Serology is the study of blood serum and the detection of antibodies and antigens. Key events in the history of serology include Karl Landsteiner's 1901 discovery of the A, B, and O blood groups. Serological tests can be classified as primary, secondary, or tertiary based on their level of sensitivity and directness of measurement. Common serological techniques include ELISA, immunofluorescence, agglutination tests, precipitation reactions, and complement fixation tests. These methods are used to detect infections and other medical conditions.

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2. Serology
BY : SHEHRYAR AHMAD
DISCIPLINE : MLT IST BATCH ,
IPMS KMU
EMAIL : SHERY9444@GMAIL.COM
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3. Objective
 Definition of serology
 History or begining
 Terminologies used in test methodology
 Serological test methods and classification
 Uses of serological test
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4. Serology
 The branch of
laboratory medicine
that studies blood
serum for evidence of
infection and other
parameters by
evaluating antigen-
antibody reactions in
vitro
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5. Serology
 Serology is the
scientific study of
blood serum. In
practice, the term
usually refers to the
diagnostic
identification of
antibodies in the
serum
We can detect antigens
too
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6. Beginning of Serology
 Serology as a science began in 1901. Austrian
American immunologist Karl Landsteiner (1868-1943)
identified groups of red blood cells as A, B, and O.
From that discovery came the recognition that cells of
all types, including blood cells, cells of the body, and
microorganisms carry proteins and other molecules on
their surface that are recognized by cells of the
immune system.
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7. Terms used in evaluating test
methodology
Sensitivity
 Analytical Sensitivity – ability of a test to detect very small amounts of a
substance
 Clinical Sensitivity – ability of test to give positive result if patient has the
disease (no false negative results)
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8. Specificity
 Analytical Specificity – ability of test to detect substance without
interference from cross-reacting substances
 Clinical Specificity – ability of test to give negative result if patient does not
have disease (no false positive results)
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9. Affinity
 Affinity refers to the
strength of binding
between a single antigenic
determinant and an
individual antibody
combining site.
 Affinity is the equilibrium
constant that describes the
antigen-antibody reaction
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10. Affinity
 Antibody affinity is the strength of the reaction between a single antigenic
determinant and a single combining site on the antibody.
 It is the sum of the attractive and repulsive forces operating between the
antigenic determinant and the combining site .
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11. Avidity
 Avidity is a measure of the
overall strength of binding
of an antigen with many
antigenic determinants and
multivalent antibodies
 Avidity is influenced by
both the valence of the
antibody and the valence
of the antigen.
 Avidity is more than the
sum of the individual
affinities.
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12. "Serial dilution" - This term is frequently
used and refers to a "multiple" dilution problem. In other
words, an initial dilution is made and then this dilution is
used to make a second dilution, and so on.
For example, a 1:2 serial dilution is made using a 1 mL
volume of serum. This expression indicates that 1 mL of
serum is added to 1 mL of H20 and then mixed. This initial
dilution is 1:2. Then, 1 mL of this dilution is added to 1 mL
of H20 further diluting the sample. This same process is
continued.
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15. Titre vs Dilution
 • Dilution is the chemical composition that can be changed easily but titre
is the exact value and cannot be changed.
 • Dilution is a simple process and can be done very easily but titre testing
requires elaborate laboratory preparations.
 • Dilution is a simple physical process and physical change occurs only
when titre value is reached.
 • Dilution cannot determine the chemical composition of the solution but
titre testing is done to determine the composition of the solution.
 • Dilution is a process that can by physical or chemical but titre is a
particular value that is expressed by many units.
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16. Prozone & post zone phenomenon:
 False negative antigen antibody reaction, either agglutination or precipitation, can
occur if antigen and antibody are not mixed in the right proportions. This can
happen if either antibody is in excess (Prozone) or when antigen is in excess (Post
zone).
 Prozone phenomenon:
Some sera when tested un-diluted, do not show agglutination. The
same sera when tested after making dilution show a positive
agglutination/precipitation reaction. This phenomenon is called
“Prozone phenomenon” in which agglutination or precipitation
occurs at higher dilution ranges of serum, but is not visible at lower
dilutions or when undiluted. Excessive levels of antibody result in
false negative reaction as antibody excess results in formation of
very small complexes which do not clump to form visible
agglutination. Prozone reaction is the probable cause of false-
negative result.
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17. Post-zone phenomenon:
 This refers to the reaction wherein excess of antigen results in
no lattice formation and a false negative agglutination
reaction. Antigen excess is also the probable cause of false-
negative antigen-antibody agglutination/precipitation
reaction
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18. Precipitation Curve 18

