Chapter 16

Laboratory Methods
Used In The Detection
of Immunological
Response (Antigen-
antibody Reactions)

Dr Sonal Saxena
Dr Arpita Saxena
 Combination of antigen and antibody in a
specific and observable manner is called an
antigen-antibody reaction
Three-stage reaction
― Primary stage: Initiation of reaction
INTRODUCT without any visible effect
ION ― Secondary stage: Demonstrable events
like agglutination, precipitation,
phagocytosis, etc.
― Tertiary stage: Initiation of chain reaction
causing neutralisation or destruction of
injurious antigens or tissue damage.
INTRODUCTION
Antigens and antibodies bind (Ag-Ab) by forming the non-covalent bonds which are as
follows:
Electrostatic forces
Hydrogen bonds
Van der Waal’s forces
Hydrophobic forces

Table 16.1 Comparative efficiency of the

immunoglobulin classes in different serological
reactions
GENERAL FEATURES AG-AB REACTION
Reaction is specific

Entire molecule is involved

No denaturation of the antigen or the antibody

Combination occurs at the surface

Combination is firm but reversible

The firmness of the bond is determined by

◦ Affinity
◦ Avidity

Participation of both antigen and antibody

Can combine in varying proportions

 Antigen–antibody reactions in vitro are known as
serological reactions.
• Sensitivity: Ability of a test to detect even
GENERAL minute quantities of an antigen or antibody.
False-negative results are minimal in high
FEATURE sensitivity.
S AG-AB • Specificity: Ability of a test to only detect
reactions between homologous antigens and
REACTIO antibodies and no other. In a highly specific
N test, false-positive reactions are absent or
minimal; only true-positive detected.
In general, sensitivity and specificity are inversely
proportional.
 Qualitative assays: Detection of
the mere presence of an antigen
or antibody
 Quantitative assays: Estimation of
the quantity of an antigen or
antibody
 Precipitation
When a soluble antigen combines with its antibody
REACTIONS ON in the presence of electrolytes (NaCl) at a suitable
WHICH temperature and pH, the antigen–antibody complex
ANTIGEN- forms an insoluble precipitate
ANTIBODY
May be qualitative or quantitative test
ASSAYS ARE
BASED Sensitive for the detection of antigens
A.
PRECIPITATION Flocculation
REACTIONS When, instead of sedimenting, the precipitate
remains suspended as floccules, the reaction is known
as flocculation
PRECIPITATION
REACTIONS
Amount of precipitate formed depends on the
amount of antigen and antibody
 Prozone phenomenon: Excess antibody
 Zone of equivalence: Optimum proportions
 Post-zone phenomenon: Excess antigen
Mechanism: Lattice formation with alternating
antigen and antibody molecules

Fig. 16.1 Lattice hypothesis—as antigen

is added to a solution containing a fixed
amount antibody, the amount of
precipitin increases as the antibody-to-
antigen ratio approaches the equivalence
zone and decreases once the proportion
of antigen exceeds the optimal ratio
 Types:
1. Immunodiffusion (precipitation reaction in gel)
- To detect antigens/antibodies
PRECIPIT - To detect purity of an antigen
ATION 2. Modifications of the immunodiffusion test:
REACTIO - Radial immunodiffusion
- Double diffusion in two dimensions (Ouchterlony
NS procedure)
3. Immunoelectrophoresis
PRECIPITATION
REACTIONS

Single diffusion in two dimensions and double

diffusion in two dimensions (Ouchterlony
technique)
PRECIPITA
TION
REACTION
S
F IG . 1 6 .3
IM M U N OE L E C T R OP H OR E S IS
 APPLICATIONS OF PRECIPITATION REACTIONS
• Grouping of streptococci by the Lancefield’s technique
• VDRL test for syphilis
• To standardise toxins and toxoids
• To test toxigenicity in diphtheria bacilli
• Forensic application in the identification of blood and
seminal stains
• Testing for food adulterants
 Agglutination: When a particulate antigen is
mixed with its antibody in the presence of
electrol ytes at a suitable temperature and
pH, the particles become clumped or
agglutinated
AGGLUTINA ◦ Slide agglutination
TION ◦ Tube agglutination
REACTION Incomplete’ or ‘monovalent’ antibodies do
not cause agglutination

- They act as ‘blocking’ antibodies, inhibiting

agglutination by the complete antibody added
subsequently
AGGLUTINATI
ON REACTION
 1. Agglutination on a slide
- Identification of bacterial isolates
- Blood grouping and cross-matching
2. Agglutination in tubes
-
AGGLUTINA -
Serial dilutions of the serum
Febrile agglutination tests
TION - Limitations of agglutination tests
REACTION - Agglutination test for brucellosis: prozone
phenomenon
- Incomplete or ‘blocking’ antibodies:false
negative results
- Cross-reacting/heterophile antigens: false
positive results
 ANTIGLOBULIN (COOMBS) TEST

Detects anti-Rh antibodies that do not

agglutinate with Rh-positive erythrocytes in
saline.
Principle
 Incomplete anti-Rh antibodies
 Coats the surface of the erythrocytes but
do not agglutinate

