Agglutination

When particulate antigen combines with its antibody in the

presence of electrolytes at an optimal temperature and pH,
resulting in visible clumping of particles

 More sensitive than precipitation for the detection of antibodies

 The agglutination reaction takes place better with IgM antibody

 Lattice formation hypothesis holds good for agglutination too

 Blocking antibodies inhibit the agglutination by the complete

antibody added subsequently
 Types of agglutination reactions

1. Side agglutination test

2. Tube agglutination test

3. The antiglobulin (Coombs) test

4. Heterophile agglutination test

5. Passive agglutination test

 Slide agglutination test
 A uniform suspension of antigen is made in a drop of saline
on a slide and a drop of the appropriate antiserum is added

 Reaction is facilitated by mixing the antigen and the antiserum

with a wire loop or by gently rocking the slide

 Clumping occurs instantly or within seconds when agglutination

test is positive

 A control consisting of antigen suspension in saline, without

adding antiserum must be included on the same slide
 Uses

1. It is a routine procedure to identify the bacterial strains isolated

from clinical specimens

(eg: Identification of Salmonella species)

2. It is also used for blood grouping and cross matching

Tube agglutination test
What is the titer of Ab?

The titer is customarily reported as the reciprocal of the

highest dilution of Ab that causes an obvious agglutination
 Uses

Used for serological diagnosis of

1. Enteric fever (Widal test)

2. Typhus fever (Weil-Felix reaction)

3. Infectious mononucleosis (Paul-Bunnel test)

4. Brucellosis (SAT)

5. Primary atypical pneumonia

(Streptococcus MG agglutination test)
 Problems related to tube agglutination

1. Prozone phenomenon

2. Blocking antibodies

 Blocking or incomplete antibodies may be detected by

performing the test in hypertonic (5%) saline or albumin saline

 Antiglobulin (Coombs) test is more reliable for detecting these

antibodies
The antiglobulin (Coombs test)

Originally devised by Coombs, Mourant and Race (1945) for the

detection of incomplete anti-Rh antibodies

There are two types of Coombs test

1. Direct Coombs test

2. Indirect Coombs test

Direct Coombs test

+ ↔

Patient’s RBCs Coombs Reagent

(Antiglobulin)
 Indirect Coombs test

Step 1
+ ↔

Patient’s Target
Serum RBCs

Step 2

+ ↔

Coombs Reagent
(Antiglobulin)

The only difference between the two is that the sensitisation of

the erythrocytes with incomplete antibodies takes place in vivo
in direct type whereas it occurs in vitro in indirect type
 Uses of Coombs test

1. For detection of anti-Rh antibodies

2. For demonstration of any type of incomplete antibody

(eg: Brucellosis)
 Heterophile agglutination test

 Heterophile antibodies have a property to react with

microorganisms or cells of unrelated species due to common
antigenic sharing

i) Weil-Felix reaction

 Some proteus (OX19, OX2, and OXK) strains are

agglutinated by sera of patients with rickettsial infections

 This is due to antigenic sharing between these Proteus

strains and Rickettsial species
ii) Paul-Bunnel test
Sheep erythrocytes are agglutinated by sera of infectious
-mononucleosis’

iii) Streptococcus MG agglutination test

It is positive in primary atypical pneumonia
 Passive agglutination test

 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

 When instead of antigen, the antibody is adsorbed on the

carrier particles for estimation of antigens, it is known as
reversed passive agglutination
Latex agglutination test

Polystyrene latex particles (0.8 – 1 µm in diameter) are widely

employed to adsorb several types of antigens
 This test is convenient, rapid and specific

 Used for detection of hepatitis B antigen, ASO, CRP, RA factor,

HCG and many other antigens

 Latex agglutination tile is used to perform this test

 Haemagglutination test

 Erythrocytes sensitised with antigen are used for detection of

antibodies

 Rose-Waaler test for detection of RA factor in patient serum

 The antigen used for the test is sheep red blood cells sensitised
with rabbit antisheep erythrocyte antibody (amboceptor)
 Coagglutination