19.  Classification of antigen-antibody interactions:
 1.Primary serological tests: (Marker techniques) e.g.
 –Enzyme linked immuono sorben assay (ELISA)
 –Immuno flurescent antibody technique (IFAT)
 –Radio immuno assay (RIA)
 2.Secondary serological tests: e.g.
 –Agglutination tests
 –Complement fixation tests (CFT)
 –Precipitation tests
 –Serum neutralization tests (SNT)
 –Toxin-antitoxin test
 3.Tertiary serological test: e.g.
 –Determination of the protective value of an anti serum in an animal.
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20. Primary binding tests
Primary binding tests are tests that directly measure the binding of antigen
and antibody (i.e.; directly measure or visualize the immune complex). They
are the most sensitive techniques in terms of the amount of detectable
antigen orantibody.
Example:
Enzyme linked Immunosorbent assay (ELISA) tests
Radioimmunoassay (RIA)
Western blotting
Primary binding tests are performed by allowing antigen and antibody to
combine and then measuring or visualizing the
amount of immune complex formed. It is usual to use radioisotopes,
fluorescent dyes, or enzymes as labels toidentify one of the reactants.
Certain examples of primary binding test are :
ELISA
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21. ELISA methods takes over
 Enzyme-linked immunosorbent assay, also called
ELISA, enzyme immunoassay or EIA, is a biochemical
technique used mainly in immunology to detect the
presence of an antibody or an antigen in a sample.
The ELISA has been used as a diagnostic tool in
medicine
 Because the ELISA can be performed to evaluate
either the presence of antigen or the presence of
antibody in a sample
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22. Enzyme immunoassay (EIA/ELISA)
 Sandwich technique”
 Monoclonal or polyclonal antibody adsorbed on solid surface
(bead or microtiter plate)
 Add patient serum; if antigen is present in serum, it binds to
antibody coated bead or plate
 Add excess labelled antibody (antibody conjugate); forms
antigen-antibody-labelled antibody “sandwich” (antibody in
conjugate is directed against another epitope of antigen
being tested)
 Add substrate, incubate, and read absorbance
 Washing required between each step
 Absorbance is directly proportional to antigen concentration
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23. ELISA Most popular technological advance in Laboratory
Medicine
 ELISA methods can
detect any infectious
disease provided if we
have antibodies and
antigen to any
infection, enzyme or
any substance
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24. Immuno flurescent antibody technique
(IFAT)
Principle
 Fluorescent dyes (fluorochromes) illuminated by UV lights are used to
show the specific combination of an antigen with its antibody. The
antigen-antibody complexes are seen fluorescing against a dark
background. Immunofluorescence tests are referred to as fluorescent
antibody tests (FAT).
 There are two types of fluorescent antibody tests (FAT):
Direct and Indirect
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25. Direct and Indirect Methods 25

