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The Biotechnology Education Company®
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279
EDVO-Kit #
Immunology
of Pregnancy Tests
Storage:
Store the entire experiment
in the refrigerator.
EXPERIMENT OBJECTIVES:
The objective of the simulation experiment is to
introduce concepts and experimental procedures
involved with enzyme linked immunosorbent assays
(ELISA) in the context of testing for pregnancy.
All components are intended for educational research only.
They are not to be used for diagnostic or drug purposes, nor
administered to or consumed by humans or animals.
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EDVO-Kit # 279
Immunology of Pregnancy Tests
Table of Contents
Experiment Components
Experiment Requirements
Background Information
Immunology of Pregnancy Tests
Page
3
3
4
Experiment Procedures
Experiment Overview
Student Experimental Procedures
Study Questions
7
8
11
Instructor's Guidelines
Notes to the Instructor
Pre-Lab Preparations
Quick Reference Tables
Avoiding Common Pitfalls
Expected Results
Study Questions and Answers
13
14
16
17
17
18
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EDVO-Kit # 279
Immunology of Pregnancy Tests
Experiment Components
A
B
C
D
E
F
G
H
hCG Antibody (simulated)
Positive Control
Urine Sample Patient 1 (simulated)
Urine Sample Patient 2 (simulated)
Anti-hCG-peroxidase conjugate
Hydrogen peroxide, stabilized
Peroxide co-substrate
Phosphate buffered saline concentrate
•
•
•
•
Microtiter strips
Transfer pipets
Microtest tubes with attached caps
15 ml plastic tubes
This experiment
is designed for
10 groups.
Store entire
experiment in the
refrigerator.
Requirements
•
•
•
•
•
•
Distilled or deionized water
Beakers
37°C Incubation oven
Disposable lab gloves
Safety goggles
Automatic micropipets (0 - 50 μl) and tips recommended
Make sure glassware is clean, dry and free of soap residue.
For convenience, additional disposable transfer pipets can be purchased for liquid removal and washing steps.
All components are
intended for educational
research only. They
are not to be used
for diagnostic or
drug purposes, nor
administered to or
consumed by humans or
animals.
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4
EDVO-Kit # 279
Immunology of Pregnancy Tests
Background Information
Immunology of Pregnancy Tests
The female reproductive organs consist of the ovaries, the ovarian tubes,
the uterus and the vagina. The principal function of the female reproductive system is to produce the ova and to provide the environment for
fertilization and nurturing of the ovum to make possible normal growth
to a mature fetus. The final step is to deliver the offspring to the outside
environment.
The human female usually releases a single ovum per ovulation cycle.
Upon ovulation the ovum is released into the ovarian tube. It is in the
ovarian tubes that the ovum may be fertilized. It then passes into the
uterus where it implants into the wall of the uterus. In the uterus, the fertilized ovum grows to become a fetus. Synchronized orchestration of a
number of hormones mediates the events that are involved in ovulation.
These hormones are produced in the pituitary and in the ovary itself and
bring about their effects by circulating in the blood stream.
During each female menstrual cycle, groups of cells (follicles) grow in the
ovary. Although many follicles will grow, only one follicle will develop to
maturity while the others will degenerate. The pituitary gland produces
two hormones, luteinizing hormone (LH) and follicle stimulating hormone
(FSH). At the beginning of the cycle, FSH stimulates growth of the follicle,
which begins to secrete estrogen. The estrogen stimulates proliferation of
the inner lining (endometrium) of the uterus and stimulates the synthesis of
progesterone receptors in preparation for higher levels of progesterone
later in the cycle.
As the menstrual cycle progresses, estrogen levels rise and reach a peak
approximately midway in the menstrual cycle. Decreasing levels of estrogen then bring about a surge in LH production, which induces ovulation.
After ovulation, the follicle forms the corpus luteum, named after its yellow appearance, that produces high levels of progesterone. This progesterone is required for the preparation and upkeep of the endometrium
that is essential for the nourishment of the implanted fetus.
The corpus luteum requires LH to function. If fertilization does not occur,
decreasing LH levels cause disintegration of the corpus luteum. The reduced progesterone levels then lead to breakdown of the surface of the
endometrium, which is released during menstruation. After 4-5 days, menstruation stops and the cycle repeats. Human female menstrual cycles
vary in length amongst individuals. Taken as an average, it is 28 days long
starting from the first day of vaginal bleeding.
