Serological Diagnostic Methods

Microbial Diseases

Allyssa Haywood
11/10/2016
Table of Contents

 Introduction to serology…………………………………………………………………..3

 Types of Serological Methods……………………………………………………………4

 Agglutination tests…………………………………………………………………………….4

 Precipitation tests……………………………………………………………………………..7

 Complement fixation tests…………………………………………………………........9

 Immunofluorescence testing…………………………………………………………..10

 Enzyme-Linked ImmunoSorbent Assay (ELISA)…………………………………11

 References……………………………………………………………………………………….13

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 Introduction to Serology

This is the branch of laboratory medicine that studies blood serum for evidence of
infection and other parameters by evaluating antigen-antibody reactions in vitro.
There are several types of techniques that are employed to detect the presence of
microbial diseases present in one’s body.
All serological techniques that detect antibody are based on the principle of adding
specific microbial antigen(s) to patient serum. If a specific microbial antibody is
present in the serum then it will bind to the antigen to form an antigen/antibody
complex. An indicator system is then used to detect whether such a complex has
been formed.
The types of serological methods used are: Agglutination tests
Precipitation tests
Complement fixation tests
Immunofluorescence testing
Enzyme-Linked ImmunoSorbent Assay
(ELISA)

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 Types of serological methods

A. Agglutination is the reaction of an antibody with an antigen and can be

detected by agglutination (clumping) of the antigen. The general term
agglutinin is used to describe antibodies that agglutinate particulate antigens.
Diseases may be diagnosed by combining the patient’s serum with a known
antigen. An agglutinin is an antibody that interacts with antigen on the
surface of particles such as erythrocytes, bacteria, or latex particles to cause
their agglutination in an aqueous environment containing electrolyte. The
most widely known agglutinogens are those of the ABO and related blood
group systems.

Agglutination is the basis for multiple serological reactions including:

 blood grouping,
 diagnosis of infectious diseases
 noninfectious diseases

Numerous techniques have been described for agglutination tests. These

techniques may be performed using: slides, test tubes, or micotiter plates,
depending on the purpose of the test. However the agglutination technique remains
the same.

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Agglutination test can be quantitative and qualitative:
 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. For example, a
patient’s red blood cells can be mixed with antibody to a blood group
antigen to determine a person’s blood type. In a second example, a patient’s
serum is mixed with red blood cells of a known blood type to assay for the
presence of antibodies to that blood type in the patient’s serum. This test can
be done on a slide.

 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.

There are various types of agglutination methods. Direct and indirect are the
two common methods.

1. Direct Agglutination reactions test patient serum for the presence of antibodies
against large, cellular antigens. Antigen is naturally present on the surface of
the cells. In this case, the Ag-Ab reaction forms agglutination, which is directly
visible.

2. Indirect Agglutination tests the patient serum for the presence of antibodies
against soluble antigens serum is mixed with latex spheres with the soluble
antigens attached. Antibodies will then cause visible agglutination of the latex
spheres with the soluble antigens attached. Alternatively, antibodies may be
attached to the latex spheres to test for the presence of soluble antigens in
patient serum.

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B. Precipitation reactions are based on the interaction of antibodies and
antigens. They are based on two soluble reactants that come together to
make one insoluble product, the precipitate. The reaction depends on the
formation of lattices (cross-links) when antigen and antibody exist in
optimal proportions. Precipitation reactions differ from agglutination
reactions in the size and solubility of the antigen which are larder and
sensitivity. There are several precipitation methods applied in clinical
laboratory for the diagnosis of disease. These can be performed in semisolid
media such as agar or agarose, or non-gel support media such as cellulose
acetate.

There are 3 types of Precipitation test:

1. Radial Immunodiffusion(Mancini):- In radial immunodiffusion

antibody is incorporated into the agar gel as it is poured and different
dilutions of the antigen are placed in holes punched into the agar. As
the antigen diffuses into the gel, it reacts with the antibody and when
the equivalence point is reached a ring of precipitation is formed.

2. Immunoelectrophoresis: - A complex mixture of antigens is placed

in a well punched out of an agar gel and the antigens are
electrophoresed so that the antigens are separated according to their
charge. After electrophoresis, a trough is cut in the gel and antibodies
are added. As the antibodies diffuse into the agar, precipitin lines are
produced in the equivalence zone when an antigen/antibody reaction
occurs.

3. Countercurrent electrophoresis: - The antigen and antibody are

placed in wells punched out of an agar gel and the antigen and
antibody are electrophoresed into each other where they form a
precipitation line. This test only works if conditions can be found
where the antigen and antibody have opposite charges. This test is
primarily qualitative, although from the thickness of the band you can
get some measure of quantity. Its major advantage is its speed.

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C. Compliment Fixation Test: - Complement fixation is a classic method for
demonstrating the presence of antibody in patient serum. It consists of two
components; sheep blood RBCs and 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. The Wassermann test is one form
of complement fixation test.

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D. Immunofluorescence testing: - Fluorescent molecules are used as
substitutes for radioisotope or enzyme labels. The fluorescent antibody
technique consists of labeling antibody with dyes such as fluorescein
isothiocyanate (FITC). After the labeling of a specific antibody with a
fluorescent molecule, it can still be reacted with its antigen and identified
microscopically. Fluorescent antibody conjugates are commonly used in
immunoassays.

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E. Enzyme-Linked ImmunoSorbent Assay (ELISA):- 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 and plant pathology, as well as a quality control check in various
industries.

 In simple terms, in ELISA an unknown amount of antigen is affixed

to a surface, and then a specific antibody is washed over the surface so
that it can bind the antigen. This antibody is linked to an enzyme, and
in the final step a substance is added that the enzyme can convert to
some detectable signal. Thus in the case of fluorescence ELISA, when
light is shone upon the sample, any antigen/antibody complexes will
fluoresce so that the amount of antigen in the sample can be
measured.

 Because the ELISA can be performed to evaluate either the presence

of antigen or the presence of antibody in a sample, it is a useful tool
both for determining serum antibody concentrations such as with the
HIV test and also for detecting the presence of antigen. It has also
found applications in the food industry in detecting potential food
allergens such as milk, peanuts, walnuts, almonds, and eggs. The
ELISA test, or the enzyme immunoassay (EIA), was the first
screening test commonly employed for HIV.

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 References

 http://www.surgeryencyclopedia.com/Fi-La/Immunoassay-Tests.html

 http://medical-
dictionary.thefreedictionary.com/immunoelectrophoresis

 http://www.slideshare.net/doctorrao/serology-11426450

 https://books.google.com.vc/books?id=nuCT5RVToN4C&pg=PA172
&dq=immunoelectrophoresis+test&hl=en&sa=X&ved=0ahUKEwin9
92luZrQAhVJ5WMKHTpyAU0Q6AEIGzAA#v=onepage&q=immun
oelectrophoresis%20test&f=false

 https://books.google.com.vc/books?id=HcgGLfxDJSQC&pg=PA110
&dq=immunoelectrophoresis+test&hl=en&sa=X&ved=0ahUKEwin9
92luZrQAhVJ5WMKHTpyAU0Q6AEIMTAE#v=onepage&q=immu
noelectrophoresis%20test&f=false

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