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Dr.

Yomna Hashem
Serology
The branch of immunology that traditionally deals with in vitro
diagnostic testing of serum.
Antibodies formed during an immune reaction are important in
combating infection, but they hold additional practical value.
Characteristics of antibodies (such as quantity or specificity) can
reveal the history of patients contact with microorganisms or
other antigens.

Plasma: is the pale yellow liquid component


of blood that normally holds the blood cells.

Serum: is the blood plasma not including


the fibrinogens.
Detection of unknown antibody can be done using known antigen
while detection or identifying unknown antigen can be done using
antibody of known specificity.

In serological diagnosis of disease, a


An unknown microbe is mixed with
blood sample is tested for the
serum containing antibodies of known
presence of antibody using an antigen
specificity, a procedure known as
of known specificity.
serotyping.
Specificity: is the property of a test to focus upon only a
certain antibody or antigen and not to react with unrelated or
distantly related ones.
Sensitivity: means that the test can detect even very
small amounts of antibodies or antigens that are the targets
of the test. Abs
Ag1
Ag2

Ab for Ag2
Ags

This test shows specificity in which Sensitivity is demonstrated by the fact


an antibody (Ab) attaches with that Ab can detect antigens even when
great exactness with only one type the antigen is greatly diluted.
of antigen (Ag)
Visualizing Antigen-Antibody Interactions
The primary basis of most tests is the binding of an antibody (Ab) to an
antigen (Ag). Tests involve some type of endpoint reaction visible to the
naked eye or with regular magnification
In the case of large antigens such as cells, Ab binds to Ag and creates
large clumps or aggregates that are visible macroscopically or
microscopically. Smaller Ab-Ag complexes will require special indicators
in order to be visualized.
The use of instruments for the measurement of the reaction increases
the sensitivity of the results e.g. Florescence, colorimetric or radiometry

An antigen-antibody reaction can be used to read a titer, or the quantity


of antibodies in serum. Titer is determined by serially diluting the sample
and mixing it with antigen. It is determined by observing the highest
dilution of serum that produces a visible reaction with an antigen.
Types of Antigen Antibody Reactions
Agglutination (Particulate Antigen e.g.: Bacteria or RBCs )
Precipitation ( Soluble Antigen )
Complement Fixation
Neutralization
Toxin-Antitoxin Neutralization
Virus Neutralization
Reaction revealed using suitable Marker
Immunofluorescence
Enzyme-linked Immunosorbent assay
Radioimmunoassay

The result of the reaction depends on: physical form of the antigen,
its concentration, the quality of antibody and its concentration,
temperature and pH.
Describes the reaction between the insoluble or particulate antigen
(e.g.. microbes, RBCs, latex, etc.) and soluble antibodies.
Antibodies are usually of the IgG or IgM class.
When antigen is mixed with specific antibody, cross-linking of Ag
particles will occur visible clumps which will then sediment.

+
Antigen Antibody

clumps
Direct agglutination
1 - Testing a patients serum for the presence of Abs to
a particular bacteria.
2 Identifying bacteria by specific Abs.

Passive agglutination
Agglutination reaction that takes place between
Abs and a soluble Ag that has been attached to an
insoluble particle e.g.. latex.
Haemagglutination reaction
Hemagglutination reactions involve agglutination
reactions using red blood cells.
classification of Blood based on the presence or absence
of inherited antigenic substances on the surface of red blood cells
(RBCs). The two most important ones are ABO and the RhD
antigen; they determine someone's blood type (A, B, AB and O,
with +, or Null denoting RhD status).

During blood transfusion, testing blood from both donors and


recipients must be done to ensure that every individual recipient is
given blood that is compatible and is as safe as possible. If a unit of
incompatible blood is transfused between a donor and recipient, a
severe acute hemolytic reaction with hemolysis (RBC
destruction), renal failure and shock is likely to occur, and death is
a possibility. Antibodies can be highly active and can attack RBCs
and bind components of the complement system to cause massive
hemolysis of the transfused blood.
ABO blood group system
The ABO system is the most important blood-group system in human-
blood transfusion. The associated anti-A and anti-B antibodies are usually
IgM, antibodies.
Rh blood group system
Direct Coombs test
For diagnose of Erythroblastosis foetalis in which the RBCs of the
newborn are coated with an incomplete antibody IgG antibody (i.e.
anti-Rh antibody) leading to Condition of a low count of RBCs
caused by immune system lysis or breaking of RBC membranes.
The individual's blood contain IgG antibodies that can specifically bind to
antigens on the RBC surface membrane, RBCs can become coated with
IgG antibodies. Complement proteins may subsequently bind to the
bound antibodies and cause RBC destruction.

