Serology Lecture

note for Medical

Laboratory students
 Chapter One
Basic principles of
Immunologic and Serologic
reactions
Acknowledgements

 Addis Ababa University

 Jimma University
 Hawassa University
 Haramaya University
 University of Gondor
 American Society for clinical Pathology
 Center for Disease Control and Prevention Ethiopia
 Learning Objectives

At the end of the lesson, the students should be

able to:

List different types of serological tests

State the principle of serological tests
Describe the application of different serological
techniques
Identify the advantages and drawbacks of different
serological techniques
Identify factors, which affect antigen-antibody
reaction.
 Out line
1.1. Introduction

1.2. Immunological technique

1.2.1. primary binding sites

1.2.2. Secondary binding sites

1.2.3. Tertiary binding tests

1.3. Factors affecting antigen antibody reactions

1.1. Introduction

 Serology is the scientific study of blood serum.

 In practice, the term usually refers to the

diagnostic identification of antibodies in the
serum.
• Such antibodies are typically formed in
response to an infection (against a given
microorganism)

 against other foreign proteins (in response, for

example, to a mismatched blood transfusion), or
to one's own proteins (in instances of
autoimmune disease).
 1.1. Introduction

 Immune system: the structures, cells, and soluble

constituents that allow the host to recognize and
respond to foreign stimulus.

 Secondary immune response: the cellular and

humoral events that occur when an antigen is
encountered for a second or subsequent time.
1.1. Introduction

 Serum: the fluid portion of the blood after the

blood clots.

 Specificity: the special affinity between an

antigen and its corresponding antibody.

 Susceptibility: having little resistance.

1.1. Introduction
Application of serologic tests

 Serological tests may be performed for diagnostic

purposes when an infection is suspected, in
rheumatic illnesses, and in many other situations,
such as checking an individual's blood type.

 Serology blood tests help to diagnose patients with

certain immune deficiencies associated with the
lack of antibodies. In such cases, tests for
antibodies will be consistently negative.
1.1. Introduction

Antibody molecules combine reversibly with

antigens to form immune complexes.

Ag+Ab Ag.Ab complex

The detection and measurements of these reactions

form the basis of serology a sub discipline of
immunology. Therefore Serology - is the science of
measuring antibody or antigen in body fluids.
1.2. Immunological techniques

 Three groups of immunological techniques are

used to detect and measure antigen-antibody
combination.

 Primary binding tests

 Secondary binding tests and

 Tertiary binding tests.

 1.2.1 Primary binding tests

E.g.
 Enzyme linked Immunosorbent assay (ELISA)
tests and
 Radioimmunoassay (RIA)
 Western blotting
 Northern blotting
 Southern blotting
 Fluorescence tests
1.2.1 Primary binding tests

 Widely used in the serological diagnosis of

 bacterial,

 viral,

 fungal, and

 parasitic diseases.

 They are usually sensitive and give reproducible

results.
1.2.1 Primary binding tests

1. Immunofluorescence tests

There are two type of fluorescent antibody tests

(FAT):

a. Direct

b. Indirect
1.2.1 Primary binding tests

a. Direct fluorescent antibody tests (Direct FAT)

Is used to detect and identify unknown antigen in

specimens
E.g. Viral, bacterial, and parasitic antigens

It is called direct test because the fluorescent dye is

attached, or labeled, directly to the antibody.
1.2.1 Primary binding tests

 The fluorochrome used is usually fluorescein

isothyocynate (FITC), which gives a yellow-
green fluorescence.

 A fluorescent substance is one that, when

absorbing light of one wavelength, emits light of
another (longer) wavelength.
1.2.1 Primary binding tests

Procedure
A tissue or smear containing the organism (antigen)
is fixed to a glass slide and incubated with the
fluorescent antibody (antibody chemically linked to
FITC).

It is then washed to remove unbound antibody.