26. Secondary binding tests
 Secondary binding tests are tests that detect and measure the
consequences (secondary effect) of antigen-antibody interaction.
 These consequences include:
 Precipitation of soluble antigens
 Clumping (agglutination) of particulate antigens
 Neutralization of bacteria, viruses, or toxins; and
 Activation of the complement system.
They are usually less sensitive than primary binding tests, but may be easier to
perform.
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27. Principle of agglutination tests
 Agglutination is the visible clumping together of bacteria,
cells, or particles, by an antigen combining with its specific
antibody. The resulting clumps are referred to as agglutinates.
 In tests used to detect antibody (agglutinin) in a patient‘
serum, a known antigen (aggutinogen) suspension is used.
 The antigen particles are agglutinated if the serum contains
the corresponding antibody. In general, to detect antibody in
patients serum a known antigen suspension is added or to
detect antigen in serum, a specific antibody is added.
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30. Agglutination tests can be performed:
A. On slides
B. In tubes
C. In microtitration plates
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31.  Slide agglutination tests
 These are rapid, easily performed techniques that give a reaction in minutes or even
seconds. They are, however, not usually as sensitive as tube or microtitration techniques
 Tube agglutination tests
 In tube tests, agglutination occurs in a larger volume of fluid and therefore, in an
environment that can be more fully controlled. Tube tests are usually more sensitive than
slide tests. In this tube agglutination test, serum is diluted serially and then antibody level
is measured by adding standard antigenic suspension.
 Microtitration agglutination tests
 These techniques are performed in microtitration plates. They have now replaced several
tube agglutination tests since they are more sensitive, more economical, easier to
perform, and usually give quicker results
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32. METHODOLOGY
 Agglutination either a qualitative or quantitative :
Method
Slide test
•Rabid
•Semiquntitative
•Room temp.
incubation
Tube tests
•Longer incubation
•Different incubation
Condition
•Semiquntitative
Microwel assay
•Modification tube
assay
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33. Classification of agglutination reaction
A. Direct agglutination :
+ ↔
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34. Direct
agglutination
RBC Antigens
ABO & Rh
I antigens
Paul Bunnell
Bacterial Ag
Febrile
Widal
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35. Cont.
RBC antigen
 ABO blood groups and Rh . .
Bacterial antigen
 Use for detect recent infection .
 Febrile agglutinin: Ab produce during bacterial infection with
fever .
 Widal test: salmonella Bactria as antigen for detection of Ab to
typhoid paratyphoid organism .
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38. Hemagglutination
Principle :
- It is a type of agglutination test performed on RBCs.
- many human viruses have the ability to bind to the surface structures on red
blood cells from different species thereby causing agglutination
Example :
 influenza virus binds to red blood cells
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39. Haemaggultination Tests:
 It has two types:
 Active: the antigen is the RBC itself.
Viruses can clump red blood cells from
one species or another (active
hemagglutination)
Example is the test used in ABO grouping.
 Passive: the antigen here is not the RBC. The
RBC absorbs it and expresses it on the surface.
It will form clumps when mixed with
antibodies.
i.e. red cells are passive carriers .
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40. Haemaggultination Tests
active passive
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42.  Indirect (passive) haemagglutination test (IHA
The indirect haemaggutination (IHA) test is a passive
agglutination test (see previous text) in which known antigen is
coated on treated red cells.
Carrier red cells
The cells are formalin fixed and treated with tannic acid to make the antigen
adhere, Antigen coated red cells are referred to sensitized cells. In the IHA
test, the sensitized red cells are added to dilutions of the patient's serum. If
the serum contains the corresponding antibody in sufficient concentration,
the red cells will be agglutinated and settle to form an even covering in the
bottom of the well. The antibody titer is the highest dilution of serum in which
agglutination can be detected. If the sensitized cells are not agglutinated they
will settle and form a red button in the bottom of the well.
 Applications of IHA tests include the Treponema pallidum
haemagglutination (TPHA) to detect treponemal antibodies and the
antisterptolysin O (ASO) titration technique used in the diagnosis of
S.pyogens infections.
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43. Hemagglutination Inhibition technique
Hemagglutination inhibition test is used to detect some viral
antibodies, for example, rubella. A known quantity of rubella
viral antigen is mixed with dilutions of the patient’s serum, to
which red blood cells are added. If the serum lucks antibody,
the virus will spontaneously attach to the red cells, link
together, and agglutinate. If antibody to the virus is present, all
of the virus particles will be bound by antibody, which
prevents or inhibits hemagglutination. The serum is therefore
positive for the antibodies. The highest dilution of serum that
totally inhibits agglutination of red cells determines the
antibody titer of the serum.
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46. Cont.
B. Passive agglutination ( latex
agglutination)
 Antigen is attached to a particulate carrier , then
react with antibody .
 Advantage: rabidity
 Carriers include : charcoal , gelatin , RBCs
+ ↔
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47. cont
Used for :
 Rheumatoid factor :
 ASO: antistreptolysin O in serum associated with
streptococcal infectioin
 SLE :detection of antinuclear antibodies in human sr.
associated systemic lupus erythematousus .
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48. Cont.
C. Reverse passive agglutination :
 Ab is attached to carrier .
 Use to detect C-reactive protein is an acute phase protein increase druing
infection and inflammation, so it an inflammatory marker .
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49. Provides a highly sensitive assay for small quantities
of an Antigen.
Example: First home pregnancy test
Agglutination Inhibition: 49

