I) Immunity

The term ‘immunity’ is defined as resistance exhibited by the host against any foreign antigen including
microorganisms. This resistance plays a major role in prevention of infectious diseases. Immunity may
be innate or acquired/adaptive immunity-

1) Innate immunity:-
Components of innate immunity
 Barrier mechanisms of skin, respiratory tract, intestinal tract, conjunctiva, genitourinary tract.
 Non-specific antibacterial substances e.g. properdin, complement, lysozyme, interferons.
 Phagocytic cells (neutrophils, macrophages)
 Natural killer (NK) cells
 Fever and inflammation
 Acute phase reactant proteins (APRs)
 Complement pathways-alternative

2) Acquired/ adaptive immunity:-

The resistance acquired by an individual during life is known acquired immunity. It is of two types,
active and passive immunity
a) Active immunity- Natural and Artificial active immunity
b) Passive immunity- Natural and Artificial immunity

Active Immunity Passive Immunity

1. Produced actively by the immune system. Received passively by the host. The host’s
immune system does not participate.
2. Induced by infection or by contact with Conferred by administration of ready-made
immunogens. antibodies.
3. Long-lasting and effective protection. Protection short lived and less effective.
4. Immunity is effective only after a lag period Immunity is effective immediately.
i.e. time required for generation of antibodies.
5. Immunological memory present, therefore, No immunological memory. Subsequent
subsequent challenge is more effective administration of antibody is less effective due to
(booster effect). immune elimination.
6. Negative phase may occur. No negative phase.
7. Not applicable in immunodeficient persons. Applicable in immunodeficient persons.
8. Used for prophylaxis to increase body Used for treatment of acute infections.
resistance e.g. BCG vaccine.
Combined Immunisation:-
A combination of active and passive immunisation is employed simultaneously which is known as
combined immunisation

Herd Immunity:-
It refers to the overall immunity in a community (or herd) towards a pathogen

II)Antigen
An antigen is a substance which, when introduced into a body evokes immune response (both humoral
and/or cell mediated) and has the ability to combine specifically with the final products of two immune
 responses (i.e. antibodies of humoral response and T-lymphocyte surface receptors in cell mediated
response).

Epitope
 The smallest unit of antigenicity on antigen is called epitope or antigenic determinant.
 The specific site of an antibody to which epitope of an antigen binds is called paratope

Types of antigen:-
a) Complete antigen:- These are substances which can induce antibody formation by themselves and
can react specifically with these antibodies
b) Haptens/incomplete antigens:- Haptens are substances unable to induce antibody formation on its
own but can become immunogenic (capable of inducing antibodies) when covalently linked to proteins,
called carrier proteins.
c) Heterophile Antigens:- The same or closely related antigens present in different tissues of more than
one species are known as heterophile antigens.

Biological classes of antigens:-

1) T-cell Dependent (TD) Antigens
 These are antigens which require T cells participation to stimulate B-cells to produce antibodies.
These antigens are called TD antigens

2) T-cell Independent (TI) Antigens

 These antigens do not require the help of T-cells for stimulation of B-cells. These antigens are
called TI antigens.
 TI antigens do not require initial antigen processing by antigen-presenting cells (e.g.
macrophages) unlike TD antigens.

Superantigens:-
 Superantigens are molecules that can interact with antigen-presenting cells (APCs) and T-lymphocytes
in a non-specific manner.
 Interaction of superantigens with MHC class II molecules of the APC and the V β domain of the T-
lymphocytes receptor activates a large number of T-lymphocytes than conventional antigens. This leads
to massive cytokine expression and immunomodulation.

III) Antibodies—Immunoglobulins
 Antibodies or immunoglobulins are specialised proteins which are formed by immune system in
response to an antigen, and react with that antigen specifically.
 Chemical nature of antibodies is globulin and they are named as immunoglobulins.
 An antibody molecule consists of two identical heavy and two identical light chains.

ImmunogloBulIn classes
B. Immunoglobulin A (IgA)
(i) IgA is the second major serum immunoglobulin (about 10-13% of serum immunoglobulins).
(ii) IgA occurs in two forms, serum IgA (monomeric form) and secretory IgA (dimeric form).
(iii) IgA does not fix complement but can activate alternative complement pathway.