The direct Coombs test is used to detect these antibodies that are bound
to the surface of red blood cells; a blood sample is taken and the RBCs are
washed (removing the patient's own plasma) and then incubated with
antihuman globulin (also known as "Coombs reagent"). If this
produces agglutinatin of RBCs, the direct Coombs test is positive, a visual
indication that antibodies are bound to the surface of RBCs.
Indirect Coombs test
The indirect Coombs test is used in prenatal testing of
pregnant women, and in testing blood prior to a blood
transfusion. It detects antibodies against RBCs that are present
unbound in the patient's serum. In this case, serum is extracted
from the blood sample taken from the patient. Then, the serum
is incubated with RBCs of known antigenicity; that is, RBCs with
known reference values from other patient blood samples. If
agglutination occurs, the indirect Coombs test is positive
It is done to detect circulating free incomplete antibody in the
serum of Rh-negative mothers sensitized by Rh antigens.
Bacterial Agglutination (Direct agglutination)

Widal test is a tube agglutination test employed in the serological


diagnosis of enteric fever (typhoid).
Principle: Patients suffering from enteric fever would possess antibodies
in their sera which can react and agglutinate serial dilutions of Salmonella
antigens in a tube agglutination test.

Cellular Ags of Salmonella are prepared from attenuated or


killed bacteria
Salmonella antigens are :
O somatic Ag of S. typi
H Flagellar Ag of S. typi
AH Flagellar Ag of paratyphi A
BH flagellar Ag of paratyphi B
The Widal test is positive if TO antigen titer is 1:160 or more indicating an
active infection.
if TH antigen titer is 1:160 or more indicates past infection or in
immunized persons.
Passive Agglutination (Agglutination Inhibition Reactions)

During pregnancy the hormone chorionic gonadotrophin is excreted


in urine.
Kits commercially available contain two reagents; one is a suspension
of HCG-coated latex particles, and the other is a solution of HCG
antibodies.
One drop of the urine is mixed with one drop of antibody solution for
one minute on a black glass slide. One drop of the HCG-coated latex
particles are added to the slide and left for one minute.
If the level of HCG is too low, the antibodies will remain to agglutinate the
HCG-coated latex particles. If agglutination occurs, the subject is not
pregnant.
If the level of HCG is high, the HCG will bind to the antibodies, and thus
no agglutination with the HCG-coated latex particles occurs. If no
agglutination occurs, the subject is pregnant
Certain viruses, e.g.. Influenza virus) can agglutinate RBCs
spontaneously. Detection of the presence of anti-viral antibody
in the serum can be done by adding the patients serum to the
virus. If antibody is present, it will react with the virus and inhibits
the viral ability to haemagglutinate RBCs added to the reaction
mixture.

Influenza virus + Red blood cells (RBCs) haemagglutination

(Influenza virus + antibody) + RBCs Haemagglutination


inhibition (no haemagglutination.)
Haemagglutination
Inhibition
Describes the reaction between soluble antigen and soluble antibodies.
Antibodies are usually of the IgG or IgM class.
The antigen is soluble, when it is mixed with the antibody precipitation
will occur when they are at their optimum concentrations.
If either the antibody or the antigen is present in excess, the
precipitation is not complete.

Types of Precipitation reactions


Precipitation in Solution
Ring Test

Precipitation in Agar Gel


Double Diffusion
Radial Immunodiffusion
Immunoelectophoresis
Simply can be done by slowly layering
small volume of soluble antigen over the
"Ab" solution, precipitation will occur at
the interface (ring test). Applied for typing
of Streptococci and Pneumococci.
plate of agar containing wells (holes) cut into the agar.

The antiserum is incorporated in the agar and antigens in the


wells.
As the antigen and antibody diffuse toward each other, a
precipitate forms which is visible as a definite ring in the agar.
This ring represents the immunocomplex that formed at equivalence
zone
Cutting wells in a gel (agar or agarose); one well contains the
antibody and the other well contains the antigens.
Antigens and antibodies diffuse towards each other
Ag and Ab form a line of precipitation (precipitin line) where they
meet at equivalence zoon after incubation time.
It is very widely used, especially for showing identity, partial
identity (cross-reaction), or non-identity of different antigenic
preparations.
Figure1: The precipitation
lines have fused
completely showing the
presence of identical
determinants between the
two Ags.
Figure2: Ag A is different
from Ag B and both react
with the Abs, the precipitin
lines cross and a double spur
is formed; this is a line of
nonidentity determinants.

Figure3: Ag A and Ag
B share a common
determinants but are
not exactly the same,
a single spur is
formed. This is the
line of partial identity.
It is used to test for toxigenicity of C. diphtheriae.

filter paper strip impregnated with diphtheria


antitoxin is placed on the surface of a special
1
agar plate. Strains to be tested, are streaked
on the agar's surface in a line across the
plate, and at a right angle to the antitoxin 2
paper strip. After 24 hours of incubation at
37, plates are examined with transmitted
3
light for the presence of fine precipitin lines
at a 45-degree angle to the streaks. The
presence of precipitin lines indicated that the
strain produced toxin that react with the
antitoxin.
Immunoelectophoresis
When a large number of different antigens
are present in solution, it is difficult to
separate the precipitation bands for each
"Ag"-"Ab" reaction by simple gel diffusion.
A combination of the electrophoretic
mobility and the diffusion is applied. A
glass plate is covered with a thin layer of
gel and the antigen (e.g. serum) is placed
in a small cup cut in the gel. A direct
current is applied for one hour. The serum
components will migrate by
electrophoresis, but are not seen. A trough
is cut longitudinally in the gel and an
antiserum against the electrophoresed
serum is placed. Both components will
diffuse and precipitation bands are
observed.

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