Download lab 17 immunological testing

MICROBIOLOGY LAB MANUAL
LAB 17 IMMUNOLOGICAL TESTING
Learning objectives, by the end of the lab period the student should be able to:
1. Name and describe the functions of each cell of the non-specific (innate) and specific
immune systems.
2. Describe the nature, origin and function of antigens and antibodies.
3. List the components of our first line of defense against infection.
4. List the second line of defense components which include: bloodborne chemicals, protective cells
and processes that inactivate or kill invaders.
5. Understand how the third line of defense differs from the first two, its components and processes.
6. Distinguish among natural active and natural passive immunity, artificial active and artificial
passive immunity.
7. Accurately perform an immunological agglutination test, and correctly interpret both negative and
positive results.
8. Successfully perform, read, and interpret results based on commercial package inserts.
9. Explain the immunological reaction that occurs in an agglutination test.
10. Explain the importance of one of the pathogens identified by the kit you chose; i.e: Sreptococcus
pyogenes Salmonella typhii, Shigella flexneri, Shigella sonnei, Cryptococcus neorformans as a
pathogen, and enumerate the diseases it can cause and the symptoms of each.
11. Some kits such as the RPR and Rheumaton factor do not target a specific organism, explain how
these kits work and why are they useful.
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12. Explain how serological kits differ from traditional identification methods and list their
advantages and disadvantages.
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Introduction
The vertebrate immune system has developed a multi-pronged mode of attack against foreign
invaders. 1. Non-specific or innate immunity, which involves phagocytosis, inflammation, and
cytotoxic responses. The non-specific cytotoxic response is mediated by Natural Killer (NK)
lymphocytes. All of these responses attack without regard to the specific identity of the foreign
invader. 2. Specific immunity involves responses by T-and B-lymphocytes against specific foreign
invaders.
Specific immunity has two arms. The first is humoral immunity, which involves antibody
production by B-lymphocytes, plasma cells and memory B-cells. After stimulation by an antigen,
B-cells become activated, which involves proliferation (clone production by rapid cell division
into thousands of daughter cells) and differentiation (conversion into active, antibody-secreting
Foreign Antigen (ag)
Fungus, Bacteria, Parasite,
Virus, Allergen, Toxin, etc
Non-specific or Innate Immunity
Phagocytosis, inflammation,
cytotoxic response
Specific Immunity
T and B Lymphocytes
Humoral Immunity =
B cells and antibodies (abs)
[Plasma cells and memory cells]
Cellular Immunity = cells
Antibodies are blood proteins, also known as immunoglobulins, which attach themselves to
antigens, identifying the foreign body as a target destruction by other immune system cells. When
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cells). Most activated B-cells become antibody-producing factories called plasma cells. A few
activated B-cells become memory B-cells, which mostly lie dormant, waiting to be activated in
large numbers, should the antigen be encountered in a subsequent infection.
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an antibody attaches to its antigen, it forms an antigen-antibody complex or immune complex.
Immune complexes result in:
a. agglutination or clumping of cells with the antigen on their surface,
b. precipitation of soluble antigens,
c. chemotaxis (attraction of phagocytes, such as neutrophils, monocytes and macrophages),
d. degranulation of basophils and mast cells (which secrete inflammation-promoting substances),
and
e. opsonization (enhancement of phagocytosis).
Other white blood cells phagocytize the immune complexes, directly attack on fungal, protozoan and
multicellular parasites, and moderate of inflammation.
Cell-mediated immunity involves T-lymphocytes of various types that orchestrate the immune
response by secretion of lymphokines (protein messenger molecules, which activate or deactivate
immune system cells). In order to do their job, T-cells must be sensitized by contact with a partly
digested or processed antigen, which is presented to them by antigen-presenting cells (APC), such
as tissue macrophages, dendritic macrophages intestinal epitheliocytes, or B-lymphocytes.
Antigen-presenting cells secrete the lymphokine, interleukin-1, which attracts T- and Blymphocytes and stimulates them to examine presented antigens.
T-lymphocytes include a variety of cells with different roles involved in hand-to- hand combat:
helper T-cells, which activate themselves upon contact with their antigen, then secrete the
lymphokine, interleukin-2 that enables other T-cells and B-cells to be activated upon exposure to
their antigen.
cytotoxic T-cells carry out direct attacks by secreting perforin, a protein molecule that
punctures cell membranes of foreign, abnormal and parasitized cells, causing them to die from
cytotoxic lysis. Cytotoxic T-cells also secrete tumor necrosis factor, a protein that causes abnormal
and parasitized cells to “commit suicide” through a process called apoptosis.
delayed hypersensitivity T-cells secrete lymphokines, which promote inflammation, and
attract, retain and activate macrophages to superior size, activity levels and killing power. Tregulatory cells (T-reg cells) secrete lymphokines, which moderate, control and eventually shut
down the immune response after it has done its job.
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It was long recognized that those who had contracted a disease and recovered were immune to
subsequent attacks of the same disease. This type of immunity is referred to as naturally-acquired
active immunity. Artificial stimulation of this protection (Immunization) is an important aspect of
present-day healthcare which protects a person without requiring them to actually catch the disease.