C. Immunoglobulin M (IgM)
(i) IgM is a pentamer consisting of 5 immunoglobulin subunits and one molecule of J chain.
(ii) It constitutes about 5–8 percent of total serum immunoglobulins.
(iii) It is heavy molecule (19S) with a molecular weight 900,000 to 1,000,000 hence also
called the ‘millionaire molecule’.
(iv) It is the first antibody to be produced in response to infection (acute infection).
(v) It cannot cross the placenta, presence of IgM antibody in serum of newborn indicates
congenital infection.
D) Immunoglobulin D (IgD)
(i) IgD resembles IgG structurally.
(ii) IgD is also present on the surface of unstimulated B lymphocytes in blood and acts as
recognition receptors for antigens.

E) Immunoglobulin E (IgE)
(i) IgE is mainly produced in the linings of respiratory and intestinal tracts.
(ii) It is also referred to as reagins
(iii) It resembles IgG in structure.
(iv) It is the only heat labile (inactivated at 560C in one hour) immunoglobulin.
(v) It has affinity for surface of tissue cells particularly mast cells of the same species
(homocytotropism)
(vi) IgE mediates type I hypersensitivity reaction.
IV) Antigen-Antibody Reactions
Antigen combines with its specific antibody in an observable manner and the reaction between antigen
and antibody is specific.

Characteristics-
1. Reaction is specific; an antigen combines only with its homologous antibody and vice-versa.
However, cross reactions may occur due to antigenic similarity.
2. A single antibody can cause different types of antigen-antibody reactions and a single antigen
can stimulate production of different classes of immunoglobulins which differ in their reaction
capacities as well as in other properties

I. Role of Immunoglobulins Classes in Serological Reactions

Serological reaction Immunoglobulin class

IgG IgM IgA
Precipitation Strong Weak Variable
Agglutination Weak Strong Moderate
Complement fixation Strong Weak Negative

II. Types of antIgen-antIbody reactIons

a. Precipitation reactions

i. Precipitation
When a soluble antigen reacts with its antibody in the presence of electrolytes (NaCl)
at an optimal temperature and pH, the antigen-antibody complex forms an insoluble
precipitate and it is called precipitation.

ii. Flocculation
When instead of sedimenting the precipitate is suspended as floccules, the reaction is
called flocculation.

Prozone Phenomenon
Absence of precipitation (false negative) in the presence of excess antibodies is known as
prozone phenomenon

i) Mechanism of precipitation
Marrac (1934) proposed the lattice hypothesis which is widely accepted.

ii) Precipitation in liquid medium

Flocculation test
It can be done in slide and in tube.
Slide test- VDRL and RPR (Rapid Plasma Reagin) tests.
Tube test- The Kahn test (tube flocculation) was done previously.
iii) Precipitation in gel
-Immunodiffusion test
These are precipitation tests in gel.
Various Immunodiffusion Tests
1. Single diffusion in one dimension (Oudin procedure)
2. Double diffusion in one dimension (Oakley-Fulthorpe procedure)
3. Single diffusion in two dimensions (Radial immunodiffusion)
4. Double diffusion in two dimensions (Ouchterlony procedure)

A special variety of double diffusion in two dimensions is the Elek’s test for toxigenicity in
C.diphtheriae

-Immunoelectrophoresis
Immunoelectrophoresis combines electrophoresis and immunodiffusion.

-Electroimmunodiffusion
Immunodiffusion can be speeded up if antigen and antibody are driven by electricity. It is
combination of electrophoresis and diffusion.

-Counterimmunoelectrophoresis
Rocket electrophoresis

b. Agglutination
It is an antigen-antibody reaction, in which a particulate antigen combines with its antibody
in the presence of electrolytes at an optimal temperature and pH, resulting in visible clumping
of particles.

Types of Agglutination Reaction

1. Slide Agglutination Test
2. Tube Agglutination Test
3. Heterophile Agglutination Test
4. Passive (indirect) Agglutination Test (for antibody detection)
 A precipitation reaction can be converted into agglutination test by attaching soluble antigens
to the surface of carrier particles such as latex particles, bentonite and red blood cells. Such
tests are called passive agglutination tests and are used for detection of antibodies.

Reverse passive agglutination test (for antigen detection)

When instead of antigen, the antibody is adsorbed on the carrier particles for estimation of
antigen, it is known as reverse passive agglutination.