If implantation does occur, the fetal trophoblast makes human chorionic
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EDVO-Kit # 279
Immunology of Pregnancy Tests
5
Immunology of Pregnancy Tests
The term “pregnancy test” is actually is a misnomer since most pregnancy
tests do not actually determine pregnancy but rather the level of hCG.
The hormone is a glycoprotein, which is made up of two subunits, the
alpha subunit which has a molecular weight of 18,000 and the beta chain
which has a molecular weight of 32,000. It should be noted that several
glycoprotein hormones such as hCG, LH and FSH share a common alpha
subunit. The pregnancy test detects only the unique beta-subunit of hCG.
Since hCG is only produced in the developing embryo (except in rare
cases of secretion of hCG by hydatidiform moles or choriocarcinoma),
and since it is detectable within a few days of implantation, it is a very
specific and early test for pregnancy.
Originally pregnancy tests required the injection of urine from suspected
pregnant women into female rabbits. The hCG in the urine brought about
physiological changes in the reproductive system of the rabbit, which
were detected by examination upon sacrificing the animal. This has long
been replaced by immunological assays based on the ELISA test.
ENZYME LINKED IMMUNOSORBENT ASSAY (ELISA)
Enzyme linked immunosorbent assay (ELISA) tests were originally developed for antibody measurement. These immunoassays have also been
adapted to successfully detect samples that contain antigens.
This ELISA simulation experiment has been designed to detect a hypothetical patient's circulating level of hCG. ELISAs are done in microtiter
plates or strips which are generally made of polystyrene or polyvinyl chloride. The plates or strips contain many small wells which are somewhat
transparent and in which liquid samples are deposited. First, the antibody
against hCG is added to the wells where some remain adsorbed by hydrophobic association to the walls after washing away the excess. There
is no specificity involved in antigen or antibody adsorption although some
substances may exhibit low binding to the microwell walls. In certain
cases the antigens can be covalently cross-linked to the plastic using UV
light. After washing away unadsorbed material, the unoccupied sites on
the walls of the plastic wells are blocked with proteins, typically gelatin or
bovine serum albumin.
In this simulation experiment, hCG present in the urine sample will bind to
the adsorbed antibody in the well and remain there after washing.
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Background Information
gonadotropin (hCG), a hormone that acts like LH and prevents disintegration of the corpus luteum and thus assures a continued supply of
progesterone. hCG is made throughout pregnancy by the placenta.
6
EDVO-Kit # 279
Immunology of Pregnancy Tests
Immunology of Pregnancy Tests
Background Information
If hCG has remained bound to the anti-hCG antibody in the well, then
the secondary antibody will bind to another antigenic determinant on
the hCG. This complex will remain attached after washing. The second
antibody to hCG is purified and covalently cross linked to an enzyme with
a high turn over number such as horseradish peroxidase. This modification does not significantly affect the binding specificity and affinity of the
antibody or the enzymatic activity of the peroxidase.
After washing the well to remove unbound secondary antibody, a
solution containing hydrogen peroxide and aminosalicylate is added
to each well. Peroxidase possesses a high catalytic activity and can
exceed turnover rates of 106 per second. Consequently, amplification
of a positive sample can occur over several orders of magnitude. Many
hydrogen donor co-substrates can be used by peroxidase. These cosubstrates include aminodiansidine, aminoantipyrine, aminosalicylic acid
and numerous phenolic compounds that develop color upon oxidation.
The substrate solution added is nearly colorless. Peroxidase converts the
peroxide to H2O + O2 using the salicylate as the hydrogen donor.
Schematic for ELISA
Substrate
(Colorless)
Enzyme
Product
(Color)
It should be noted that polyclonal antibody
preparations to a given antigen can have
variable binding affinities due to differences
in the immunological responses between
animals. Different immunizations with the
same antigen in the same animal can also
produce variable binding affinities. The use
of monoclonal antibodies directed against
a single epitope eliminates this variability
and makes the ELISA highly specific of hCG
detection.
In this experiment, students will use the ELISA
test to determine pregnancy of four hypothetical patients.
Secondary Antibody,
conjugate to hCG
Antigen to hCG
Antibody to hCG
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EDVO-Kit # 279
Immunology of Pregnancy Tests
7
Experiment Overview
EXPERIMENT OBJECTIVE:
LABORATORY SAFETY
Wear gloves
and safety
goggles
Gloves and goggles should be worn routinely as good
laboratory practice.
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Experiment Procedures
The objective of this simulation experiment is to introduce concepts and
experimental procedures involved with enzyme linked immunosorbent assays (ELISA) in the context of testing for pregnancy.
