The Biotechnology Education Company ®
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Revi nd
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ated
Upd
EDVO-Kit #
278
Quantitative
ELISA Laboratory Activity
Storage: See Page 3 for
specific storage instructions
EXPERIMENT OBJECTIVE:
The objective of this experiment is to perform and master
the experimental concepts and methodology involved
with enzyme linked immunosorbent (ELISA) assays.
This ELISA experiment is designed to detect circulating
IgG directed towards two antigens. Observations in
this experiment include specificity of antibodies,
the effect of dilution on ELISA reactions, color
development and quantitation.
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Quantitative ELISA Laboratory Activity
Table of Contents
Page
Experiment Components
Experiment Requirements
3
3
Background Information
4
Experiment Procedures
Experiment Overview and General Instructions
The Enzyme Linked Immunosorbent Assay (ELISA)
Study Questions
6
8
14
Instructor’s Guidelines
Notes to the Instructor
Pre-Lab Preparations
Quick Reference Tables
Avoiding Common Pitfalls
Expected Results
Study Questions and Answers
15
16
18
18
19
20
Material Safety Data Sheets
21
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.
THIS EXPERIMENT DOES NOT
CONTAIN HUMAN DNA. None
of the experiment components are
derived from human sources.
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Quantitative ELISA
Laboratory Activity
278
Experiment #
Experiment Components
This experiment is
designed for 6 groups.
Upon receipt, store the
perishable components
(A-I) in the refrigerator.
Now with
NEW
Substrate!
A
B
C
D
E
F
G
H
I
Antigen 1
Antigen 2
Primary Antibody 1
Primary Antibody 2
Secondary Antibody
Gelatin (blocking agent)
Hydrogen Peroxide, stabilized
Phosphate buffered saline concentrate
Aminosalicylic acid (Peroxide co-substrate)
•
•
•
•
Microtiter plates
Transfer pipets
Microcentrifuge tubes
Plastic tubes (15 ml and 50 ml)
None of the components have been prepared from human sources.
Requirements
•
•
•
•
•
•
Distilled or deionized water
Beakers or flasks
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 (Cat. #632) can be purchased
for liquid removal and washing steps.
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Quantitative ELISA Laboratory Activity
Experiment
Background Information
Antibodies are specific human and animal proteins that are produced by white blood cells
in response to foreign material. Examples of such foreign material, known as antigens,
include infectious agents and various environmental "non-self" materials. Biological antigens are high molecular weight biomolecules such as proteins, carbohydrates and nucleic
acids which can be circulating freely or as part of a complex such as part of a virus coat or
bacterial cell surface. Antibodies are made in response to antigens. They bind to antigens
and play a significant role in the subsequent removal of such materials from circulation.
For example, exposure to an infectious agent causes the individual to mount an antibody
response which eventually results in plasma antibody molecules that bind to different
viral proteins (and/or different areas of the same polypeptide).
When an antibody binds to a specific biological antigen, it can recognize specific chemical
charges, sequences or structural conformational elements. These structural binding characteristics make up the specific fingerprint for an antigen. Each antibody molecule can
bind two antigen molecules. This recognition and binding is highly specific and makes
possible the differentiation between two circulating viruses that may be very closely
related, as in the case of two strains of the same virus.
When an antigen and its antibodies form insoluble complexes, this highly specific binding
reaction is known as immunoprecipitation. Precipitation of the complex is the result of
various polyclonal antibodies binding to the antigens to form a network. In the traditional immunoprecipitation assay, antibodies are obtained from the serum of an animal
exposed to the specific antigen. The serum, also known as plasma, is prepared by the
removal of red blood cells. It contains the specific proteins for that particular animal and
antibodies against a "non-self" antigen that is introduced in the animal by either design
or an infection. Antibodies are purified from animal sera samples and can be used to
detect particular antigens, such as human infectious agents.
Description of the Immunological Screening Test
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. ELISAs are done in microtiter plates which are generally
made of polystyrene or polyvinyl chloride. The plates are somewhat transparent and
contain many small wells, in which liquid samples are deposited. First, the antigens are
added to the wells where some remain adsorbed by hydrophobic association to the walls
after washing away the excess. The antigens can be the whole infecting agent, such as a
virus or lysate, specific proteins, or a mixture of the two. There is no specificity involved
with the adsorption process although some substances may exhibit low binding to the
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 gelatin, milk proteins or bovine serum albumin.
In this experiment, positive samples will have antibodies that will bind to the preadsorbed
antigens in wells. If the primary antibody has remained in a well, then the secondary antibody will bind to it and also remain attached after washing. These secondary antibodies are usually raised in rabbits and goats immunized with "non-self" IgG fractions. The
second IgG antibodies are purified and covalently cross- linked to horseradish peroxidase.
This modification does not significantly affect the binding specificity and affinity of the
antibody or the enzymatic activity of the peroxidase.
Duplication of this document, in conjunction with use of accompanying reagents, is permitted for classroom/laboratory use only.
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Quantitative ELISA Laboratory Activity
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Background Information
After washing, 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 co-substrates include o-diansidine, 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. The oxidized salicylate is brown and can
be easily observed in positive wells.
