Transcript Agglutination Reaction

Antigen-antibody
reactions
Let’s start
• The immune system is a system of biological
structures and processes within an organism that
protects against disease.
• To function properly, an immune system must
detect a wide variety of agents, from viruses to
parasitic worms, and distinguish them from the
organism’s own healthy tissue.
Antigens
o Antigens are any substances that are capable, under
appropriate conditions, of inducing the formation of
antibodies and reacting specifically with the antibodies
so produced.
o These antigenic molecules may have several antigenic
determinants, called epitopes, and each epitope can
bind with a specific antibody. Thus, a single antigen
can bind to many different antibodies with different
binding sites
Chemical Nature of Antigens
(Immunogens(
A. Proteins
The vast majority of immunogens are proteins. These may be
pure proteins or they may be glycoproteins or lipoproteins. In
general, proteins are usually very good immunogens.
B. Polysaccharides
Pure polysaccharides and lipopolysaccharides are good
immunogens.
C. Nucleic Acids
Nucleic acids are usually poorly immunogenic. However, they
may become immunogenic when single stranded or when
complexed with proteins.
D. Lipids
In general lipids are non-immunogenic, although they may be
haptens.
Antibodies
• An antibody is a protein produced by the body’s
immune cells “ activated B-lymphocytes” when it
detects a foreign antigen.
• Classes of antibodies:
IgG: The main antibody in blood(70%), made in 2ry
immune response and has a long half life”up to 20
years”.
IgM: accounts for(10%) of Igs, has a key role in 1ry
immune response.
IgA: (20%) of Igs present in serum and secretions.
IgE: (0.001%), involved in allergy and parasitic
infections.
IgD: (1%) present on the surface of B cells, so that it
plays a role in induction of Ab production
Nature of antigen-antibody
reactions
• A. Lock and Key Concept
The combining site of an antibody is located in the Fab portion of the
molecule and is constructed from the hypervariable regions of the
heavy and light chains
• B. Non-covalent Bonds
The bonds that hold the antigen to the antibody combining site are all
non-covalent in nature. These include hydrogen bonds, electrostatic
bonds, Van der Waals forces and hydrophobic bonds.
• C. Reversibility
Since antigen-antibody reactions occur via non-covalent bonds, they
are by their nature reversible.
Affinity and avidity
A. Affinity
Antibody affinity is the strength of the reaction between
a single antigenic determinant and a single combining
site on the antibody.
B. Avidity
Avidity is a measure of the overall strength of binding of
an antigen with many antigenic determinants and
multivalent antibodies.
Specificity and cross reactivity
Specificity
Specificity refers to the ability of an individual antibody combining
site to react with only one antigenic
Cross reactivity
Cross reactivity refers to the ability of an individual antibody
combining site to react with more than one antigenic determinant
Sensitivity
Cross reactivity refers to the ability of an individual antibody to locate
antigen even when it is greatly diluted.
Factors affecting measurement of
antigen-antibody reactions
•
The only way that one knows that an antigen-antibody
reaction has occurred is to have some means of directly
or indirectly detecting the complexes formed between
the antigen and antibody.
• The ease with which one can detect antigen-antibody
reactions will depend on a number of factors.
1. Affinity
The higher the affinity of the antibody for the antigen, the
more stable will be the interaction. Thus, the ease with
which one can detect the interaction is enhanced.
2. Avidity
Reactions between multivalent antigens and multivalent
antibodies are more stable and thus easier to detect.
Factors affecting measurement of
antigen-antibody reactions
3. Antigen to antibody ratio
The ratio between the antigen and antibody influences the
detection of antigen-antibody complexes because the size of
the complexes formed is related to the concentration of the
antigen and antibody.
4. Physical form of the antigen
The physical form of the antigen influences how one detects
its reaction with an antibody. If the antigen is a particulate,
one generally looks for agglutination of the antigen by the
antibody. If the antigen is soluble one generally looks for the
precipitation of the antigen after the production of large
insoluble antigen-antibody complexes.
Types of Antigen – Antibody Reaction
• The types of antigen – antibody reactions are:
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Precipitation Reaction.
Agglutination Reaction.
Complement Fixation.
ELISA – Enzyme Linked ImmunoSorbent Assay
Immunofluorescence.
Agglutination
• Agglutination is the clumping of particles.
• Agglutination occurs if an antigen is mixed with its
corresponding antibody called isoagglutinin.
