Lecture 2-Serology and Vaccination For Biotechnology 4 Stage

Lecture 2- Serology and Vaccination for
Biotechnology 4th stage

Prf. Dr. Rajwa Hasen Essa
Prf.Dr. Alia Essam Mahmood
Dilution is the act of making a weaker solution from a stronger solution, by
adding water or saline, dilution is usually expressed as one unit of the
original solution to the total number of units of final solution. Since the
dilution-fold is the same in each step, the dilutions are a geometric series
(constant ratio between any adjacent dilutions). Serial dilution is decreasing
the volume of serum progressively by maintaining a constant volume of
fluid. Most commonly serial dilutions are two-fold or ten-fold range of
dilutions, but finally the total volume in each tube is the same.
A general rule for calculating the concentration of solutions
M1V1=M2V2
A titer is a way of expressing concentration, through employs serial dilution
to obtain approximate quantitative information. The titer corresponds to the
highest dilution factor that still yields a positive reading.
Antibody titer is a measurement of how much antibody an organism has
produced that recognizes a particular epitope, expressed as the inverse of the
greatest dilution (in a serial dilution) that still gives a positive result.
The endpoint titer is defined as the reciprocal of the highest analyte
dilution that gives a reading above the cutoff, or the point in a titration at
which a reaction is complete, often marked by a colour change.
Cutoff defines as a level or limit at which something stops.
Cross-reactivity is the ability of an antigen to bind with an antibody that
was raised to a different antigen. It may arise by one of two mechanisms:
shared epitopes on multivalent antigens, or conformational similarity of
epitopes.
Serology-Techniques
Serology refers to using antigen-antibody reactions in the laboratory for
diagnostic purposes. Its name comes from the fact that serum, the liquid
portion of the blood where antibodies are found is used in testing. Serologic
testing may be used in the clinical laboratory in two distinct ways:
a. To identify unknown antigens (such as microorganisms). This is called
direct serologic testing. Direct serologic testing uses a preparation known
antibodies, called antiserum, to identify an unknown antigen such as a
microorganism.
b. To detect antibodies being made against a specific antigen in the
patient's serum. This is called indirect serologic testing. Indirect serologic
testing is the procedure by which antibodies in a person's serum is made by
that individual against an antigen associated with a particular disease are
detected using a known antigen.
Antigen-antibody reactions may be detected in the laboratory by a variety of
techniques. Some of the commonly used techniques for observing in vitro
antigen-antibody reactions are briefly described below.
1- A precipitation reaction is based on the principle of “Antigen-Antibody
Reaction”, which occurs at the zone of equivalence. At the region of
equivalence, the ratio or concentration of both antigen and antibody are
equal which bring out the formation of “Immunocomplex” or lattice or
cross-linked structure at the zone of equivalence where the concentration of
both are equal, both antigen and antibody diffuse either in one dimension or
two dimensions in liquid or semi-solid media, which results in the formation
of the antigen-antibody complex as a visible ring or line of a precipitate.
Precipitin is the antibodies which react with their respective antigens to
form a “Precipitate”, the free antigens and antibodies will remain as the
“Supernatant”. For the precipitation reaction, polyclonal
or multivalent antibodies and antigens are used because if it is not so, the
formation of the lattice will not occur. For the appearance of a precipitin ring
or band, the reaction may take a few hours to days.
2- Agglutination
The reaction between a particulate antigen as bacteria, RBC, latex
particles and an antibody results in visible clumping called agglutination.
Antibodies that produce such reactions are known as agglutinins. The
principle of agglutination reactions is similar to precipitation reactions; they
depend on the cross-linking of polyvalent antigens.
There is no agglutination can be observed when the concentration of the

antibody is high, (lower dilutions), and then the sample is diluted,
agglutination occurs. Prozone effect is defined as the invisibility of
agglutination at high concentrations of antibodies. It is due to the reason that
excess antibody forms very minute complexes that do not clump to form
visible agglutination.
3- Complement fixation test is based on the principle that when antigen and
antibodies of the IgM or the IgG classes are mixed, complement is “fixed” to
the antigen-antibody complex. If this occurs on the surface of RBCs, the
complement cascade will be activated and hemolysis will occur.
4- Radioimmunoassay: A specific laboratory test (assay) that uses
radiolabeled and unlabeled substances in an immunological (antibody-
antigen) reaction, and it is a test procedure that integrates immunologic and
radiolabeling techniques to measure minute quantities of a substance, as a
protein, hormone, or drug, in a given sample of body fluid or tissue.
Basically, radioimmunoassay is based on three principles which give it high
sensitivity:
• A strong immune binding reaction: Antigen vs Antibody reaction
• The competitive binding reaction which gives specificity
• The radio emission gives the specificity
The major advantages of RIA, are higher sensitivity, easy signal detection,
and well-established, rapid assays.
The major disadvantages are the health and safety risks posed by the use of
radiation and the time and expense associated with maintaining a licensed
radiation safety and disposal program. For this reason, RIA has been largely
replaced in routine clinical laboratory practice by other serology techniques.
5- Fluorescent antibody technique

A fluorescent dye is chemically attached to the known antibodies. When the
fluorescent antibody reacts with the antigen, the antigen will fluoresce when
viewed with a fluorescent microscope.

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Lecture 2- Serology and Vaccination for

Biotechnology 4th stage

There is no agglutination can be observed when the concentration of the

5- Fluorescent antibody technique

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