MICROBIOLOGY 101 LABORATORY MANUAL
EXERCISE #21: IMMUNOLOGY, AGGLUTINATION OF CELLS
NAME, ID #:_______________________________________________.
TA Name __________________________
REVISED: 08/04/99
The definition of an antibody and an antigen are MUTUALLY
INTERDEPENDENT in that you can not have one without the other.
Figure 1. An antigen with 4 epitopes. Each epitope is a unique grouping of amino acids that induce the
formation of a unique/specific antibody that will only bind to that epitope on the one
antigen.
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- An ANTIBODY is a specific type of protein that is
produced by an organism in response to the presence of a specific ANTIGEN
(which also is usually a protein).
- An ANTIGEN is any group or arrangement of molecules
that induces or elicits the production of the special proteins that comprise that
collection of proteins known as ANTIBODIES.
- Antigens & antibodies are defined by the ability of the antibody to SPECIFICALLY BIND to an antigen. The particular arrangement of
atoms on the antigen to which the antibody binds is called the EPITOPE.
A given antibody will usually only bind to one epitope.
Antigen/antibody reactions are EXACTLY LIKE
enzyme/substrate and virus attachment proteins/cell receptor interactions in that they are
the result of SPECIFIC BINDING
of a protein to another molecule. ALL ANTIGEN/ANTIBODY
reactions are so specific that a change in a SINGLE ATOM
in either the binding protein or its target molecule can results in a loss of binding. As
explained in #Chapter 16, the human immune system can make over
100 million unique and equally discriminating antibodies. Antibodies are produced by
specific blood cells, B-cells, that are selected for (CLONAL
SELECTION) by the presence of the antigen to which they specifically bind.
After a series of complex differentiation's the B-cells turn into #PLASMA cells that are
factories for the production of ONE SPECIFIC ANTIBODY.
Antibodies, because of their specificity, are wonderful tools for the identification of
antigens. Since their discovery antibodies have been used to diagnose or detect disease
antigens. The antigen/antibody detection system can work in either direction. For example,
if you suspect that a certain antigen may be present, say in a blood or urine sample,
antibody that binds specifically to that antigen can be used to detect its presence.
Conversely, if you suspect that a certain antibody is present in a sample you can use its
target antigen to collect that antibody and detect its presence.
Figure 2. Basic IgG antibody structure. Click here for
the best description of antibodies on the Internet (You will need to download Chime
first).
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The uses of antibodies in the identification of disease organisms, in
cancer cell identification, and in a plethora of other identifications is growing. New
techniques using antibodies are being developed that allow the detection of as few as 5
HIV virus particles in a patient's sample. Techniques for isolating B-cell cell-lines that
produce large quantities of a single, specific antibody have been available for >20
years. The antibodies produced in this way are called MONOCLONAL
(mono = one) antibodies. They are beginning to be used to fight cancer. In this procedure
an individual's cancer cells are isolated and a cancer antigen is used to select for a
B-cell line that produces antibodies against an antigen specific to that cancer. The
B-cells are propagated and the large quantities of the unique anticancer monoclonal
antibody produced are used to treat the original cancer. Reports of success using this
technology were reported in 1995.
The instructors will describe how blood typing (A, B, O & Rh) is carried out and
will explain its significance.
PURPOSE OF LABORATORY:
 Figure 3. An example of antibodies clumping
antigens. The interaction of antibodies, with their dual binding arms reacts with
antigen to produce large aggregates that result in agglutination. If you imagine that the
"blue stars" are bacteria or red blood cells you will understand how
agglutination occurs.
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- To demonstrate the use of the agglutination test (antibodies) in the identification of
specific cells.
- Students who wish so may have their blood typed under the supervision of Ms. K. Malm who
has been certified to work with human blood.
RELATIONSHIP TO LECTURE MATERIAL
- INTERNET LECTURE: CHAP. XVI , Specific immunity, blood
testing.
MATERIALS
- Certified Human blood for determining O, A, B, AB blood types.
- Anti-A and -B antibody
- Bacterial cultures; positive and negative controls, labeled appropriately
- Antibody for determining bacterial coagulase characteristic
- Toothpicks
- Clean slides and marking pens
- An individual certified to test human blood.
- Agglutination material for testing for Staphylococcus
GENERAL INSTRUCTIONS:
- Please place all appropriately labeled drawings on the back of the manual so the
instructor can identify them.
PROCEDURE:
BACTERIAL TESTING
- Read pg. 106-9 in A Photographic Atlas for the Microbiology
Laboratory.
- Per pair, obtain a sample of the appropriate bacterial cultures.
- Using a wax pencil draw 3 circles, about 1.5 cm in diameter on a clean slide and label
the middle one "C" for control and the outer two the same as the two cultures.
- Place a loop of the control bacterial culture in the central control circle. Place a
loop of each of the two unknown bacteria in the labeled outer circles, being sure to FLAME
the loop between the samples.
- Add a drop of the antisera to each of the outer bacterial drops. Mix the drops with the
loop, being sure to FLAME & COOL it between each mixing.
- Examine the suspensions in the three circles and compare them, looking for agglutination of
the latex particles
as shown in the Atlas.
- From these results determine which of the bacterial samples contains antigen that reacts
with the anti-coagulase antibody. Place your results on the board by your names.
BLOOD TESTING
- Per pair, obtain the sample of human blood (screened to be free of HIV) and the antibody
kits containing anti-A and anti-B antibody.
- Using a clean slide, divide it in half and mark on the bottom A and B.
- Place one drop of the anti-A antiserum on side A and one drop of anti-B on side B.
- Using an eye-dropper, mix one drop of blood into each drop of antiserum, being careful
to not TOUCH the dropper to the serum and contaminate it.
- Mix the serum and the blood gently with clean toothpicks, using a fresh one for each
sample. Discard the toothpicks appropriately.
- Continue to mix the sample by gentle rocking as demonstrated by the Instructor for about
2 minutes.
- Examine the results for agglutination as shown in the Atlas. Determine the blood type of
the blood sample(s). Complete this before doing your own blood typing.
- If you desire to have your own blood typed follow the instructions of Ms Malm.
SAMPLE QUESTIONS: You should be able
to answer these questions at the conclusion of this laboratory.
- Explain how the change of a single atom in an epitope can prevent the binding of an
antibody?
- Explain how antigen/antibody associations are related to enzyme/substrate associations?
- Define B-cells and Plasma-cells and their role in antibody production.
- Explain the "clonal selection theory" of antibody formation.
- Explain what a "universal donor" and a "universal recipient" are
with regards to the A, B, O blood groupings.
- Explain what blood types persons with A, B, AB and O can receive and why?
- Explain what can happen is an Rh- woman carries a Rh+ baby and why.
- What is the importance of determining the "COAGULASE" characteristic of S.
aureus?
Copyright © Dr. R. E. Hurlbert, 1999.
This material may be used for educational purposes only and may not be duplicated for
commercial purposes.
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PHONE: 509-335-5108
FAX: 509-335-1907
E-mail address: hurlbert@wsu.edu or hurlbert@pullman.com
OFFICE HOURS: Mon., W ed.2-4 PM.
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