• This term is commonly used in blood grouping.
Agglutination
This occurs in biology in several examples:
• The clumping of cells such as bacteria or red blood cells in
the presence of an antibody or complement. The antibody
or other molecule binds multiple particles and joins them,
creating a large complex. This increases the efficacy of
microbial elimination by phagocytosis as large clumps of
bacteria can be eliminated in one pass, versus the
elimination of single microbial antigens.
• Another example occurs when people are given blood
transfusions of the wrong blood group.
Characteristics
• Is the aggregation of particulate matter due to
combination with specific antibody.
• Takes place on the surface of the particle and thus
antigen must be exposed and able to bind with
antibody
• Antigens may be:
▫ On a cell (direct agglutination)
▫ Attached to latex spheres (indirect or passive agglutination)
• Agglutination reaction is aided by elevated
temperature (37-56°C) and by movement which
increases the contact between antigen and antibody.
• Clear supernatant.
• Clumps aggregate and settle as large visible clumps.
Steps in Agglutination
1- Sensitization
Involves antigen-antibody
combination through single
antigenic determinants on the
particle surface
Antibody molecules attach to
their corresponding Antigenic
site (epitope) on the red blood
cell membrane. There is no
visible clumping.
2- Aggregative Stage
(lattice formation)
▫ Represents the sum of
interaction between antibody
and multiple antigenic
determinants on a particle
▫ Dependent on environmental
conditions as well as the
relative concentrations of
antigen and antibody
Antibody molecules
crosslink RBCs forming a
lattice that results in visible
clumping or agglutination.
Uses of Agglutination Reactions
1. Aid in the identification, by means of
known antisera (serum containing
antibodies specific for a given antigen),
microorganisms cultured from clinical
specimens.
2. Help estimate the titer of antibacterial
agglutinins in the serum of patients with
unknown diseases.
Types of Agglutination Reactions
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Direct Agglutination
Passive Agglutination
Reverse Passive Agglutination
Agglutination inhibition
Hemagglutination-inhibition
Coagglutination/Conglutination
Direct agglutination
• Occurs when antigens are
found naturally on a particle
(Serotyping of Salmonella)
• e.g. identification of bacterial
types , O antigen (somatic) compact, fine and granular
agglutination , H antigen
(flagellar) - form a loosely
woven network of clumped
cells (coarse and floccular),
called snowflake agglutination
Hemagglutination
• Hemagglutination, is agglutination that
involves red blood cells (RBCs). It has two
common uses in the laboratory:
1. Blood typing
2. Quantification of virus dilutions in
a Haemagglutination assay.
Blood typing
• Blood type can be determined by using antibodies that bind
to the A or B blood group antigens in a sample of blood.
• For example, if antibodies that bind the A blood group are
added and agglutination occurs, the blood is either type A or
type AB.
• To determine between type A or type AB, antibodies that
bind the B group are added and if agglutination does not
occur, the blood is type A.
• If agglutination does not occur with either antibodies that
bind to type A or type B antigens, then neither antigen is
present on the blood cells, which means the blood is type O.
Blood typing
Viral hemagglutination assay
• Hemagglutination phenomenon is almost commonly
used for diagnosis of infection produced by some
viruses.
• The presence of virus in infected cell cultures can be
detected by hemagglutination
• The identity of the virus or of antibodies in a patient’s
serum can be determined by specific inhibition of that
hemagglutination.
Viral hemagglutination assay
• The basis of the HAI assay is that antibodies
to that particular virus (for example-influenza
virus) will prevent attachment of the virus to
RBC. Therefore hemagglutination is inhibited
when antibodies are present.
Hemagglutination and hemagglutination inhibition
Passive agglutination
• Employs particles that are coated with antigens not
normally found on their surfaces.
• Inert materials commonly used:
1. Bentonite
2. Colloidion
3. Latex particles
4. Colloidal charcoal
• Passive agglutination tests have been used to detect
rheumatoid factor and antinuclear antibody.
Reverse Passive agglutination
• Reverse Passive agglutination antibody rather than antigen is
attached to a carrier particle.
• Several kits are available today
for rapid identification of such
antigens from such infectious
agents as group A and B
streptococci, Staph. , Neisseria,
and others.
Passive and reverse passive
Agglutination inhibition
• Agglutination inhibition based on competition
between particulate and
soluble antigens for
limited antibody
combining sites, and a
lack of agglutination is an
indicator of a positive
reaction.
