Different types of Ag- Ab

reactions

DR.SYADA MONIRA HOQUE

Types of antigen- Antibody
reactions in vivo

1. Agglutination
2. Precipitation
3. Complement fixation
4. Neutralization
5. Antibody dependant cell mediated
cytotoxicity (ADCC)
6. Immobilization
Types of antigen antibody
reactions used in vitro
► 1.Agglutination
► 2.Haemagglutination
► 3.Precipitation:
► a. Precipitation in solution
► b. Precipitation agar:
►Single(SRID)
►Double diffusion(Ouchterlony)
► c.Precipitation in agar with an electric
field
►Immunoelectrophoresis.
►Counter-immunoelectrophoresis
► 4. Radioimmunoassay(RIA)
► 5. Enzyme linked immunosorbent assay
(ELISA)
► 6. Immunofluorescence
► 7. Complement Fixation Test(CFT)
► 8. Neutralization test
► 9. Immunoblotting(western blot)
Agglutination
► Theterm agglutination came from glu-
which means adhesion
► Theact of adhesion of different parts is
agglutination
► When an antibody reacts with a
multivalent particulate (insoluble)
antigen, lattice formation occurs due to
cross linking of various antigen particles
by the antibody
Types of Agglutination
► Direct agglutination
1. Slide – Blood grouping, Serotyping of bacteria
2. Tube –Widal test (Classical)
► Indirect or Passive agglutination
1. Hemagglutination
2. Latex agglutination
3. Particle agglutination
4. Co-agglutination
► Flocculation tests
► Coombs test
► Direct – to detect antibody bound to fetal RBC
surface
► Indirect – To detect circulating antibody in
serum in mother
Advantages and disadvantages
of agglutination

► Advantages
► Most widely used
► Very simple
► No instrument is required
► Cheap

► Fairly sensitive

► Disadvantages
► Not highly specific
► Not highly sensitive
 Direct agglutination

►Occurs when the

antigenic
determinant is
inherent to the
particle itself.
(naturally)
►Example #1 –
Using group A rbc’s
to detect anti-A in
serum
Direct agglutination..2

► Example # 2 – Using
bacteria (Ag) looking
for Ab in serum
Indirect or Passive agglutination
• Results when inert
particles are coated with
soluble Ags which may
react with Ab. Particles
include latex, rbc’s,
charcoal, etc.
• Example – Ag attached to
latex particle (known) +
serum looking for
(unknown) Ab. If Ab
present, you get visible
agglutination.
Passive
Agglutination/Hemagglutination
► Definition - Agglutination test done with a
soluble antigen coated onto a particle

• Applications
– Measurement of antibodies to soluble antigens
Latex agglutination
►In latex agglutination
procedures, Ag
molecules can be
bound to the surface of
latex beads
►If Ab is present in the
test specimen, the Ag
will combine with the
Ab and form visible
aggregates
Latex agglutination

►Latex particles can

be coated with Ab,
and in the presence of
Ag can form visible
aggregates
Hemagglutination

►Agglutination of rbc’s
as a result of Ab
interaction with
antigenic determinants
on rbc’s surfaces
►Example – using group
A rbc’s to detect anti-A
in serum
 Coombs (Antiglobulin)Tests
• Incomplete Ab
• Direct Coombs Test
– Detects antibodies on erythrocytes in fetus

Patient’s RBCs Coombs Reagent

(Antiglobulin)
 Coombs (Antiglobulin)Tests
► Indirect Coombs Test
► Detects anti-erythrocyte antibodies in serum in
mother

Step 1
+
Patient’s Target
Serum RBCs

Step 2

+
Coombs Reagent
(Antiglobulin)
Coombs (Antiglobulin)Tests
► Applications
► Detection of anti-Rh Ab
► Autoimmune hemolytic anemia
Flocculation tests

► Flocculation tests for Ab detection are

based on the interaction of soluble Ag with
Ab, which results in the formation of a
precipitate of fine particles. (Ag consists of
lipid type particles)
► Examples VDRL & RPR’s
Precipitation
► When soluble antigens and antibodies are mixed
together at optimum concentration, lattice formation
occurs
► In this test, the antigen is in solution. The antibody cross-
links antigen molecules in variable proportions, and
aggregates (precipitates) form. In the zone of
equivalence, optimal proportions of antigen and
antibody combine; the maximal amount of precipitates
forms, and the supernatant contains neither an excess
of antibody nor an excess of antigen. In the zone of
antibody excess, there is too much antibody for efficient
lattice formation, and precipitation is less than maximal.2
In the zone of antigen excess, all antibody has
combined, but precipitation is reduced because many
antigen–antibody complexes are too small to
precipitate; i.e., they are "soluble
Types of precipitation
1. Precipitation in gel
■ Single radial immunodiffusion
■ Double diffusion
2. Precipitation in Electrophoresis
■ Immune electrophoresis
■ Counter current Immune electrophoresis (CIE)
Advantages and disadvantages
of precipitation

► Advantages
► Fairly sensitive
► High specificity

► Disadvantages
► Time consuming
► Some costly instruments are required
► High technical skill required
Complement
fixation test (CFT)