Examined by dark-field illumination under a

microscope with UV light source.
 The antigenic particles that have bound the
labeled antibodies are seen to fluoresce brightly.
1.2.1 Primary binding tests

 Direct FAT can be used;

 To identify bacteria when the numbers are very
low,
 It may also be used to detect viruses growing in
tissue culture or tissues from infected animals
such as rabies virus in the brains of infected
animals or antigens of HIV on the surface of
infected cells.
1.2.1 Primary binding tests

b. Indirect Fluorescent Antibody Test (IFAT)

 In the IFAT, unlabelled antibody combines with

antigen and the antigen antibody complex is
detected by attaching a fluorescent-labeled
antispecies globulin to the antibody. The antibody,
therefore, is labeled indirectly. Fluorescent-labeled
antihuman globulin is used if the antibody is of
human origin.
1.2.1 Primary binding tests

 The indirect FAT is used in two main ways:

I. To detect and identify unknown antigen in

specimens

II. To detect antibodies in a patient's serum using a

known antigen (microorganism).
1.2.1 Primary binding tests

I. Indirect FAT to detect antigen

 A slide preparation of the specimen is made and
unlabelled specific antibody is added.

 After allowing time for the antigen and antibody to

combine, the preparation is washed leaving only
antibody that has combined with the antigen on
the slide.
1.2.1 Primary binding tests

 A fluorescent labeled anti-species globulin is

added and allowed to combine with the antibody.
The excess is washed from the slide.

 The preparation is examined by fluorescence

microscopy using the correct filters. The antigen
antibody complex will be seen fluorescing
brightly.
1.2.1 Primary binding tests

II. Indirect FAT to detect Antibody

 In this test, a known antigen is placed on the slide
and the patient's serum is added.

 The preparation is then washed and fluorescent-

labeled antihuman globulin is added to
demonstrate the antigen-antibody reaction. The
preparation is examined by fluorescence
microscopy using the correct filters.
1.2.1 Primary binding tests

2.Enzyme Linked Immunosorbent Assay (ELISA)

 Used in the diagnosis of microbial infections
 They are specific, sensitive, and require only a
small amount of specimen
 Reagents used in the ELISA are stable and have
a long shelf life which makes for easy
distribution to district laboratories
1.2.1 Primary binding tests

 The results of qualitative ELISA techniques can

be read visually

 Large numbers of specimens can be tested at

one time and the ELISA can be easily
automated for use in epidemiological surveys.
1.2.1 Primary binding tests

There are two main ways of performing ELISA

a. Double antibody technique, to detect antigen

b. Indirect technique, to detect and assay

antibody.
1.2.1 Primary binding tests

a. Double antibody ELISA

Specific antibody is coated on the surface of the well
of a microtitration plate (or a test tube), and the
specimen is added.
After a period of incubation during which the
antibody takes up (captures) the antigen from the
specimen, the well is washed leaving the antigen
attached to the antibody.
1.2.1 Primary binding tests

 Enzyme labeled specific antibody (often the same

antiserum as that coating the well except it is
enzyme linked) is added to detect the presence of
the antigen.
 After a further period of incubation during which the
enzyme labeled antibody combines with the antigen,
the well is washed and a substrate is added. The
enzyme acts on the substrate to give a color change
in the fluid.
1.2.1 Primary binding tests

 The enzyme activity is stopped by altering the pH

of the reaction or denaturing the enzyme.
 By measuring the color produced, the amount of
attached antibody and therefore antigen in the
specimen can be estimate.

Application
 In developing countries, an important application of
the double antibody ELISA is in the diagnosis of
 Rotavirus infection in young children.
1.2.1 Primary binding tests

Double antibody ELISA (antigen test)

Source: Kuby Immunology 2007, 5th ed

1.2.1 Primary binding tests

Procedure
 Prepare all reagents and necessary materials
 Prepare serum from non-hemolyzed blood.
 Take micro titration well plates, which are coated
with specific antibody.
 Add positive and negative control in 4 micro
titration well.
 Add specimens to the micro titration well of 96.
If specimen containing antigen combines with
antibody.
1.2.1 Primary binding tests

 Wash the plate by using automatic washer more

than 4 times.
 Add enzyme labeled antibody, which attaches to
the antigen.
 Wash the procedure.
 Add the substrate, which is hydrolyzed (broken
down), gives color changes.
 Read the result.
1.2.1 Primary binding tests

b. Indirect ELISA

•In this technique, known antigen is attached to the

inside surface of the well and patient’s serum is added.
After incubation and washing, enzyme labeled
antihuman globulin is reacted with the antibody that
has attached to the antigen.
1.2.1 Primary binding tests

• The presence and concentration of antibody

that has reacted with the antigen is shown by a
change in color when the substrate is added.