8
EDVO-Kit # 279
Immunology of Pregnancy Tests
Student Experimental Procedures
Experiment Procedures
GENERAL INSTRUCTIONS AND PROCEDURES
Labeling the Microtiter Strip:
Equilibrate a 37°C
incubation oven before
starting the experiment.
•
•
•
Place the microtiter strip as shown in Figure 1.
Carefully mark the strip with your initials or lab group number and
number the wells 1-4 down the side.
Do not separate the reaction wells.
Labeling the Plastic Transfer pipets:
Label 5 transfer pipets as follows:
•
•
•
•
•
Figure 1
( - ) (negative)
(+ ) (positive)
USP 1 (Urine Sample Patient 1)
USP 2 (Urine Sample Patient 2)
PBS (Phosphate Buffered Saline)
Use the appropriately labeled plastic transfer pipet for sample additions,
removals, and washes as outlined in the experimental procedures. If using transfer pipets to add reagents to the wells, label 3 additional pipets
"hCG Ab", "hCG Ab2" (hCGAb-HRP conjugate), and "substrate".
INSTRUCTIONS FOR ADDING LIQUIDS AND WASHING WELLS
Adding Reagents to Wells:
If available,
reagents should
be dispensed with an
automatic micropipet
using disposable tips.
For adding reagents to the wells, use the labeled transfer pipets or use an
automatic micropipet and disposable tips.
Liquid Removal and Washes:
When instructed in the experimental procedures, remove liquids with the
appropriately labeled transfer pipet, and then wash the wells as follows:
A. Use the transfer pipet labeled "PBS", to add PBS buffer to each of the
wells. Add PBS buffer until each well is almost full.
The capacity of each well is approximately 200 μl. Do not allow the
liquids to spill over into adjacent wells.
B.
With the appropriately labeled transfer pipet, remove all the liquid
(PBS buffer) from each of the wells. Dispose the liquid in the beaker
labeled "waste".
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EDVO-Kit # 279
Immunology of Pregnancy Tests
9
Student Experimental Procedures
ENZYME LINKED IMMUNOSORBENT ASSAY (ELISA)
1.
To all 4 wells, add 50 μl or 3 drops of “hCG Ab” (human chorionic
gonadotropin antibody). If using a transfer pipet, use the one
labeled "hCG Ab".
2.
Incubate for 5 minutes at room temperature.
3.
Remove all the liquid (hCG antibody) with a transfer pipet.
4.
Wash each well once with PBS buffer as described above ("Liquid
Removal and Washes").
Adding Reagents:
Be sure to use a fresh tip for each
reagent (Steps 1, 5, 9, & 13).
Alternatively, use the appropriately
labeled transfer pipet for each
reagent.
Liquid Removals:
In research labs, following this step, all sites on the microtiter strip
are saturated with a blocking solution consisting of a protein mixture, such as BSA. We have designed this experiment to eliminate
this step to save time.
Use the appropriately labeled transfer
pipet to remove all liquid from each of
the wells. (steps 3, 7, & 11) and after
washes (steps 4, 8 & 12)
Transfer pipet
Transfer pipet
Transfer pipet
Transfer pipet
(-)
(+)
USP 1
USP 2
Well 1
Well 2
Well 3
Well 4
5.
Remember to use the appropriately labeled transfer pipets provided with this experiment for adding a new reagent. If you are
using automatic micropipets, use a clean micropipet tip for each
reagent.
Dispose the liquid into a beaker
labeled "waste".
Washes:
For all wells, use the transfer pipet
labeled "PBS" to add PBS until each
well is almost full. (Steps 4, 8, & 12)
Quick Reference:
The positive control contains
hCG. Negative urine samples will
not contain hCG. Urine samples
obtained from a pregnant patient
will contain hCG and thus have a
positive result.
To Test Patient 1, add reagents as outlined below:
•
Add 50 μl or 3 drops of PBS Buffer to the first well.
(This is the negative control.)
•
Add 50 μl or 3 drops of "+" (positive) to the second well.
(This is the positive control.)
•
Add 50 μl or 3 drops Urine Sample from Patient 1 “USP 1” to
the third well.
•
Add 50 μl or 3 drops Urine Sample from Patient 2 “USP 2” to
the fourth well.
6.
Incubate at 37°C for 15 minutes .
7.
Remove all the liquid from each well with the appropriately labeled transfer pipet.
8.
