Antigens

Antigens

 Definition: Are the substances of various chemical types when introduced

parentally into the body, stimulate the production of antibodies with which it
reacts specifically and in an observable manner
 (Complete antigen -Immunogenicity: Ability to induce an immune response
 Immunological reactivity: Ability to react with antibody in specific and
observable manner)
 Immunogens: Substances that are able to generate an immune response by
themselves
 Antigens and Immunogens are the interchangeable terms
 HAPTEN: ( Incomplete antigen)
 Hapten is a low molecular weight substance that is not immunogenic by itself
but can react with specific antibody.
 Haptens can be made complete antigens on combining with a larger molecule
called carrier proteins
 Examples
 Dinitro chlorobenzene (DNCB)
 Picryl Chloride
 ANTIGENIC DETERMINANTS
 The smallest unit of antigenicity – antigenic determinant or epitope
 The combining area on the antibody molecule corresponding to the epitope ,
is called PARATOPE.
 Epitopes and paratopes determine the specificity of immunological reactions
FACTORS DETERMINING THE ANTIGENICITY

 1. Foreignness – Only antigens which are foreign (non-self) to the

individual induce an immune response.
 2. size -Antigenicity is related to molecular size. Very large molecules
(hemocyanins) are highly antigenic and particles with low molecular
weight (less than 5000) are non antigenic or poor antigenic.
 3. Chemical nature -Protein> Carbohydrate>Lipid> Nucleic acid
 Most of the naturally occurring antigens are proteins and
polysaccharides
 4. Susceptibility to tissue enzymes -Only substances which are
susceptible to the action of tissue enzymes behave as antigens.
Classification of antigens

 I. Based on origin
 1. Tissue antigens Blood group antigens, Human leukocyte antigens
 2. Microbial antigens
 Somatic ‘O’ antigen , Flagellar ‘H’ antigen, Capsular ‘K’ antigen
 II. Based on the types of immune response induced
 1. T cell Independent (TI) antigen – Directly stimulate antibody production by
B cells without the participation of T cells
 2. T cell Dependent (TD) antigen
 Antigen which activates B cell only with the help of T cell
Antibodies
Antibody

 Definition : Is an immunoglobulin which is produced as a result

of antigenic stimulation with which it reacts specifically and in
some observable manner
 IMMUNOGLOBULINS : Are proteins of animal origin endowed
with known antibody activity and also include certain other
proteins related to them by chemical structure
 Antibody activity is mainly associated with the Gamma globulin
fraction. So gamma globulin is used as synonym for antibody
 Immunoglobulin constitute 20-25% of the total serum proteins
Structure of Immunoglobulin
STRUCTURE OF IMMUNOGLOBULIN

 Immunoglobulins are glycoproteins

 Each molecule consisting of two pairs of polypeptide chains
of different sizes. The smaller chains are called Light (L)
chains and the larger ones Heavy (H) chains with mol. Wt
of 25,000 and 50,000 respectively
 The L chain is attached to the H chain by a disulphide bond
 The two H chains are joined together by 1- 5 disulphide (S-
S) bonds depending on the class of Igs.- Interchain
disulphide bonds..
 The H chains are structurally and antigenically distinct for each class
and there are 5 classes of Igs ( based on H chain) which are
designated by the Greek letter
 Gamma (γ) - Ig G, Alpha (ά) – Ig A,Mu ( μ) – Ig M,Delta ( δ) – Ig D and
 Epsilon (ε) – Ig E
 The L chains are similar in all classes of Igs. They occur in 2 types –
Kappa(κ) and Lambda(λ)
 A molecule of Ig may have either Kappa or Lambda chain but never
both together
 Each basic Ig molecule is ‘Y’ shaped and consists of 2 heavy chains
and 2 light chains with molecular formula H2 L2.
 Ig molecule can be broken or it can be digested by proteolytic
enzymes like pepain at one particular point called Hinge region
( between CH1 and CH2. After digestion there will be 2 pieces of Fab
(Fraction of antigen binding) and one piece of Fc (Fraction of
crystallisation)
 Function of Fab fragment is antigen binding
 Function of Fc fragment - various biological activities like placental
transfer, complement activation, attachment to various cells
 Each L and H chains are subdivided into variable and constant regions.
 Variable region is in aminoterminal end and constant region is in
carboxyterminal.
 In each region there are three dimensionally folded repeating
segments called domains produced by intrachain disulphide bond.
 An L chain consists of 1 variable (VL) and 1 constant (CL) domain.
Most H chains consist of 1 variable (VH) and 3 constant (CH) domains.
Each domain is approximately 110 amino acids long
 The variable regions are responsible for antigen binding where as
constant regions are responsible for various biological functions
 Properties of human immunoglobulins