• The intensity of the color is directly proportional

to the concentration of antibody in the serum.
1.2.1 Primary binding tests

(Specimen)

Source: Kuby Immunology 2007, 5th ed

1.2.1 Primary binding tests

Procedure
 A micro titration well plate is coated with known
antigen.
 Add patent’s serum. If the serum contains
antibody it combine with antigen.
 Wash carefully by using automatic washer more
than 4 times.
1.2.1 Primary binding tests

 Add enzyme labeled antihuman globulin, which

attaches to the antibody.
 Wash carefully.
 Add the substrate, which is hydrolyzed (broken
down) by the enzyme to give a color change.
 Read the result.
 1.2.1 Primary binding tests
Reading ELISA results
If it is qualitative ELISA
 Read by naked eye.

• The presence or absence of antigen is seen as a

simple color change.

If it is quantitative antibody techniques

 Read either

• by measuring the intensity of color in a

spectrometer (spectrophotometer) or
• by testing dilutions of the test serum and
determining the highest dilution that shows a color
change.
1.2.1 Primary binding tests

3. Radio Immunoassay (RIA)

One of the most sensitive techniques for detecting
antigen or antibody is RIA
1st developed by two endocrinologist called

S.A. Berson and

Rosalyn Yalow, in 1960 to determine levels of

insulin-anti-insulin complexes in diabetics
1.2.1 Primary binding tests

a. Conventional RIA
• Is a competitive immunologic procedure
• It measures very low concentrations of
antigens (or antibodies) by using radioactively
labeled antigens as competitors.
• Radioactive isotopes such as 3H , 14
C, 35
S, 30
P
or 125I can be used for labeling
• Radioactive isotopes are molecules with
unstable nuclei and therefore emit radiation
spontaneously.
1.2.1 Primary binding tests
 It is highly sensitive method to detect low
concentration of unknown (unlabeled) antigen
 RIA is used to assay:
 Hormones,
 Drugs,
 Enzymes,
 Microbial antigens
e.g.
• hepatitis B antigen, carcinoembryonic and
α-feto protein antigen.
 It also used to the detection of antibody
1.2.1 Primary binding tests
 RIA technique utilizes three components

 Patient antigen
the specific compound we wish to
determine.

 Labeled antigen
the same compound as above to which is
attached a radioactive label.

 Antibody
specific for the sample and labeled
antigen.
1.2.1 Primary binding tests

 There are two assay approaches in conventional

RIA

i. Liquid phase Assay

ii. Solid phase Assay

1.2.1 Primary binding tests

i. Liquid phase assay:

 The sample, labeled antigen and the specific

antibody are added to the mixture in a
solution form.

 After completion of incubation with the

ligand of interest (analyte), a bound-free
separation step is performed using different
techniques.
 1.2.1 Primary binding tests
ii. Solid phase assay
In this assay,
 the specific antibody is added either in a
suspension or the antibody is covalently bound to
the inside wall of the reaction tube.

 Separation of the bound-free fraction is realized by

centrifugation or magnetic separation followed by
decanting the supernatant or by simply pouring off
the reaction mixture if coated tube is used.

