied to wells;Separation by charge

Antibody in trough, Protein diffusion in gel

Precipitin complex indicating identity and recognition

Immunoelectrophoresis Result1 2

3

Example: Human sera probed with rabbit anti-human antibodies

AgAg

Electroimmunodiffusion

• Development of precipitin lines can be speeded up by electrically

Various methods

A. One dimensional double electroimmunodiffusion

• Counter immunoelectrophoresis

B. One dimensional single electroimmunodiffusion

• Rocket electrophoresis

A. One dimensional double electroimmunodiffusion [Counter immunoelectrophoresis]

Clinical application: detecting various Ags; α-fetoprotein in serum& specific Ags of Cryptococcus & Meningococcus in the CSF

ROCKET ELECTROPHORESIS

AGGLUTINATION REACTIONS

Interaction between antibody and a particulate antigen resulting in visible clumping

Slide agglutination Tube agglutination Antiglobulin test Passive agglutination Haemagglutination

Applications

+

Patient’s RBCs Coombs Reagent(Antiglobulin)

Patient’s Serum

TargetRBCs

+ Step 1

+ Coombs Reagent

(Antiglobulin)

Step 2

Complement fixation test

• The ability of Ag-Ab complex to fix complement is made use of in CFT

• Looking for evidence of infection.

• The test requires five reagents and is carried out in two steps.

• The Wassermann test is one form of complement fixation test: serodiagnosis of syphilis.

NEUTRALISATION TESTS

• Based on the concept that antibodies can neutralize biological activity of many pathogens and their toxins

Viral neutralisation test Toxin neutralisation test

Opsonisation

Heat labile substance

Heat stable substance

Complement Bacteriotropin

Opsonin

Opsonisation

Immunofluorescence

Coons -1942Albert Hewett Coons (1912–1978)

Direct Immunofluorescence test

Indirect Immunofluorescence test

Radioimmunoassay [RIA]

• 1957 – Berson and Yalow

• Application – Hormone - Drugs - Tumour markers - IgE - Viral antigens

Nobel Prize 1977

Radioimmunoassay [RIA]

Enzyme immunoassay

• Enzyme labeled conjugate

• Advantage – Sensitivity - Simplicity - Economy - Absence of radiation - Availability of test kit - Facility for automation

Enzyme immunoassay [EIA]

• Includes all assays based on measurement of enzyme labeled antigen, hepten or antibody

2 types

Homogeneous EIA Heterogeneous EIA

EMIT ELISA

One step Multistep

ELISA

• Technique involves the use of an IMMUNOSORBENT

• Usually done using 96-well microtitre plates suitable for automation

• Application: – Rota virus - Anti -HIV antibody test

ELISA

Enzyme-linked Immunosorbent Assay

Chemiluminescence Immune Assay

• Refers to a chemical reaction emitting energy in the form of light

• Chemiluminescent compounds - LUMINOL - ACRIDINIUM ESTERS

• The method has been fully automated

Immunoelectroblot technique

• Combine the sensitivity of EIA with much greater specificity

• Combination of 3 separate procedures

Separation Blotting EIA or RIA

Immunochromatographic test

• One step• Qualitative • Wide application - Simplicity - Economy - Reliability • HBSAg detection

Immune response

THE IMMUNE SYSTEM

We are constantly exposed to an incredible diversity of bacteria, viruses, and parasites

How does the immune system protect us?

The key isour ability to produce more than 108 distinct antibodies and more than 1012 T-cell receptors

The immune system has 2 arms

Innate immunity

Adaptive immunity

The Immune System is the Third Line of Defense Against Infection

The immune system of the body consists of two major components

B lymphocytes T lymphocytes

Mainly derived from bone marrow

T lymphocytes are of thymic origin

Responsible for the synthesis of circulating, humoral antibodies, also known as immunoglobulins.

Cellmediated immunologic processes

Immune response

• The specific reactivity induced in a host by an antigenic stimulus

2 TYPES:

1. Humoral (AMI)

2. Cellular (CMI)

• The production of antibodies consists of 3 steps

Humoral Immune response(AMI)

Entry Processing Secretion

Antibody production follows a characteristic pattern

Lag phase Log phase Plateau or steady state

Phase of decline

ANTIBODY RESPONSE AFTER EXPOSURE TO ANTIGEN

Fate of antigen in tissues

• Antigens are removed from circulation in 2 phases

Non-immune phase Phase of immune elimination

PRODUCTION OF ANTIBODIES

• Immune response to an antigen is brought about by 3 type of cells

Lymphocytes APC Effector cells

© Ranjith’s

Central Role of Helper T Cells

© Ranjith’s

Cytotoxic T Cells Lyse Infected Cells

Humoral Response to Antigens

Factors influencing antibody production

Genetic Age Nutrition

Route of administration

Size & number of doses

Multiple antigens

Adjuvants Immunosuppressive drugs

Effect of antibody

CELLULAR IMMUNE RESPONSE (CMI)

Specific immune response that do not involve antibodies.

Delayed hypersensitivity

Immunity in infections

Transplantation immunity

Immunological surveillance

Autoimmune disease

Scope of CMI

Induction of CMI

• Antigenic stimulus• Only T-cell dependant antigens lead to CMI• T-cell bears specific receptor on its surface• T cells recognize antigens only when

presented with MHC molecules• Helper T-cells• Cytotoxic T-cells

T Cells Only Recognize Antigen Associated with MHC Molecules on Cell Surfaces

Central Role of Helper T Cells

CYTOTOXIC T CELLS LYSE INFECTED CELLS

Cytokines • Peptide mediators or intracellular messengers

which regulate immunological, inflammatory and reparative host responses

• They are potent hormone like substances

• Produced by lymphocytes, macrophages, platelets & fibroblasts

CYTOKINES - CLASSIFICATION

Interleukins

Colony stimulating factors

Tumour necrosis factors

Interferons

Others

• Source: Macrophages & other cells• IL-1α and IL-1β• Functions: Proliferation & differentiation of T, B and other

cells Pyrogenic Induce acute phase proteins Bone marrow cell proliferation

Interleukin-1

• Source: T-cells• Powerful modulator of the immune response• Functions: Promote growth and differentiation of T and B

cells Cytotoxicity of T and NK cells Secretions of other lymphokines

Interleukin-2[TCGF]

• Stimulates the growth & differentiation of pleuripotent stem cells in the bone marrow

• Responsible for adjusting the rate of production of blood cells

• Treating hematopoietic dysfunctions in infections & malignancies

Colony stimulating factors

• Induce hemorrhagic necrosis in certain tumours

• 2 types: TNFα and TNFβ• Cachectin is TNFα

Tumour necrosis factors

• Originally identified as antiviral agentsα-Produced by leucocytesβ-Produced by fibroblastsγ-Produced by T-cells• Functions: Macrophage activation Antitumour activity

Interferons