Property Ig G Ig A Ig M Ig D Ig E

Heavy chain symbol γ ά μ μ ε

Serum conc. mg/ml 8-10 0.5 - 3 1.5 0.03 0.00003

% of total Ig in serum 75 15 9 <1 <1

Mol. Wt (x1000) 150 170/400 900 180 190

Sedimentation 7 7/11 19 7 8
coefficient (S)
Property IG G Ig A Ig M Ig D Ig E
Serum half life (days) 23 6 5 2-8 1-5

Transplacental + - - - -
passage
Found in secretion - + - - -

Complement fixation + - + - -

Mediation of allergic - - - - +
responses
Structure Monomer Monomer/ Monomer/ Mono Monom
dimer pentamer mer er
Functions IgG

 Is a general purpose antibody. Protective against those infective agents which

are active in the blood and tissues
 Is the predominant Antibody in the secondary immune response and provides
important defense against bacteria and viruses
 It opsonises bacteria and making them easier to phagocytise. Neutralises
bacterial toxins and viruses
 Fixes complement which enhances bacterial killing
 Provides natural passive immunity to the child, can protect the same for 3-6
months of age
 Functions
 sIg A is selectively concentrated in secretion and on mucus surfaces forming
an antibody paste and play an important role in local immunity.
 It prevents attachment of microorganisms (Bacteria, viruses) to mucus
membrane
 Functions
 Main Ig produced early in the primary immune response
 It is present as a monomer on the surface of B cells where it functions as an
Ag – binding receptor for Antigen recognition
 It is the most efficient Ig in agglutination, complement activation and other
Antibody reaction and is important in defence against bacteria and viruses
 Earliest Ig to be synthesised in the fetus or new born indicates intrauterine
infection, its detection is useful in the diagnosis of congenital infections
 It is short lived and their demonstration in serum indicates recent infection
 Responsible for protection against blood invasion microorganisms
 Functions
 Is medically important for 2 reasons
 It is a cytotropic Ab. It can fix with mast cells and basophils through Fc . It
mediates Type I hypersensitivity reaction – Anaphylaxis, Atopy
 It participates in host defense against certain parasites – helminthes – Ascaris,
Hook worm
 Serum Ig E level increases in both the above conditions
Antigen antibody
reactions
Introduction

 By definition Ags and Abs combine with each other specifically and in an
observable manner.
 This Ag-Ab reactions occur continuously in vivo, form the basis of humoral
immunity in case of infectious diseases
 Given proper conditions and necessary regents Ag-Ab reaction can be made to
occur in vitro
 These are called serological reactions and the study is known as “Serology”
Uses of serological reactions

 Serodiagnosis of infectious diseases

 As confirmation of additional proof of infectious diseases
 To evaluate the prognosis and efficacy of treatment
 Diagnosis of non-infectious diseases
Classification of serological/Ag-Ab
reactions
 Precipitation test
 Agglutination test
 Complement fixation test
 Neutralization test
 Immunofluorescence test
 Radio Immuno Assay
 Enzyme Immuno Assay
AGGLUTINATION
Definition

 When particulate Ags combine with their specific Abs at optimal proportions,
in the presence of electrolytes and at a suitable temperature and pH, the Ag-
Ab complexes are agglutinated or clumped – such reactions are known as the
agglutination reactions
CLASSIFICATION

 Based on the principle:

 Direct (Active)agglutination reaction
 Indirect (Passive) agglutination reaction
 Reverse passive agglutination reaction
Direct/active agglutination reactions

 When particulate Ags themselves take part in the reaction and get
agglutinated , then these reactions are known as active/direct agglutination
reactions
 Two types
 Slide agglutination tests
 A drop each of the Ag and Ab is mixed on a slide, resulting in agglutination.
 E.g. Bacterial slide agglutination tests for identification of clinical isolates
such as Salmonella, Shigella, Vibrio, Streptococcus etc. using specific antisera
 Blood grouping test
Slide agglutination test
Slide agglutination test – Blood grouping
Tube agglutination

 Here serial dilutions of the serum( clinical sample) containing the Abs are
made in test tubes and to fixed amount of this serially diluted sample, equal
volumes of Ag are added
 These tubes are then incubated in an incubator at a suitable
temperature(37oC) for an appropriate period of time after which is observed
for agglutination
 Because of the serial dilution of the serum the amount of Abs present can be
estimated – quantitative tests
Applications

