Download ( R.PM) for 5 minutes. Then serum separate and incubate at 56 ºC

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SEROLOGY
Serological tests: mean the measurement of antigen-antibody
interaction for detection of the causes of disease.
Antibodies (immunoglobulins ):are large Y-shaped proteins. They are
used by the immune system to identify and neutralize foreign objects
like bacteria and viruses. The basic structure of all antibodies are same.
There are four polypeptide chains held together by disulfide bonds.
These four polypeptide chains form a symmetrical molecular structure.
There are five classes of immunoglobulins including IgM, IgG, IgA, IgD,
and IgE.
Antigen: a substance that when introduced into the body stimulates the
production of an antibody. Antigens may include virus, parasites ,
bacteria, foreign blood cells, and the cells of transplanted organs(part or
whole cell).
Antibody titer: the highest dilution of serum samples give positive result.
Importance of serological tests:
For diagnosis of infection by antibody detection (indirect evidence of
infection) specially the infectious agents not have available antigen assays
or culture techniques making the detection of specific antibodies
diagnostically useful.
Some serological tests consider screening tests and other consider
confirmative tests the screen test is a simple, rapid ,don’t detect titer of
antibodies and the positive sample only should retest with confirmative
test while the negative sample don’t retest.
The confirmative not simple and need time to read the result(positive,
negative and suspected) also detect the titer of Ab.
In confirmative test only suspected result should retest after 2-4 weeks.
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Serum samples collection:
Venous blood obtain from the jugular vein of animal in sterile glass
tubes without anti-coagulant. Each sample label describing the animal
species, gender and age. Blood left at room temperature to clot then
centrifugation at 1500 revolutions per minute ( R.P.M.) for 5 minutes.
Then serum separate and incubate at 56 ºC for 30 minutes in a water
bath to impair the complement activity(if the test not CFT) and
transferred into labeled eppendrof tubes then stored with deep freeze
at (-18ºC) until used.
If the tube contain anticoagulant blood components will sediment and
the liquid then called (plasma).
Examples of some simple serological tests:
Latex agglutination test)LAT)
Screening test for diagnosis of toxoplasmosis,in this test, latex particles
are sensitized with inactivated Toxoplasma gondii antigens when the
patient serum is reacted with sensitized latex particles, and if toxoplasma
specific antibodies are present, latex particles allow visual observation of
antigen-antibody reaction.
Procedure:
 After well mixing of the latex reagent, 30 µl of antigen add into a
slide black area.
 30 µl of serum place over antigen drop .
 Mix both drops by wooden stick and shaken the slide.
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Examine the presence or absence of visible agglutination after 5
minutes , the presence of agglutination indicates an antibody
concentration equal or greater than 4 international units IU\ml.
Rose Bengal Plate Test(RBPT):
This test is a simple spot agglutination test, and considered the most
popular screening test for determining brucellosis. All positive cases to
RBPT are retested by other confirmatory tests such as tube agglutination
test(TAT)
Procedure
-All serum samples and antigen of Rose Bengal put at room
temperature before use.
- 30 µl of antigen and 30 µl of the tested serum placed alongside -on
a white plate , and then mixed with wooden stick.
- The plate shaken for 4 minutes .
- Observe the agglutination reactions , sample classify positive if
agglutination particle appeared and negative if no agglutination
detected.
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Milk ring test(MRT)
A simple and effective method, but can only be used with cow’s milk .
A drop of haematoxylin-stained antigen is mixed in tube with a
small volume of milk(1 ml) which should store for at least 24 hours at
4°C. The milk/antigen mixtures are incubate at 37°C for 3 hour . If
specific antibody is present in the milk it will bind to the antigen and
rise with the cream to form a blue ring at the top of the column of
milk.
The test is considered to be negative if the color of the underlying
milk remains homogeneously dispersed in the milk column.
False-positive reactions may occur in recently vaccinated cattle (less
than 4 months) or in samples containing abnormal milk, such as
colostrum or that due to mastitis and direct examine the sample of
sample without leave it at 4°C for 24 hours.
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Tube agglutination test(TAT):
Confirmatory test for diagnosis of brucellosis ,the test is performed in
plastic tubes by placing 0.8 ml of phenol saline into the first tube and
0.5 ml of phenol saline in the remaining tubes of a series of five or ten
tubes. A volume of 0.2 ml serum is added to the first tube, mixed, and
then 0.5 ml is transferred to the next tube. Further volumes of 0.5 ml are
transferred to subsequent tubes to give a series of doubling dilutions. An
equal volume of standard B. abortus agglutination suspension, diluted
in phenol saline, is then added to each tube, and the tubes are incubated
at 37 °C for 20 hours.
To determine the amount of international units IU of antibodies we
depend on :
1 - degree of agglutination
- 2- titer of antibodies
The positive result may take one of the four degrees of turbidity or
clearance supernatant of agglutination tubes (1+ = 25% clear, 2+ = 50%
clear, 3+ = 75% clear, 4+ = 100% clear).
Formula for each degree of agglutination to determine the amount of
international units IU of antibodies :
+1 𝐼𝑛𝑣𝑒𝑟𝑡𝑒𝑑 𝑑𝑖𝑙𝑢𝑡𝑖𝑜𝑛*1.56 - (
𝐼𝑛𝑣𝑒𝑟𝑡𝑒𝑑 𝑑𝑖𝑙𝑢𝑡𝑖𝑜𝑛
)
4
+2 Inverted dilution*1.56
+3 Inverted dilution*1.56 + (
𝐼𝑛𝑣𝑒𝑟𝑡𝑒𝑑 𝑑𝑖𝑙𝑢𝑡𝑖𝑜𝑛
)
4
+4 Inverted dilution*1.56 + (
𝐼𝑛𝑣𝑒𝑟𝑡𝑒𝑑 𝑑𝑖𝑙𝑢𝑡𝑖𝑜𝑛
4
For cattle, titer equivalent to ≥ 80 IU.
For goats, titer equivalent to ≥ 50 IU.
For sheep, titer equivalent to ≥ 40 IU.
∗ 2)
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Prozone effect: lack of agglutination in due to antibody with high
concentration resulting in very small complexes that do not clump to
form visible agglutination.
Mercapto-ethanol tube agglutination test(2-ME):
The 2-mercapto-ethanol test is carried out by diluting 0.1 ml of serum
in 0.4 ml of (physiological saline) and adding 0.5 ml of 2-mercaptoethanol . The serum is then incubated at 37 °C for one hour, followed by
serial doubling dilutions in saline. Volumes of 0.5 ml of antigen diluted
in saline (without phenol) are then added to each tube . It is more
closely correlated with active infection of brucellosis but no more
sensitive than the TAT.
This test is useful in the differentiate IgM agglutinins resulting from
recent infection or exposure to cross-reacting antigens from the IgG
agglutinins associated with active or long-standing infection because 2Mercaptoethanol to reduce IgM antibodies .
Complement fixation test(CFT): a test for diagnosing an infectious
disease by detecting the presence of antibody in the blood, based on the
fixing of a known quantity of complement to the antigen being tested
and the specific antibody that combines with it.
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Enzyme-linked immunosorbent assay(ELISA) :a sensitive immunoassay
that uses an enzyme linked to an antibody or antigen as a marker for the
detection of a specific protein, especially an antigen or antibody. It is
often used as a diagnostic test to determine exposure to a particular
infectious agent.
Polymerase chain reaction(PCR): molecular technique(not serologic)
for rapidly producing many copies of a fragment of DNA for diagnostic
or research purposes.