Wash each well once with PBS buffer (as described under "Instructions for Adding Liquids and Washing Wells" on page 8).
Optional Stopping Point: The experiment can be stopped after step 8,
but requires that PBS be added to all the wells for overnight storage at room
temperature. The experiment can be resumed during the next lab period.
Remove the PBS and continue with step 9.
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Experiment Procedures
REMINDERS:
10
EDVO-Kit # 279
Immunology of Pregnancy Tests
Student Experimental Procedures
Experiment Procedures
ENZYME LINKED IMMUNOSORBENT ASSAY, CONTINUED
9.
Add 50 μl or 3 drops of the anti-hCG peroxidase conjugate
(hCG Ab2) to all 4 wells of each strip. If using a transfer pipet, use the
one labeled "2°Ab".
10. Incubate at 37°C for 15 minutes.
At this time you can obtain the substrate to be used in step 13. Since
the substrate must be prepared just prior to use, your instructor will
prepare it towards the end of the incubation in step 10.
11. Remove all the liquid from each well with the appropriately labeled
transfer pipet.
12. Wash each well once with PBS buffer (as described under "Liquid
Removal and Washes").
13. Add 100 μl or 5 drops of the substrate to all 4 wells. If using a transfer
pipet, use the one labeled "substrate".
14. Incubate at 37°C for 5 minutes.
15. Remove the strip for analysis.
16. If color is not fully developed after 5 minutes, incubate at 37°C for a
longer period of time.
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EDVO-Kit # 279
Immunology of Pregnancy Tests
11
Experiment Results and Study Questions
LABORATORY NOTEBOOK RECORDINGS:
Before starting the experiment:
• Write a hypothesis that reflects the experiment.
• Predict experimental outcomes.
During the Experiment:
• Record (draw) your observations, or photograph the results.
Following the Experiment:
• Formulate an explanation from the results.
• Determine what could be changed in the experiment if the experiment were repeated.
• Write a hypothesis that would reflect this change.
STUDY QUESTIONS
Answer the following study questions in your laboratory notebook or on a
separate worksheet.
1.
Why is the ELISA reaction so sensitive?
2.
Why is it important to have a positive control?
3.
What is being detected in the standard home pregnancy test?
4.
What is the difference between poyclonal and monoclonal antibodies ?
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Experiment Procedures
Address and record the following in your laboratory notebook or on a
separate worksheet.
12
EDVO-Kit # 279
Immunology of Pregnancy Tests
Experiment Procedures
Notes:
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EDVO-Kit # 279
Immunology of Pregnancy Tests
13
Notes to the Instructor
1.
Pre-lab preparation of biologicals and reagents takes approximately
one and one-half hours.
2.
The student experimental activity requires approximately 60 minutes.
If you do not find the answers to your questions in this section, a variety of
resources are continuously being added to the EDVOTEK® web site. In
addition, Technical Service is available from 9:00 am to 6:00 pm, Eastern time zone. Call for help from our knowledgeable technical staff at
1-800-EDVOTEK (1-800-338-6835).
Safety Data Sheets can be found on our website:
www.edvotek.com/safety-data-sheets
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Instructor's Guide
APPROXIMATE TIME REQUIREMENTS FOR PRE-LAB AND
EXPERIMENTAL PROCEDURES
14
EDVO-Kit # 279
Immunology of Pregnancy Tests
PreLab Preparations
Instructor's Guide
PREPARATIONS BEFORE THE LAB
Microtiter Plates
1.
As shown in the figure below, orient the microtiter plates so that the
numbers 1 - 12 are at the top and the letters A - H are on your left.
2.
Cut each plate on the dotted lines as shown in the figure. Each piece
will be two rows of four wells. Each lab group will receive one piece
and use one row.
1
Row 1
A
Row 2
B
Row 3
C
Row 4
D
Row 5
E
Row 6
F
Row 7
G
Row 8
H
2 3
4
5
6 7
8 9 10 11 12
Cutting guide depicted by dashed lines
Dispensing Components A through D:
Use a clean pipet for dispensing each of the components A-D directly
from the component tubes provided in this experiment kit. Label
microtest tubes and dispense volumes as outlined in the chart "Quick
Reference: Preparation of Experiment Reagents" .
Note: This experiment is ideal for problem-based experimentation.
Instructors may wish to dispense the patient samples so the various
student groups get different results.
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EDVO-Kit # 279
Immunology of Pregnancy Tests
15
Pre-Lab Preparations
Preparation of Phosphate Buffered Saline
1.