 Widal test: Serodiagnosis of enteric fever

 Standard agglutination test : Brucellosis:
 Weil Felix test: Rickettsial fever
Indirect/Passive agglutination test

 When soluble Ags are attached onto the surfaces of carrier particles, thus
converting them into particulate Ags, they then react with their Abs leading
to the agglutination of carrier particles
 Here, the carrier particles are said to be passively agglutinated as they
themselves are not involved in the reaction
Examples for passive agglutination test

 Using latex beads – Latex agglutination tests

 ASO test – Rheumatic fever
 RA test – Rheumatoid arthritis
 Using RBC’s – TPHA - Syphilis
 Streptozyme test –Streptococcal infection
Latex agglutination test
Reverse passive agglutination

 Principle : Here instead of the Ags, Abs are attached onto carrier particles
and this complex is used to detect different types of Ags in biological fluids
 Carrier particles used
 RBC’s – RPHA – HBs Ag detection in Hepatitis B
 Latex particles – RPLA – Detection of capsular polysaccharide antigens in CSF –

 C. neoformans, N. meningitides, H. influenzae

 CRP test – C Reactive Protein
IMMUNOFLUORESCENCE

 Fluorescent antibody test(FAT)

 Definition – A serological reaction/test used for the detection of either
Ags/Abs using fluorescent labeled/conjugated antibodies
Principle

 Fluorescent dyes/fluorochromes are the substances have the ability to adsorb

the light rays of shorter wavelength and emit the light rays of longer
wavelength
 While doing so they become highly luminous/fluoresce.
 Certain fluorescent dyes can be conjugated to antibodies and such labeled
antibodies can be used to demonstrate either Ags or Abs in the clinical
samples and the technique known as Immunofluorescence
 Commonly used fluorochromes
 Fluorescein isothiocyanate
 Lissamin - rhodamine.
Classification

 1. DIRECT IMMUNOFLUORESCENCE
 2. INDIRECT IMMUNOFLUORESCENCE
 Direct Immunofluorescence Assay

Steps Explanation
Step-1 Sample containing cells carrying surface antigens is smeared on a
slide.
Step-2 Primary antibody specific to the antigen, tagged with fluorescent
dye is added.
Step-3 Slide is washed to remove the unbound antibodies and then viewed
under a fluorescence microscope.

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 Indirect Immunofluorescence Assay

• Detects antibodies in sample. Slides smeared with cells carrying known antigens are
commercially available.
Steps Explanation
Step-1 Test serum containing primary antibody is added to the slide.
Step-2 Slide is washed to remove the unbound antibodies. A secondary antibody
(anti human antibody conjugated with fluorescent dye) is added.
Step-3 Slide is washed and then viewed under a fluorescence microscope

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Publishers
Indirect Immunofluorescence
Applications of IFT

 To detect Ags in clinical samples of various bacterial, viral and parasitic

infections.
 e.g. Syphilis, Chlamydial infections, Legionella infection, Toxoplasmosis, Kala
azar, Rabies etc.
 Detect antibodies in patient serum using a known antigen.
 To detect Abs against various infectious diseases
 Bacterial - (T. pallidum - FTA Abs Test),
 Viral - (HSV, Rubella, EBV)
 Parasitic - (Toxoplasma)
 Diagnosis of autoimmune diseases
 Antinuclear antibody in SLE
Enzyme Immuno Assay
(EIA)
Principle

 This serological test uses enzyme system to demonstrate

the specific combination of Ag and its Ab and thereby
helps in the detection of the Ag or Ab
 The enzyme system consists of
 1. An enzyme which is linked or attached onto Antibodies
also called conjugate
 Important enzymes used are
 Alkaline phosphatase
 Horse radish peroxidase
 2. A substrate which is added after the Ag-Ab reaction.
 Paranitrophenyl phosphate
 Ortho phenylene diamine dihydrochloride
 This substrate is acted on by the enzyme molecules attached onto the Abs in
the Ag Ab complex to give a colour change
 Stop solution – to stop the reaction, usually week acids are used
 Reading of the change in colour is done by using spectrophotometer (ELISA
reader)
 The intensity of the colour change (OD value) is directly proportional to the
amount of bound Ag or Ab whose presence and concentration need to be
detected
Types of EIA

 Two types
 Homogenous Assay – here no need to separate the bound and free fractions
and carried out in liquid phase
 This is mainly used for the detection and assay of substances such as drugs,
antibiotics, hormones etc.
 Not commonly used for microbiological Ag/Ab detection
Heterogenous Assay

