Lab 7
The Lymphatic System and Immune Response - Exercise 35A
Lecture in Lab - Lymphatic and Immune Systems
Lymphatic System and Immune Response
Activity 1: Identifying the Organs of the Lymphatic System
Activity 2: Studying the Microscopic Anatomy of a Lymph Node…, 1.
Use the ileum with Peyer's Patches slide and identify Peyer's Patches.
Use Plate 35 as a reference.
ELISA Simulation
Follow directions in the handout.
ELISA Simulation
(Adapted from Instructor’s Guide – Carolina Biological)
This kit explores how the principles of antibody-based human immunity apply
to a common laboratory test called ELISA (enzyme-linked immunoabsorbant
assay). ELISA is commonly used to test blood serum for the presence of
antibodies against disease-causing pathogens such as viruses and bacteria. This
activity will investigate 6 patients for either HIV, Lyme Disease, Avian Influenza
(Bird Flu) or West Nile virus. There is no risk of infection from any materials in
this kit.
Antibodies are a group of serum proteins (the Igs) that are in the blood or bound
to cell membranes. Antigen binding sites fit the shapes of specific antigens.
They bind in a lock-and-key manner to form antigen-antibody complexes (see
Figure 1).
ELISA is based on the principle that antibodies attach to their antigen targets
with great specificity to form antigen-antibody complexes. This particular test is
called indirect ELISA and tests the patient’s blood for antibodies to the infectious
agent. The basic steps are outlined in Figure 2.
1. Proteins from the infectious agent are added to the wells of plastic microtiter
plates. These antigens bind to the bottom of the plastic well. Unbound material
is removed with a buffer wash.
2. Blood serum from the patient is added to the wells. If antibodies to the
antigens are present they will bind to the antigen and form antibody-antigen
complexes. The wells are again washed with buffer. You cannot see the antigenantibody complexes by eye, so detection steps are used to visualize them.
3. An antibody (secondary antibody) that recognizes human antibodies is added
to the wells. If human antibodies are present the secondary antibody binds to
the human antibodies (which in this case are the antigen!) and forms antigenantibody complexes. The wells are rinsed again with buffer.
4. The secondary antibody has an enzyme attached to it. This is called a
conjugated antibody. When a special chemical called a chromogen is added to
the wells, if the enzyme is present the chromogen changes color. The intensity of
the color is related to the amount of antigen-antibody complex present.
5. No change in color indicates either no infection or no antibody response to the
infection.
6. Samples are usually tested in triplicate to ensure reproducibility. Samples in
clued positive and negative controls. The washes are very important and it is
also important to use a clean pipet for each new sample or reagent.
An Example of an ELISA test. Dark-colored wells indicate more antigenantibody complexes present than in lighter colored wells. Clear solution means
no antigens present in the sample or no antibodies present in the serum.
Name____________________________
Review Sheet
Lab Section ________________
Turn in at the next Lab
A
B
C
D
E
F
1. Match the labels on the figure with the terms below.
___ Axillary nodes
___ Inguinal nodes
___ Cervical nodes
___ Right lymphatic
___ Cisterna chyli
duct
___ Thoracic duct
A
B
C
D
2. Match the labels on the figure with the terms below.
___ Peyer's patches
___ Spleen
___ Thymus
___ Tonsils
3. What is the source of lymph in the lymphatic system?
4. How is lymph propelled through the lymphatic vessels?
5. Name two veins where lymph is returned to the circulatory system.
a. ____________________________________
b. ____________________________________
6. Describe two ways that lymph vessels are structurally similar to veins.
a.
b.
7. Why is lymph often cloudy or milky looking in the cisterna chyli?
________________________________________________________________________
C
A
D
E
B
F
8. Match the labels on the figure with the terms below.
___ Afferent vessels
___ Capsule
___ Cortex
___ Efferent vessels
___ Germinal center
___ Medullary cords and sinuses
9. Why are there fewer efferent than afferent vessels?
10. If you are looking for the following immune system cells in a lymph node, where
would you look?
B cell
____________________________
T cell
____________________________
Macrophage
____________________________
11. Match the following structures with the correct function.
___ B cells
___ Lymph node
___ Macrophage
___ Spleen
___ Thymus
A. T cells develop self-tolerance and immunocompetence here
B. Major site for presentation of antigens to B and T cells
C. Removes damaged red blood cells and other debris from circulation
D. Produce clones of antibody- producing plasma cells
E. Large phagocyte which also acts as an antigen presenting cell
ELISA
Record your Group Data Here
Questions
1. In what way is the ELISA assay similar to blood typing?
2. Why is this called an indirect method? In other words, how does it detect infection
by a disease-causing agent?
3. What is the function of the secondary antibody and chromogen in an ELISA?
4. Why did you perform three identical tests for the control and patient samples?
5. Why might some positive results be lighter than others?
6. Describe the disease-causing pathogen you assayed for. Include its mode of
transmission to people. 2 points
7. Evaluate your data. What is the diagnosis for each of the patients tested?
A.
B.
C.
D.
E.
F.
8. Did any of the outcomes surprise you? If so, why?
9. For patients who tested positive, how might they have prevented infection?
Name __________________________________
Pre-Lab Questions – Respiratory System
1. The major role of the respiratory system is
2. Upper respiratory structures include the nose, the larynx and the
a. epiglottis
b. lungs
c. pharynx
d. trachea
3. The most prominent (largest) of the laryngeal cartilages is the ___________________
cartilage.
4. Air flows from the trachea into the right and left _________________________ .
5. What is the role of the respiratory epithelium in the trachea?
6. The main structural and functional units of the lungs are the _______________ .
7. In the human there are __________ lobes in the right lung and __________ lobes in
the left lung.
8. When air passes out of the lungs it is called
(circle one)
inspiration
expiration .
9. During normal quiet breathing about _________ ml of air moves into and out of the
lungs.
10. The maximal amount of air that can be exhaled after a maximal inhalation is called
______________________________ .