Fig. 16.4 (a) slide agglutination, (b) tube

agglutination and (c) blood grouping
 • It is washed free of all unattached
protein and treated with a rabbit
antiserum against human gamma
globulin, the cells agglutinate
ANTIGLOBU • Two types:
LIN i) Direct Coombs test
ii) Indirect Coombs test
(COOMBS) • Uses of Coombs test
TEST  Detection of anti-Rh antibodies
 Demonstrating any type of
incomplete or non-agglutinating
antibody, as, for example, in
brucellosis
 Passive agglutination test: When a soluble antigen is
attached to the surface of carrier particles, it is possible
AGGLUTINA to convert precipitation tests into agglutination tests
Examples : Hemagglutination
TION Latex agglutination
REACTION Reversed passive agglutination
Co-agglutination test
• The ability of the antigen–
antibody complexes to ‘fix’
complement is made use of
in the CFT
• Used for the detection of
both antibodies and antigens
• Requires five reagents:
Antigen, antibody,
complement, sheep
erythrocytes and
amboceptor (rabbit antibody
to sheep red cells)

Fig. 16.5 Complement fixation test

COMPLEMENT FIXATION
TEST (CFT)
COMPLEMENT FIXATION TEST (CFT)

Applications of complement fixation test

 Detect and quantify an antibody that does not
agglutinate or precipitate with its antigen
 Detect low levels of antibody (less than 1 μg per
millilitre) during early infection
 Screen numbers of viral or bacterial infections to
detect IgG antibodies (for some respiratory virus and
Coxiella burnetii antibodies, and occasionally
antibodies from CSF)
NEUTRALISATION TESTS

Based on the action of antibodies to neutralise an end product or

the outcome of the action of an antigen
Types:
◦ Virus neutralisation tests: E.g., plaque inhibition test
◦ Toxin neutralisation: Schick test (in vivo)
Anti-streptolysin O
Nagler’s reaction
RADIOIMMUNOASSAY (RIA)
RIA is a competitive binding assay in which fixed
amounts of antibody and radiolabelled antigen react in
the presence of an unlabelled antigen
Applications in quantitation of
– Hormones
– Drugs
– Tumour markers
– IgE
– Viral antigens
• Limited use due to radioisotope hazard
EIA : Enzyme-labelled antigens and antibodies are
used as serological reagents for the assay of
antibodies and antigens

EIAs are of two basic types

1) Homogeneous:

• Do not require the bound and free fractions to be separated;

the test can be done in one single step
• For example, enzyme-multiplied immunoassay technique (EMIT)

ENZYME IMMUNOASSAY
(EIA)
 2) Heterogeneous: Require the
ENZYME separation of the free and bound
IMMUNOA fractions either by centrifugation or
SSAY (EIA) by adsorption on solid surfaces and
washing
ENZYME-LINKED IMMUNOSORBENT ASSAY (ELISA)

• An immunosorbent—an absorbing material

specific to one of the components of the reaction,
i.e., the antigen or the antibody
• Enzyme–substrate conjugates

• Enzyme converts colourless substrates to a

coloured product bound to the antibody
TYPES OF
ELISA
Indirect ELISA
Competitive ELISA
Sandwich ELISA
Capture ELISA
Cylinder or cassette ELISA
Enzyme-linked immunospot assay (ELISpot)
IgG avidity ELISA
 ENZYME-LINKED
IMMUNOSORBENT
ASSAY (ELISA)
16.6The indirect ELISA is used to quantify
antigenspecific antibodies in patient serum for
disease diagnosis serum. (Source:
https://openstax.org/books/microbiology/
pages/20-4-eias-and-elisas)
TYPE
S OF
ELISA
FIG. 16.7
M O D I F I C AT I O N S O F
ENZYME-LINKED
IMMUNOSORBENT
A S S AY ( E L I S A )
 USES OF ELISA
Diagnosis of Infectious diseases like
• Hepatitis, EBV, cytomegalovirus IgM/IgG, dengue IgG,
influenza, TORCH panel, hepatitis B, C, etc.
• Rotavirus detection in fecal specimens and enterotoxin of E.
coli in feces
• Syphilis IgG/IgM, H. pylori IgG and antigen detection
• Food toxins like chloramphenicol, streptomycin, penicillin,
aflatoxins, etc.
• Food adulterants including E. coli, Campylobacter and
Salmonella antigens
• Human allergen-specific IgE and IgA ELISA
 Chemiluminescence immunoassay (CLIA)
Immunoelectroblot or western blot
Immunochromatographic assay/lateral flow
OTHER assay
IMPORTANT
IMMUNOLOG Immunoelectron microscopic assays
ICAL ASSAYS Immunofluorescence assay
Immunohistochemical techniques
Flow cytometry
FIG. 16.10
L AT E RA L F LO W
I M M U N O A S S AY
 OTHER IMPORTANT
IMMUNOLOGICAL ASSAYS
OTHER
IMPORTANT
IMMUNOLOGI
CAL ASSAYS
Fig 16.12 A green fluorescent
monoclonal Ab against L.
pneumophila is used to
visualise and identify bacteria
from a smear of a sample
from the respiratory tract of a
pneumonia patient
 • Fluorescence technique to identify
and enumerate cells bearing a
particular antigen(s) or the surface
markers by suspending them in a
stream of fluid and passing them
through an electronic detection
apparatus
FLOW • Differentiated based on size and
CYTOMET shape using forward and right-
RY angled light scatter.
• Applications of flow cytometry
 Size, granularity, DNA or RNA
content, cellular antigens, receptor
levels of cells
 Wide use in research and
diagnostics
DETECTION OF CELL
MEDIATED IMMUNITY (CMI)
• T cell counts
• T cell subsets
• Skin test is used for delayed
hypersensitivity
• Lymphocyte transformation
test
• Target cell destruction
• Migration inhibitory factor
test
Fig 16.14 Detection of cell-mediated
immunity (CMI) by leukocyte
migration inhibition test
Table 16.4 Summary of common immunological tests
performed in a clinical laboratory