 Some strains of Staphylococcus aureus (especially Cowan 1

strain) possess protein A on their surface

 When specific IgG molecule is coated on these strains, Fc

portion of IgG molecule binds to protein A whereas antigen
combining Fab terminal remains free

 When the corresponding antigen is mixed with these coated

cells, Fab terminal binds to antigen resulting in agglutination

 This test is used for detection of bacterial antigens in blood,

urine and CSF
(eg: Gonocooci, Streptococcus pyogenes
and Haemophilus influenzae)
 Complement fixation test

 Complement is a protein (globulin) present in normal serum

 Whole complement system is made up of nine components:

C1 to C9

 Complement proteins are heat labile and are destroyed by

heating at 56°C for 20 – 30 minutes
 Principle

 The antigen-antibody complexes have ability to fix complement

 This reaction has no visible effect

 To detect the fixation of complement, an indicator system

consisting of sheep erythrocytes coated with amboceptor
is used
 Components of CFT

Test System
 Antigen: It may be soluble or particulate.
 Antibody: Human serum (May or may not contain Antibody
towards specific Antigen)
 Complement: It is pooled serum obtained from 4 to 5
guinea pigs. It should be fresh or specially preserved as the
complement activity is heat labile (stored at -30 °C in small
fractions). The complement activity should be initially
standardized before using in the test

Indicator System (Haemolytic system)

 Erythrocytes: Sheep RBC
 Amboceptor (Hemolysin): Rabbit antibody to sheep red
cells prepared by inoculating sheep erythrocytes into rabbit
under standard immunization protocol.
 Controls

 Antigen and serum controls are included in the test

 Complement control is used to ensure that the desired amount

has been added

 Cell control to make sure that sensitised erythrocytes do not

undergo lysis in the absence of complement
 Positive Test

 Step 1:
At 37°C
Antigen + Antibody + Complement Complement gets fixed
(from serum) 1 Hour

 Step 2:
At 37°C
Fixed Complement complex + Haemolytic system No Haemolysis
1 Hour (Test Positive)
 Negative Test

 Step 1:
At 37°C
Antigen + Antibody absent + Complement Complement not fixed
1 Hour

 Step 2:
At 37°C
Free Complement + Haemolytic system Haemolysis
1 Hour (Test Negative)
 Results and Interpretations:

 No haemolysis is considered as a positive test

 Haemolysis of erythrocytes indicative of a negative test

1 2 3 4

 Microtiter plate showing Haemolysis (Well A3, A4 and B4)

and No Haemolysis (Well A1, B1, B2, and B3)
 Indirect complement fixation test

 Certain avian (eg: duck, parrot) and mammalian (eg: horse, cat)
sera cannot fix guinea pig complement

 Test is done in duplicate and after the first step, the standard
antiserum known to fix complement is added in one set
 Positive test

 First step: Antigen + test serum (positive for antibody) +

guinea pig complement

 Second step: Standard antiserum cannot react with antigen

because has been used up by antibody in the first step, hence,
complement is free

 Indicator system: Haemolysis occurs because complement is

free
 Negative test

 First step: Antigen + test serum (negative for antibody) +

guinea pig complement

 Second step: Standard antiserum will react with antigen and

fix the free complement

 Indicator system: No haemolysis because complement is not

free
 Conglutination
 This is an alternative method for systems which do not fix
guinea pig complement

 Uses horse complement which is non-hemolytic

 The indicator system is sheep erythrocytes sensitised with

bovine serum

 Bovine serum contains a a beta globulin component named

conglutinin, which acts as antibody to complement

 Conglutinin can cause agglutination of sensitised sheep

erythrocytes, if these are combined with complement

 No agglutination – Positive result

 Agglutination – Negative result
 Complement dependent serological tests

1. Immobilisation test

2. Immune adherence

3. Cytolytic or cytocidal tests

Neutralisation test

Opsonisation
 Immunofluorescence

 Fluorescence is the property of certain dyes which absorb rays

of one particular wavelength (UV light) and emit rays with a
different wavelength (visible light)