LATTICE FORMATION NOT REQUIRED

CFT

► Principle: Antigen- antibody (IgG,

IgM) complex activates the
complement which can lyse
target (RBC)
► Components of test:
1. Sensitised sheep RBC (Sheep RBC+
Anti sheep RBC)
2. Complement- (Guniea pig serum)
3. Known Ag / known Ab
Complement Fixation Reaction
• Antibody titer may be too low for agglutination/precipitation

• Can detect presence based on ability to deplete complement from

serum (complement fixation)

• Antigen added to serum with complement

• If antibodies against antigen present, activates and depleted

complement

• Detected by addition of opsonized SRBC

• If complement not depleted, SRBC are lysed

Steps of CFT:

1. Heat inactivate the test serum (to detect presence

or absence of Ab) to get rid of the native
complement (560 C for 30 minutes)
2. Then add measured amounts of Ag (known) and
complement (known), to the serum (unknown Ab)
3. If Ab specific for the known Ag is present in the
serum, Ag-Ab complexes will form and bind all
complement. (reaction is invisible)
►Steps…
► If Ab (unknown) specific for the known Ag is not
present in the serum, then the known Ag and
complement remain unbound.
► Indicator system: add sheep rbc’s coated with
known Ab specific for known Ag
► Results:
► If all of the complement has been fixed, none will
be free to lyse the sheep rbc’s. (No hemolysis,
indicates a positive complement fixation test;
positive for the unknown Ab in the serum)
► Interpretation of CFT
► If no Ab is present in the patients serum, the complement is
not fixed and is free to interact in the indicator system and
lyse the rbc’s. (Hemolysis indicates a negative test; negative
for the unknown Ab in the patients serum. The only things
reacting are the knowns)

► Ag/Ab/C + Ab-coated rbc’s = no hemolysis

(positive)
► Ag/C + Ab-coated rbc’s = hemolysis
(negative)
Complement fixation test

► Pos ► Neg
Advantages and disadvantages of CFT

► Uses
► CFT for kalazar, Filaria
► Gonoccal CFT
► CFT for many viral infections
► Advantages
► Fairly sensitive
► Wide application- can be used for variety of
diseases
► Disadvantages
► Time consuming
► Very difficult to standardize
► High technical skill required
 Complement Fixation
• Methodology
► Ag mixed with test serum to be assayed for Ab

– Erythrocytes coated with Abs is added

– Amount of erythrocyte lysis is determined

Ag No Ag
Ag
Patient’s
serum
Ag
Radioimmuoassays (RIA)
Enzyme-Linked Immunosorbent Assays
(ELISA)
 Detection principles

• Radiolabelled isotopes
– 125I, 14C, 32P, 35S

• Enzymes
– Peroxydase
• Chromophores
– Fluorogenic probes, fluorescent proteins
Improved Diagnostics

► Radioimmunoassay: A very sensitive, specific

laboratory test (assay) using radiolabeled
(and unlabeled) substances in an
immunological (antibody-antigen) reaction.
RIA: radio immuno assay
 ELISA Formats
Direct sandwich ELISA – antibodies (Ab) are
coated to micro wells. Antigen (Ag) is added and
binds with antibody. Excess antigen is washed away.
Enzyme conjugate (Ab-E) is added and binds with
antigen to form the double antibody sandwich. Wells
are washed to remove any excess (Ab-E). Substrate is
added and color development is observed. The
enzyme conjugate binds ‘directly’ to the antigen.

Ab + Ag + Ab-E
 Types of ELISA

• Three different methods used to perform ELISAs

• Direct method (different from book)

• Indirect method

• Capture method (called direct method in

book)
 ELISA 38

► Patient’s serum containing unknown Ag(or Ab) is added

to known Ab(or Ag), incubated(eg.1 hour) for Ag/Ab
reaction and washed for removing excess Ag/Ab.
► Ab-labelled with an enzyme (e.g. HRP)is
added,incubated(eg.1h) and then washed
► A chromogenic substrate is added, incubated(e.g.½
h), stopping reagent added and colour development is
measured by a spectrophotometer
► Example: for HBsAg, AntiHBs, Anti-HAV, AntiHIV etc.
Sandwich Elisa
 Components
Enzyme:
•Alkaline phosphatase
•Horse radish peroxidase

•Substrate :
•Hydrogen peroxide
Western blot
41

► Proteins are separated electrophoretically in to a gel,

resulting discrete bands of protein formed
► These proteins are transferred from the gel or blotted
from the gel in to filter paper
► Patient’s serum is added
► If Abs present against these protein in patient’s serum
they bind with viral protein
Western blot
42
► These can be detected by adding antibody to human IgG
tagged with enzyme .
► Enzyme substrate added ,
► Visible color change appear.
► Discrete band appear
Use: confirmatory test for HIV if patient having screening test
ELISA is positive. Detection of HIV protein specially gp1
and p24 done by this method
Immunofluorescence
43

► Fluorescent dyes, e.g., fluorescein and

rhodamine, can be covalently bind with Ab
molecules and made visible by UV light in
fluorescence microscope. Such “labeled” Ab can
be used for detection of Ag
► Two type
► 1. direct
► 2. indirect