Add all of the Phosphate Buffered Saline concentrate (Component H)
to 135 ml of distilled water. Mix.
2.
Label this diluted Phosphate Buffered saline as “PBS”.
3.
Dispense 10 ml into small beakers or tubes for each of the 10 lab
groups.
Preparation of Anti-hCG Peroxidase Conjugate
(Secondary Antibody)
Note: Prepare on same day as needed for the experiment.
The sample volume
of the secondary
antibody is very small
- the tube can be
centrifuged to collect
the sample at the
bottom.
Quick Reference:
The substrate is
prepared for the
peroxidase enzyme,
which is attached to the
anti-hCG peroxidase
conjugate (secondary
antibody).
Prepare the substrate
15 - 30 minutes before
students require it for
plate development (last
incubation).
4.
Add 0.3 ml of diluted Phosphate Buffered Saline (PBS) to the tube of
concentrated Anti-hCG peroxidase conjugate (Component E). Mix
thoroughly by tapping and inverting the tube.
5.
Transfer 6 ml of diluted Phosphate Buffered Saline (PBS) to one of the
15 ml plastic tube provided.
6.
Transfer the entire contents of tube E prepared in step 4 to the 15 ml
tube containing 6 ml of PBS. Label the tube "2°Ab" (Secondary Antibody). Mix.
7.
Dispense 0.5 ml of the diluted Anti-hCG peroxidase conjugate for
each of the ten lab groups.
PREPARATION OF PEROXIDASE SUBSTRATE
DURING THE LAB EXPERIMENT
Prepare 15 - 30 minutes before the last incubation:
1.
Dispense 9 ml of diluted Phosphate buffered saline (PBS) to the second 15 ml tube provided.
2.
Add Peroxide co-substrate (Component G) to the 9 ml of PBS. Cap
and mix thoroughly by shaking and/or vortexing. There is usually undissolved material remaining.
3.
Then add 1 ml of Hydrogen peroxide (Component F). Cap and mix.
4.
Dispense 0.8 ml of the peroxidase substrate for each of the 10 groups.
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Instructor's Guide
PREPARATIONS ON THE DAY OF THE LAB
16
EDVO-Kit # 279
Immunology of Pregnancy Tests
Instructor's Guide
Quick Reference Tables
PREPARATION OF EXPERIMENT REAGENTS
Label
A*
B*
C*
D*
E + PBS
PBS + G + F
H + water
hCG Antibody
Positive control
Urine Sample Patient 1
Urine Sample Patient 2
Anti-hCG-peroxidase-conjugate
Peroxidase-enzyme substrate**
Phosphate Buffered Saline
hCG Ab
+
USP 1
USP 2
2°Ab
Substrate
PBS
Dispense for
each group
0.5 ml
75 μl
75 μl
75 μl
0.5 ml
0.8 ml
10 ml
* Components A - D can be dispensed before the actual day of the lab and stored in
the refrigerator. If these components are dispensed on the day of the lab, leave at room
temperature.
** Peroxidase-enzyme substrate should be prepared 15-20 minutes before the last
incubation.
STUDENT MATERIALS
Each Lab Group Should Receive:
1
1
1
1
1
1
1
10
1
1
1
Piece of microtiter plate
Tube labeled "hCG Ab"
Tube labeled "+"
Tube labeled "USP 1"
Tube labeled "USP 2"
Tube labeled "2°Ab"
Automatic micropipet with tips (optional)
Transfer pipets
Beaker or tube containing PBS
Empty beaker labeled "waste"
Tube labeled "Substrate" (just before the last incubation)
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EDVO-Kit # 279
Immunology of Pregnancy Tests
17
Avoiding Common Pitfalls
Students should be advised to be very careful when transferring solutions into and out of the microtiter strip wells.
2.
Use only clean or appropriately labeled pipets and avoid contaminating adjacent wells.
3.
Do not attempt to empty the microtiter wells by shaking it out. This will
not work - it will result in contaminating adjacent wells.
4.
Wash the wells gently and slowly.
Expected Results
Pregnancy tests together with positive and negative controls as represented in the figure below. Actual liquid amounts in the wells are not drawn to
scale, but the color of the positive patients should look similar to the positive control, which is in the second well.
(-)
(+)
USP 1
USP 2
Problem-based Individualized Experiment
Students may be given combinations of positive and negative samples so
the various student groups obtain different results. Instructors can code
samples to represent various numbers of samples.
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Instructor's Guide
1.
Please refer to the kit
insert for the Answers to
Study Questions