(i) Latex agglutination test

(ii) Haemagglutination test

(iii) Coagglutination

c. Complement Fixation test (CFT)

 The antigen-antibody complexes have ability to ‘fix’ complement. This reaction
has no visible effect.
 To detect the fixation of complement, an indicator system is used. Earlier it was
commonly used method but is almost not in use now.

d. Immunofluorescence
1. Direct Immunofluorescence Test
The specific antibodies tagged with fluorescent dye (i.e. labelled antibodies) are used
for detection of unknown antigen in a specimen. If antigen is present, it reacts with
labelled antibodies and fluorescence can be observed under ultraviolet light of
fluorescent microscope

2. Indirect Immunofluorescence Test

A known antigen is fixed on a slide. The unknown antibody (serum) is applied to the
slide. If antibody (globulin) is present in the serum, it attaches to known antigen on the
slide. For detection of this antigen-antibody reaction, fluorescin-tagged antibody to
human globulin is added. In positive test, fluorescence occurs under ultraviolet light.
e. Enzyme Linked Immunosorbent Assay (ELISA)
The principle of ELISA is almost same as that of immunofluorescence, the only difference
being, an enzyme is used instead of fluorescent dye. The enzyme acts on substrate to
produce a colour in a positive test.

General features
 ELISA can be used for detection of antigen or antibody.
 Tests for specific immunoglobulin classes (e.g. IgM ELISA) are also available.

Types of ELISA
a) Direct ELISA
Antigen (test serum)
+
Primary antibody (antibody to antigen) labelled with enzyme
+
Substrate
+
Colour detection

b) Indirect ELISA
Indirect ELISA is used for detection of antibody or less commonly antigen.
Principle of indirect ELISA is shown in box below.
c) Sandwich ELISA
Sandwich ELISA detects the antigen in serum. It is so named because the antigen
gets sandwiched between two antibodies.

d) IgM antibody capture [Mac] ELISA

This is an enzymatically amplified sandwich-type of immunoassay. This type of
ELISA is routinely used for the diagnosis of dengue, chikungunya and Japanese
encephalitis

e) Competitive ELISA
Competitive ELISA can be used for antibody or antigen detection.
Uses of ELISA
Antigen detection
Hepatitis B surface antigen (HBsAg)
NS1 antigen for dengue
Rotavirus in faeces

Antibody detection
Hepatitis B
Hepatitis C

f. Rapid tests
Principles
Two principles of rapid tests are available.
 Lateral flow assay e.g. immunochromatagraphic test (ICT).
 Flow through assay e.g. TRI-DOT assay.

1. Immunochromatography

2. Flow Through Assay

Flow through assay is another type of rapid test which differs from ICT as follows
(i) Protein A is used to label antibody.
(ii) The specimen flows vertically through the nitrocellulose membrane instead of
lateral flow in ICT.
HIV-TRI DOT is one example of flow through assay to detect antibodies to HIV-1 and
2 separately in patient’s serum
The test is present in a cassette format.
 (a) HIV-TRI DOT test (b) Diagramatic representation

g. Applications of antigen-antibody reactions

 Antigen-antibody reaction (test used) Disease

1. Slide flocculation (VDRL test) Syphilis

2. Double diffusion (Elek’s toxigenicity test) Diphtheria

3. Slide agglutination Salmonella infections, Shigella infections, Cholera

4. Tube agglutination (widal test) Enteric fever

5. Tube agglutination (serum agglutination test) Brucellosis

6. Hetrophile agglutination (Weil-Felix reaction) Typhus fever

7. Hetrophile agglutination (Paul-Bunnel test)

8. Hetrophile agglutination (Streptococcus MG Primary atypical pneumonia

agglutination test)

9. Latex agglutination Meningitis

10. Latex agglutination (RA factor) Rheumatoid arthritis

11. Latex agglutination (ASO test) Rheumatic fever

12. Latex agglutination (CRP test) Inflammatory diseases

13. Direct immunofluorescence Rabies

14. Indirect immunofluorescence (FTA-ABS test) Syphilis

15. ELISA HIV infection

16. ELISA Hepatitis B infection

17. ELISA Rotavirus diarrhoea

18. Immunochromatographic test (ICT) Meningitis

19. Immunochromatographic test (ICT) HIV infection