• The classic example of agglutination inhibition is
the early types of home pregnancy test kits
included latex particles coated with human
chorionic gonadotropin (HCG) and antibody to
HCG
• The addition of urine from a pregnant woman,
which contained HCG, inhibited agglutination of
the latex particles when the anti-HCG antibody
was added; thus the absence of agglutination
indicated pregnancy.
Coagglutination/Conglutination
• Coagglutination/Conglutination name given to systems using
bacteria as inert particles to which
antibody is attached (S. aureus).
• The Fc region of antibody
attaches to protein A of
staphylococcal cell leaving the Fab
region to combine with the
antigen
• Killed staphylococcal cells coated
with antibody can be used to
identify bacteria and detect
soluble extracellular bacterial
antigens in specimens and body
fluids.
Antiglobulin mediated agglutination
• The antiglobulin test (Coomb’s test) can be used to detect red
cells sensitized with IgG antibodies
• In order for agglutination to occur an additional of antiantibody , which reacts with the Fc portion of the IgG
antibody
• This will form a "bridge" between the antibodies or
complement coating the red cells, causing agglutination.
Instrumentation
• Several systems were developed to increase sensitivity of
results reading, many of these utilize turbidimetry
1. Particle Enhanced Turbidimetric Inhibition Assay
(PETINA)
2. Particle counting immunoassay (PACIA)
Particle Enhanced Turbidimetric Inhibition Assay
(PETINA)
• This method uses the creation of light
scattering particles to measure drug
levels.
• The latex particle-bound drug binds to
the drug-specific antibody, forming
insoluble light- scattering aggregates.
• This causes an increase in the turbidity
of the reaction mixture.
• Non-particle- bound drug in the
patient sample competes with the
particle-bound drug for antibody
binding sites, inhibiting the formation
of insoluble aggregates.
• Therefore the rate of increase of
absorbance (hence the rate of the
increase in turbidity,) is inversely
proportional to the concentration of
the drug.
Particle counting immunoassay
(PACIA)
• Involves measurement of the number of residual
non-agglutinating particles in a specimen.
• Latex particles are coated with whole antibody
molecule, if antigen is present complexes will form
and will screened out by counter .
• An inverse relationship exists between the number
of unagglutinated particles counted and the amount
of unknown in the patient specimen.
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Qualitative Agglutination Test
• Agglutination tests can be used in a qualitative
manner to assay for the presence of an antigen or
an antibody.
• The antibody is mixed with the particulate
antigen and a positive test is indicated by the
agglutination of the particulate antigen.
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Quantitative Agglutination Test
• Agglutination tests can also be used to quantitate
the level of antibodies to particulate antigens.
• In this test
▫ one makes serial dilutions of a sample to be tested for
antibody
▫ and then adds a fixed number of red blood cells or bacteria
or other such particulate antigen
▫ and determines the maximum dilution, which gives
agglutination.
▫ The maximum dilution that gives visible agglutination is
called the titer.
▫ The results are reported as the reciprocal of the maximal
dilution that gives visible agglutination. This can be done
using a microtiter plate.
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Neg.
Pos.
1/1024
1/512
1/256
1/128
1/64
1/32
1/16
1/8
1/4
Patient
1/2
Quantitative Agglutination Test
Titer
1
2
3
64
8
512
4
5
<2
32
6
7
8
128
32
4
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Determining Antibody titer
• Titer is the quantity of a substance required to
produce a reaction with a given volume of another
substance.
• Antibody titer is the highest dilution of the biological
sample that still results in agglutination, with no
agglutination being observed at any higher dilution.
• The term is used in serological reactions and is
determined by preparing serial dilutions of antibody
to which a constant amount of antigen is added.
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Determining Antibody titer
Post
Zone
Equivalence
Zone
Prozone
Serum Dilution
1:10
1:20
1:40
1:80
1:160
1:320
1:640
1:1280
1:2560
1:5120
Antigen Conc.