 The bound fraction is then washed adequately with

appropriate buffered wash solution and made
ready for counting.
1.2.1 Primary binding tests
b. Immunoradiometric Assay (IRMA) (Sandwich
Immunoassay)

 Developed with the objective of solving the

problems associated with conventional RIA

 Reading assignment (Read more about

IRMA)
 1.2.1 Primary binding tests

4. Western blot test (WB)

• Used as a confirmatory test and very specific for
HIV
• Antibodies to only a selection of viral proteins may
yield an indeterminate Western blot
• Bands corresponding to p24 and p55 are detected
early in sero conversion followed by glycoprotein
bands
1.2.1 Primary binding tests
5. Southern blotting

 Named in E.M. Southern, who 1st described it in

1975

 Used to detect specific DNA sequences

 Used in clinical lab to detect gene mutations

known to cause disease

 Used to detect carriers of the fragile X syndrome

and sickle cell anemia for genetic counseling
1.2.1 Primary binding tests

6. Northern blotting

• Very similar with southern blotting

• Used to detect RNA

• Not used in clinical laboratory mostly

1.2.2. Secondary binding tests
1. Agglutination tests

In district laboratories, agglutination tests are

frequently used because compared with other
serological tests, they are

 Simpler to perform
 Require no special equipment
 Usually less expensive.
1.2.2. Secondary binding tests

 The fundamental and most commonly used reaction in

medical serology laboratory
 Mostly used in clinical lab to demonstrate the interaction
of antigen and antibody
 By broad definition, agglutination is simply the
clumping of cells into aggregates, often as a result
of the combination of an antibody's binding sites
with antigen binding sites of the cells
1.2.2. Secondary binding tests

 Agglutination tests can be performed:

i. On slides or tiles
ii. In tubes
iii. In micro titration plates
1.2.2. Secondary binding tests
i. Slide or tiles agglutination tests

Agglutination can be either

Rapid and easily performable techniques
Gives a reaction in minutes or even seconds.
Not usually as sensitive as tube or micro titration
techniques.
Specificity depends on the reagent used.
a. active or
b. passive.
1.2.2. Secondary binding tests
a. Active agglutination slide tests

 Direct agglutination of bacterial antigen with its

corresponding antibody.

E.g. the slide agglutination of salmonella,

shigella or vibrio cholera using specific antibody,
or the agglutination of leptospiral antigen by
leptospiral antibodies present in a patients
serum in acute leptosprosis.
1.2.2. Secondary binding tests

Slide agglutination tests are used:

To identify bacteria from cultures are difficult to

standardize and control.

To check auto-agglutination (false agglutination)

1.2.2. Secondary binding tests
b. Passive agglutination slides and tile tests

 Specific antibody or known antigen is attached to inert

particles or cells.

 When the known antigen or antibody combines with its

corresponding-antibody or antigen in the specimens the
particles or cells are used only to show that an antigen
antibody reaction has occurred. Their role in these
reactions is therefore passive.
1.2.2. Secondary binding tests

The substances and cells used as carriers in passive slide

agglutination test include:
 Latex particles
 Carbon particles
 Stabilized staphylococcal cells
1.2.2. Secondary binding tests

ii. 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.

iii. Micro titration agglutination tests

 Are performed in micro iteration plates
 Now replaced several tube agglutination tests
because they are more sensitive, more economical,
and easier to perform, and usually give quicker
results.
1.2.2. Secondary binding tests
Haemagglutination inhibition antibody test (HAI)

This technique is used:

• To detect antibodies against
• arboviruses,

• influenza viruses,

• measles, viruses, and

• rubella virus.

These viruses are able to agglutinate red cells because

they posses heaemagglutinins on their outer surfaces.
 1.2.2. Secondary binding tests

 Reverse passive haemagglutination test (RPHA)

• This technique is used to identify viruses that do not

haemagglutinate.
• It performed by reacting viral specimens with red
cells coated with specific viral antibody.
• If the corresponding antigen is present, the red cells
will be agglutinated.
1.2.2. Secondary binding tests
2. Precipitation tests

A precipitation reaction may be defined as the visible

result of an antigen antibody reaction between a
soluble antigen and its antiserum.