 The major type of heterogeneous EIA is Enzyme Linked Immunosorbant Assay

(ELISA)
 This is a type of EIA where Ags or Abs are bound to a solid phase and which
requires separation of Ag-Ab complex from free Ags and Abs
 It is named because the technique involves the use of an immunosorbant – an
absorbing material
 The solid phase used – usually made of polystyrene in the form of micro wells,
tubes or beads
 Most commonly a 96 – well microtitre plate is used which is suitable for
automation
 (Ag-Ab complex)-enzyme + substrate → activates the
chromogen → color change → detected by
spectrophotometry
 ELISA kits are commercially available; contain all the
necessary reagents (such as enzyme conjugate, dilution
buffer, substrate/chromogen etc).
 Procedure involves a series of steps done sequentially; at
each step, a reagent is being added, and then incubated
followed by washing of the wells (manually or by automated
ELISA washer).
Microtitre plate
ELISA Washer
ELISA Reader
Types of ELISA

 Competitive ELISA – here serum antibody and enzyme labeled antibody

compete for the binding sites on the antigen. More specific but tedious
 Non competitive ELISA – most commonly used in diagnostic microbiology
Non competitive ELISA

 Direct ELISA – Ag detection, not practical because there is need for separate
enzyme labelled antibody for each Ag to be detected
 Indirect ELISA – Ab detection
 Sandwich ELISA – Ag detection
 Direct ELISA
 Used for detection of antigen in test serum.
 Primaryantibody (targeted against the serum antigen) is
labeled with the enzyme.
Steps Explanation
Step 1 Wells of microtiter plate are empty, not precoated with Ag or Ab.
Step- Test serum (containing antigen) is added into the wells. Antigen
2 becomes attached to the solid phase by passive adsorption.
Step- After washing, the enzyme-labeled primary antibodies are added.
3
Step- After washing, a substrate- chromogen system is added and color is
4 measured.
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 Well + Ag (test serum) + primary Ab-Enzyme + substrate- chromogen → Color
change.
 Indirect ELISA – Antibody detection
 It differs from the direct ELISA in that the secondary antibody is labeled
with enzyme instead of primary antibody.

Steps Explanation
Step 1 Solid phase of the wells of microtiter plates are precoated with the Ag.
Step- Test serum (containing primary Ab specific to the Ag) is added to the
2 wells. Ab gets attached to the Ag coated on the well.
Step- After washing, enzyme-labeled secondary Ab (anti-human
3 immunoglobulin) is added.
Step- After washing, a substrate-chromogen system is added and colour is
4 developed.
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Indirect ELISA

 Wells are coated with Ag + primary Ab (test serum) + secondary Ab-Enzyme +

substrate- chromogen → development of color.
Applications

 Detection of Antibodies in patient serum – bacterial, viral, parasitic, fungal

infections
 Bacterial infections – Chlamydia infection, Tuberculosis
 Viral – HIV, HCV, HBV, HSV infection
 Parasitic – Toxoplasmosis,
 Sandwich ELISA
 Detects the antigen in test serum.
 Sonamed because the antigen gets sandwiched between a capture
antibody and a detector antibody.
 There are two types of sandwich ELISA:
o Direct - detector antibody is a primary antibody (Not common).
o Indirect - detector antibody is a secondary antibody.

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 Wells coated with capture Ab + Ag (test serum) + primary Ab + secondary Ab-
enzyme + substrate- chromogen → color.
 Applications

 Detection of Antigens in various clinical samples

 Detection of HBs Ag in serum
 Detection of Rotavirus in stool
 Detection of chlamydial Ag in urethral discharges
Special types/modification of ELISA

 IgM Capture ELISA

 Detects the presence of only IgM
 Mainly used in the diagnosis of congenital infection and recent infections –
TORCH complex
Cylinder/cassette ELISA

 Here each specimen is tested in a separate disposable cassette

 This test is rapid , taking only about 10 minutes
 There is no need for microplate washer and reader
 The result is read visually
 Inbuilt positive and negative controls are usually provided for validation of
the test procedure
 E.g. HIV, HCV Abs, HBs Ag – HIV Tri dot, Retroquic HIV, HCV Tri dot,
Advantages of ELISA

 Sensitive and specific

 Both qualitative and quantitative test
 Reproducible
 Relatively inexpensive
 Require very small quantity of serum -100ul
 Standardized commercial kits are available for most of the infectious disease
– the most popular serological test
Disadvantage of ELISA

 Small laboratories with less sample load - ELISA is less preferred than rapid
tests as the later can be done on individual samples.
 More time (2-3 hours) compared to rapid tests which take 10-20 min.
 Needs expensive equipments such as ELISA washer and reader