50. C. Complement fixation test
 The complement fixation test is an immunological medical test
looking for evidence of infection. It tests for the presence of either
specific antibody or specific antigen in a patient's serum. It uses
sheep red blood cells (sRBC), anti-sRBC antibody and complement,
plus specific antigen (if looking for antibody in serum) or specific
antibody (if looking for antigen in serum).
 If either the antibody or antigen is present in the patient's serum,
then the complement is completely utilized, so the sRBCs are not
lysed. But if the antibody (or antigen) is not present, then the
complement is not used up, so it binds anti-sRBC antibody, and the
sRBCs are lysed.
 Complement fixation tests are used in the diagnosis of rickettsial
infections and several viral and parasitic infections.
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52. Precipitation Reaction:
When a soluble Ag combines with its Ab in the
presence of an electrolyte (NaCl) at a particular
temperature and pH, it forms an insoluble precipitate
of Ag-Ab complex. The Ab causing precipitation is
called Precipitin and the reaction is called as
precipitation reaction.
Antibodies Antigens Ag-Ab complex

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54. Precipitation occurs in two media:
Liquid.
Gel.
Precipitation in Liquid:
Antigen – Antibody reaction perform by placing a
constant amount of antibody in a series of tubes and
adding increased amount of antigen. Antigen –
Antibody reacts together resulting in precipitation.
Plotting the amount of precipitate against increasing
antigen conc. Yeilds a precipitation curve. The lab test
for its use is called nephlometry ...

55. Precipitation in gel:
Radial Immunodiffusion (Mancini) :
In these methods agar gel or similar gels are used on
plates or petriplates . Both Ag and Ab diffuse freely in
the gel system in all directions. At a certain point
depending on the rate of diffusion and concentration
of the reactants, a zone of equivalence will be formed,
which is seen as a visible precipitation.
Precipitation curve shows three zones:
1. Zone of Ab axis.
2. Zone of equivalence.
3. Zone of Ag axis.

56. If Ag or Ab preparations are
complex, multiple bands
form. These are again of 2
types- Single diffusion
methods
Mancini test.) and
doublediffusion methods.
(ouchterlony method )
Precipitation reactions in gels

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 In immunoelectrophoresis, diffusion is combined with
electrophoresis

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59. Some serological test examples 
 Anti-Streptolysin O (ASO) Test
 It is a rapid latex agglutination test for the qualitative and semi-
quantitative determination of anti-streptolysin-O antibodies (ASO) in
serum. In infections caused by β-haemolytic streptococci, streptolysin-O
liberated from the bacteria that stimulates production of ASO antibodies
in the human serum.
 The RapidTex ASO latex reagent is a stabilised buffered suspension of
polystyrene latex particles that have been coated with Streptolysin O.
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60.  Materials used in the ASO Test
 ASO Antigen: A stabilized buffered suspension of polystyrene latex
particles coated with Streptolysin O and 0.1% sodium azide as
preservative. Shake well prior to use.
 ASO Positive Control: Human serum containing more than 200 IU/ml
ASO and 0.1% sodium azide as preservative.
 ASO Negative Control: Human serum containing 0.1% sodium azide
as preservative.
 Sample Collection and Handling:
 Only fresh serum specimens should be used. Plasma must not be
used since fibrinogen may cause non-specific agglutination of the
latex. It is preferable to test samples on the same day as collected.
Serum samples may be stored at 2-8o C for up to 48 hours prior to
testing. If longer storage is necessary, sera should be stored frozen
at -20ºC.
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61.  Procedure:
 Bring all reagents and specimens to room temperature. Place one drop
(50 µl) of the positive control and 50 µl of the patient serum into
separate circles on the glass slide. Shake the ASO latex reagent gently
and add one drop (45 µl) on each circle next to the sample to be tested
and control. Mix well using disposable stirrer spreading the mixture over
the whole test area and tilt the slide gently. Agitate for about 2 minutes
with rotator or by hand and observe for the presence or abscence of
agglutination.
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Negative result:
No agglutination of the latex particles suspension within two
minutes.
Positive result:
An agglutination of the latex particles suspension will occur within
two minutes, indicating an ASO level of more than 200

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