 In addition to these two substances, electrolytes are

necessary to bring the process to its desired conclusion
and pH and temperature of the mixture also have an
effect.
1.2.2. Secondary binding tests

• Antigen and antibody molecules are bound

together in lattice of alternate molecules if the
reaction is successful.
• Unlike agglutination reactions precipitation reaction
involve a small, soluble antigen.
• When the antibody antigen rxn occurs, a few small,
soluble lattice complexes form followed by a long
period and are less visible than agglutination.
1.2.2. Secondary binding tests

 Are used to detect and identify antigens in

 specimens

 extracts and

 cultures

 Are used to detect and quantify antibodies in

serum.
 Compared with aggt. tests, ppt techniques require
more experience in their performance and
interpretation.
 Some tests have a low sensitivity.
1.2.2. Secondary binding tests

Types of precipitation test

There are three main types of precipitin techniques:

a. Tube precipitation test

b. Gel diffusion tests

c. Counter immunoelectrophoresis tests

1.2.2. Secondary binding tests

a. Tube precipitation

a clear solution containing the test antigen is

carefully layered on to a clear antiserum in a
precipitin tube or capillary tube (micro hematocrit).

Following a period of incubation, if the

corresponding antigen to antibody is optimal, a line
of visible precipitating antibody and antigen will form
between the two layers of fluid.
1.2.2. Secondary binding tests

Disadvantage
Needs large amount of antiserum
The test is not very sensitive.

Use
To detect extra cellular antigens in CSF, especially
H-influenza type b antigen
To group - hemolytic streptococci
1.2.2. Secondary binding tests
b. Gel diffusion tests
 When an antibody and its antigen are placed in
different regions of an agar gel, they diffuse
toward each other and form an opaque band of
precipitate at the junction of their diffusion fronts.

 When both antigen and antibody diffuse through

the agar this is referred to as double diffusion.

 When only the antigen or antibody diffuse, with

the corresponding antigen or antibody is being
contained in the agar, this is called single
diffusion.
1.2.2. Secondary binding tests

Double gel diffusion (Ouchterlony)

 Antigen and antibody diffuse towards each other
and where they meet in optimal proportion a
visible line of precipitation forms.

 The thickness of the line of precipitation is a semi

quantitative measure of the amounts of antigen
and antibody that combine.
1.2.2. Secondary binding tests
Example:

 Elek gel technique

+ used to detect toxogenic strains of

 C. diphtheria

 Biken test
 used to detect toxin- producing fecal

E. coli (ETEC)
1.2.2. Secondary binding tests

Single gel diffusion

 In this technique, specific antibody is incorporated
into the agar gel and wells are cut to contain the
antigen, which diffuses radially.
 A ring of precipitation forms around a well that
contains the corresponding antigen.
 The higher the concentration of antigen, the larger
the ring of precipitation will be formed.
1.2.2. Secondary binding tests

c. Counter immunoelectrophoresis (CIE)

Also called

 countercurrent-electrophoresis (CEP)

 Immuno electroosmophoresis (IEOP)

 Electro immunodiffusion.
1.2.2. Secondary binding tests

3. Complement fixation tests

In general, complement fixation tests (CFT) are best

performed in reference laboratories where facilities

exist for the careful standardization and control of

reagents, which these tests require.

1.2.2. Secondary binding tests
 Method
 Ag mixed with test serum to be assayed for Ab

 Standard amount of complement is added

 Erythrocytes coated with Abs is added

 Amount of erythrocyte lysis is determined

Ag No Ag
Ag
Patient’s
serum
Ag
1.2.3. Tertiary binding tests

 Tertiary binding tests measure the consequences of

immune responses in vivo.
 These tests are much more complex than primary
and secondary tests but their results reflect the
practical significance of the immune response.
 E.g. measurement of the protective effects
of antibody.
1.3. Factors affecting antigen antibody reaction

 specificity
 cross reactivity
 temperature
 pH
 ionic strength
 concentration
 intermolecular specificity
Review questions
 Write the difference between precipitation and
agglutination tests
 How does the zonal reaction affects test
results?
 Write the advantage and disadvantage of
serological test compared with other laboratory
techniques for infectious disease.
Reference

1. Naville J. Bryant Laboratory Immunology and

Serology 3rd edition. Serological services
Ltd.Toronto,Ontario,Canada,1992

2. Tizard. Immunology an introduction,4th

edition ,Saunders publishing,1994

3. Mary Louise .Immunology and Serology in

Laboratory medicine 3rd edition