 Coons and his colleagues showed that fluorescent dyes can be

conjugated to antibodies and these labelled antibodies can be
used to detect antigens in tissues

 The commonly used fluorescent dyes are

i) Fluorescin isothiocyanate (Blue green fluorescence)

ii) Lissamine rhodamine (orange red fluorescence)

 Immunofluorescence test is of two types

1. Drect immunofluorescence test

2. Indirect immunofluorescence test

Direct immunofluorescence test
 Uses
1. It is commonly employed for detection of bacteria, viruses or
other antigens in blood, CSF, urine, faeces, tissues and other
specimens

2. It is a sensitive method to diagnose rabies by detection of the

rabies virus antigens in brain smears

Disadvantage
 Separate specific fluorescent labelled antibody has to be
prepared against each antigen to be tested
Indirect immunofluorescence test
Advantages

 A single antihuman globulin fluorescent conjugate can be

employed for detection of antibody to any antigen

 All antibodies are globulin in nature, therefore, antihuman

globulin attaches to all antibodies

 This has overcome the disadvantage of direct immuno-

-fluorescence test
 Radioimmunoassay (RIA)

 Berson and Yallow (1959) first described

 Since then it has been utilised for quantitation of hormones,

drugs, HBsAg, IgE and viral antigens

 Radioimmunoassay is widely-used because of its great

sensitivity

 Using antibodies of high affinity, it is possible to detect a few

picograms (10−12 g) of antigen

 The greater the specificity of the antiserum, the greater the

specificity of the assay
The principle involves competitive binding of radiolabeled Ag
and unlabeled Ag to the limited supply of a high affinity Ab

FIGURE 6-9
A solid-phase radioimmunoassay (RIA) to detect hepatitis B virus in blood
samples & A standard curve to determine the conc. of HBsAg in unknown serum.
 Disadvantages

 Radiation hazards: Uses radiolabelled reagents

 Requires specially trained persons

 Labs require special license to handle radioactive material

 Requires special arrangements for

 Requisition, storage of radioactive material

radioactive waste disposal
 Enzyme Linked Immunosorbent Assay (ELISA)

 Is a biochemical technique used mainly in immunology to

detect the presence of an antibody or an antigen in a sample

 Term was coined by Engvall and Pearlmann in 1971

 Similar to RIA, except no radiolabel

 Very sensitive, pg/mL

 Different types of ELISAs

1. Indirect ELISA

2. Sandwich ELISA

3. Competitive ELISA

4. Cassette or cylinder ELISA

Indirect ELISA

Two commonly used enzymes

1. Horseradish peroxidase (HRP)

2. Alkaline phosphatase (AP)

Sandwich ELISA

 Two antibodies required; must recognize different epitopes

 1st Antibody is referred to as capture Ab

 2nd antibody as detection Ab

Competitive ELISA
 The labelled antigen competes for primary antibody binding
sites with the sample antigen (unlabelled)

 The more antigen in the sample, the less labelled antigen is

retained in the well and the weaker the signal).
 Cassette or cylinder ELISA
 It is a simple modification of ELISA for testing one or few
samples of sera at a time

 The test is rapid (about ten minutes) as compared with the

2 - 4 hours taken for conventional ELISA
 Uses of ELISA
 Used for detection of antigens and antibodies for various
microorganisms

1) Detection of HIV antibodies in serum

2) Detection of mycobacterial antibodies in tuberculosis

3) Detection of rotavirus in faeces

4) Detection of hepatitis B markers in serum

5) Detection of enterotoxin of E. coli in faeces

Western blotting (Immunoblotting)
 Western Blot for detection of HIV antibody

HIV-1 Western Blot

 Lane1: Positive Control
 Lane 2: Negative Control
 Sample A: Negative
 Sample B: Indeterminate
 Sample C: Positive