X
X
X
X
X
X
X
X
X
X
Agglutination
–
–
+
++
+++
++++
+++
++
+
–
Advantages and disadvantages
Advantages of agglutination tests:
1. Low individual test cost.
2. Ability to obtain semi quantitative results.
3. Short time to obtain result.
4. Don’t need expensive instrument.
5. Agglutination of insoluble native antigens or antigencoated particles simple to read with or without the aid
of a microscope
6. Increased degree of sensitivity
7. Great variety of detectable substances
8. If the sample contain micro-organisms, it does not need
to be viable
Agglutination Requirements
• Availability of stable cell or particle suspension
• Presence of one or more antigens close to the
surface
• Knowledge that ‘incomplete’ or nonagglutinating
antibodies are not detectable without
modifications, e.g. antiglobulin (Coomb’s)
technic
Advantages and disadvantages
Disadvantages of agglutination tests:
1. It must kept in mind that agglutination reaction are
screening tests only, and negative result doesn't
rule out disease; the quantity of antigen or antibody
may be below the sensitivity of the test system
2. May give false positive or negative results
False positive results in agglutination reactions
• Overcentrifugation
▫
Button is packed too tight and is difficult to resuspend.
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Dust, dirt or fingerprints may cause cells to clump.
▫
Test cells clump without specific AB present, mainly
problematic with RBCs.
• Contaminated Equipment
• Autoagglutination
• Delay in Reading Tests
▫ Dried out Ag may look like agglutination
• Saline Stored in Glass Bottles
▫ Colloidal Silica may leach out and cause agglutination
• Using plasma instead of serum, hemolysed or lipemic
sample.
• Cross reaction
False negative
• Undercentrifugation
Cells may not be close enough to interact
• Inactive Reagents
May cause improper storage
• Incorrect Incubation Temperature
May result in the lack of association for AGs and ABs
• Insufficient Incubation Time
AGs and ABs may not have time for association
• Prozone Phenomenon
Too much patient AB for amount of testing
• Delay in Testing Procedure
AB may be eluted from RBCs
Experiment 1
Determination of the titer of blood
group cold antibody
Materials
1. Isotonic saline.
2. Volumetric pipettes, or equivalent: 0.1- to 0.5mL delivery, with disposable tips.
3. Red cells with the antigen to be tested.
4. The tested serum.
Procedure
1.
Using 0.5-mL volumes, prepare serial twofold dilutions of
serum in saline. The initial tube should contain undiluted
serum and the doubling dilution range should be from 1 in 2 to
1 in 2048 (total of 12 tubes).
2.
Place 0.1 mL of each dilution into appropriately labeled test
tubes.
3.
Add 0.1 mL of the 2% suspension of red cells to each dilution.
4. Gently agitate the contents of each tube; incubate for 15 min.
5. Centrifuge for 15 sec.
8. Examine the red cells macroscopically; grade and record the
reactions.
Preparation of serial dilution
Tube #
1
2
3
4
5
6
7
8
Dilution
1/2
1/4
1/8
1/16
1/32
1/64
1/128
1/256
Serum
500
500
500
500
500
500
500
500
500
500
500
500
500
500
500
Saline
Results
Interpretation
• The titer is reported as the reciprocal of the highest
dilution of serum at which 1+ agglutination is observed.
Exercise
Preparation of serial dilution
Dilutions
◌Dilution is decreasing the concentration of a
solution by a calculated factor using an approved
diluent.
◌As well, dilution is used to prepare samples,
buffers, and controls.
◌In serology tests it is Used to detect the titer of a
specific Ab.
◌When a strong positive reaction is encountered,
dilution should be made to detect the titer.
Volumes used in dilutions mustn't be below 10ul or more
than 1000ul .
◌ for example: (1:4)
◌ 1 express the volume of sample
◌ 4 express the total volume( sample + reagent)
◌ This is done by mixing 100 ul of sample with 300 ul of reagent.
◌ Serial dilution :
100ul
sample
100ul
100ul
From 1
From 2
1
2
3
300
ul
300
ul
300
ul
saline
saline
1:4
1:16
sample
Total Dillution
=1/4 * 1/4
Dillution of tube 1
saline
1:64
=1/4 * 1/16
Dillution of tube 2
 Another example on serial dillution :
100ul
sample
100ul
100ul
From 1
From 2
1
2
3
900ul
saline
900ul
saline
900ul
saline
1:10
1:100
sample
Total Dillution
= 1/10 * 1/10
Dillution of tube 1
1:1000
=1/10 * 1/100
Dillution of tube 2
• 9:250??
It can be done only by serial dilution…
9:25 * 1:10
9 : 250
9:25 * 1:10
100ul
From 1
90ul
1
2
160ul
900ul
sample
Total Dillution
9:25
1:10
9:25
0