Immunization has been used since ancient times to activate the immune system and induce
immunity to disease; artificially-acquired active immunization was used by traditional healers in
China, India and Europe to hopefully infect a person with a “mild” case of smallpox by injecting
them with the exudate from active smallpox lesions. In actual practice, this was a crap shoot,
because as often as not, the patient would end up with a severe form of the disease. In the late 1700s,
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MICROBIOLOGY LAB MANUAL
Edward Jenner in England used cowpox (vaccinia) to induce immunity to smallpox (variola), thus
introducing the practice of vaccination, or artificially-acquired active immunity. Vaccination
uses a dead or attenuated germ, or even a portion of a germ or its secretions, to induce an immune
response without producing serious disease. Artificially-acquired passive immunity can be
conferred by injecting a non-immune person with blood plasma or gamma globulin from an immune
person. Naturally-acquired passive immunity is transferred with antibodies from the mother’s
blood to the baby’s blood in utero, and in the mother’s milk to the nursing infant. In each of these
cases, active immunologic responses provide longer term protection from re-infection, measured in
years, whereas passively acquired immunization provides only short term protection from infection
usually effective only for a few months.
Immunology started during the 1800s, when it was recognized that blood transfusion reactions
sometimes killed patients, but sometimes did not. Using the agglutination reaction, it became
possible to type blood for safe transfusion as early as World War I. A host of other diagnostic
immunological tests have been developed to identify the presence of microbial antigens and to
identify the presence of antibodies in a patient’s blood.
A vast number of in vitro tests have been developed, which involve testing clinical or cultural
samples in the laboratory. These include agglutination tests (such as the blood typing tests referred
to above), in which suitable ratios of antibodies are present with antigens. Either the antigen or the
antibody may coat red blood cells or latex spheres, which will normally form a suspension when
dispersed in aqueous liquid. When the complementary antibody or antigen is added, the suspension
particles will stick together to form clumps, which are visible to the naked eye, or by low-power
magnification.
LAB ACTIVITIES
Your activities for this lab are divided into 3 parts:
I. Preliminary Activities - Complete the definitions, questions and tables before coming
to class on the day this lab begins.
II. Laboratory Activities - Complete these activities during the first half of lab class.
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III. Body Defenses Video - View the video and answer the questions during the second
half of lab class.
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MICROBIOLOGY LAB MANUAL
I. Preliminary Activities
A. Define the following terms:
1. Agglutination test
2. Innate or nonspecific immune system
3. Antigen (include origin and function)
4. Antibodies (include origin and function)
5. List the components of our first line of defense against infection.
6. List the second line of defense components which include: bloodborne chemicals, protective
cells and processes that inactivate or kill invaders.
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7. Explain how the third line of defense differs from the first two, its components and
processes.
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B. You will be examining a variety of kit in this lab, but each group will look at just one kit. Each
group will report out to the rest of the class concerning the following information:
Identify the Name of your kit: _________________________________________
Name the major diseases caused by the syndrome or condition or pathogen identified by your kit.
Use your text to describe the typical signs and symptoms a patient would describe to the doctor that
would indicate this kit should be used.
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This kit was manufactured because it is important to have a specific clinical diagnosis for this
particular condition, syndrome or etiologic agent. Why is a specific diagnosis necessary?
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C. Distinguish between the different types of immunity:
Type of
Immunity
Description
How is it
produced?
How long does it last?
INNATE
SPECIFIC
NATURALLY
ACQUIRED
ACTIVE
ARTIFICIALLY
ACQUIRED
ACTIVE
NATURALLY
ACQUIRED
PASSIVE
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ARTIFICIALLY
ACQUIRED
PASSIVE
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MICROBIOLOGY LAB MANUAL
II. Laboratory Activities
A. Materials
1. Choose a kit from those provided. All necessary materials will be included with the kit.
B. Procedure
1. Follow the directions on the package insert carefully. Perform the test on the controls provided
with the kit. Record your results:
C. Answer these questions about your kit. Answer the first set of questions if your kit identifies an
etiologic agent and the second set of questions if your kit identifies a condition or syndrome.
Etiologic Agent Questions
1. If your kit identifies an etiologic agent, describe the size, staining, morphology and grouping of
cells for a pathogen identified by your kit.
2. Draw a picture of the pathogen.
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4. List advantages and disadvantages of using this kit as compared to traditional identification
methods.
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3. Describe in detail how the test works, what is the principle?
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5. Discuss sensitivity and specificity for this immunological method.
6. Discuss how you can obtain a false positive and a false negative result.
Condition or Syndrome Kits
1. What are the characteristics exhibited by a patient that would be positive with this kit?
2.
Why does the kit include positive and negative controls?
3.
Describe in detail how the test works, what is the principle?
4. List advantages and disadvantages of using this kit as compared to traditional identification
methods.
5. Discuss sensitivity and specificity for this immunological method.
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6. Discuss how you can obtain a false positive and a false negative result.
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III. Body Defenses Video
Key Concepts
I. Three main Lines of defense
A.
Barrier – blockading the portal of entry
B.
Non-specific response -
C.
Specific Response – detailed and precise response to pathogens that “make it
through the lines.”
II. Vaccinations
A.
Attenuated
B.
Dead organisms
C.
Bioengineered subunits
Video Concepts
1. List the major barriers (first lines of defense) microorganisms must overcome to invade the body.
2. What cells are responsible for the second line of defense? What action do they take against foreign
objects? What happens to the invaders?
3. The second line of defense actually initiates a response from the third line. It identifies the
specific antigen that must be attacked. What cells are important to the third line of defense? What is
the strategy used here?
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4. Normally the body is not capable of both identifying and specifically attacking an invader before
symptoms of the assault (illness) occur. Explain the theory behind the use of vaccines and the type of
immunity found in vaccinated people.
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