Figure 1 illustrated the ELISA assay. 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. Western blot
analysis of positive samples can be used to confirm the presence and size of antibodies.
This ELISA experiment is designed to detect two separate circulating IgG molecules directed towards two distinct antigens. Observations in this experiment include specificity of
antibodies, the effect of dilution on ELISA reactions, color development and quantitation.
Human
Serum
(IgG)
Horseradish
peroxidase
Anti-IgG
(Rabbit)
well
Substrate (S)
(Reduced)
(colorless)
HIV
Antigen
Product (P)
(Oxidized)
(color)
2H2O2 –> H20 + 2O2
Human
Serum
(IgG)
well
Horseradish
peroxidase
HIV
Antigen
Anti-IgG
(Rabbit)
Human
Serum
(IgG)
well
HIV
Antigen
Figure 1:
ELISA “Sandwich”
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This document, or any part, may not be reproduced or distributed for any other purpose without the written consent of EDVOTEK, Inc.
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Quantitative ELISA Laboratory Activity
Experiment
Experiment Overview and General Instructions
EXPERIMENT OBJECTIVE:
The objective of this experiment is to perform and master the experimental concepts and
methodology involved with enzyme linked immunosorbent (ELISA) assays. This ELISA experiment is designed to detect circulating IgG antibodies directed towards two antigens.
Observations in this experiment include specificity of antibodies, the effect of dilution on
ELISA reactions, color development and quantitation.
LABORATORY SAFETY
1.
Gloves and goggles should be worn routinely as good
laboratory practice.
2.
Exercise extreme caution when working with equipment
which is used in conjunction with the heating and/or
melting of reagents.
3.
DO NOT MOUTH PIPET REAGENTS - USE PIPET PUMPS
OR BULBS.
4.
Always wash hands thoroughly with soap and water
after handling contaminated materials.
Wear Gloves
and Goggles
LABORATORY NOTEBOOK RECORDINGS:
Address and record the following in your laboratory notebook or on a separate worksheet.
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.
Duplication of this document, in conjunction with use of accompanying reagents, is permitted for classroom/laboratory use only.
This document, or any part, may not be reproduced or distributed for any other purpose without the written consent of EDVOTEK, Inc.
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278
Experiment Overview and General Instructions
1.
Equilibrate a 37° C incubation
oven before starting the experiment.
1
2
5
4
3
B
2.
3.
Place the microtiter plate as
shown in the figure at right and
carefully mark the plate with
your initials or lab group number
Using a permanent marker, label
the columns 1-5 across the top
and the rows A-H down the side.
C
D
E
F
G
H
4.
As shown in the figure, draw
lines across the plate between
Rows B and C, Rows D and E, and
Rows F and G. This will create
four sections on the plate.
5.
Label 4 large transfer pipets as follows (these are to be used
for addition of reagents to the wells):
•
•
•
•
6.
7.
PBS
Ag1
Ag2
block
Microtiter plate
for Phosphate Buffered Saline
for Antigen 1
for Antigen 2
for blocking agent
Label 2 small transfer pipets Ag1 and Ag2.
These are to be used for removal of liquid from
the wells.
Proceed to dilution of Primary Antibodies 1
and 2 on pages 8 and 9.
STUDENT MATERIALS:
Experiment Procedure
CAUTION:
To avoid crosscontamination and
false results, use
the appropriately
labelled plastic
transfer pipet for
liquid removals and
washes as outlined
in the experimental
procedures.
A
Each Lab Group Should Receive the
following components prior to the start
of the experiment procedure.
1
1
1
1
1
1
1
1
11
11
1
1
1
8
Half piece of microtiter plate
Tube labeled "Ag1"
Tube labeled "Ag2"
Tube labeled "block"
Tube labeled "Ab1 - 1"
Tube labeled "Ab2 -1"
Tube labeled "2°Ab"
Automatic micropipet with tips (optional)
Transfer pipets (large)
Transfer pipet (small)
Beaker containing PBS
Empty beaker labeled "waste"
Tube labeled "Substrate"
(just before the last incubation)
Microcentrifuge tubes
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This document, or any part, may not be reproduced or distributed for any other purpose without the written consent of EDVOTEK, Inc.
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278
Quantitative ELISA Laboratory Activity
Experiment
Quantitative Enzyme Linked Immunosorbent Assay (ELISA)
DILUTION OF PRIMARY ANTIBODIES 1 AND 2
Experiment Procedure
Primary Antibody 1
If using a micropipet for diluting the
antibodies, dilute the
1:400 antibody solution 1:4 by combining
110 μl 1:400 antibody
with 330 μl diluted
PBS. Continue diluting
the antibody 4-fold, up
to 1:102,400.
1.
Label four tubes as follows:
“Ab1 - 2”. This is your 1:1600 dilution.
“Ab1 - 3”. This is your 1:6400 dilution.
“Ab1 - 4”. This is your 1:25,600 dilution.
“Ab1 - 5”. This is your 1:102,400 dilution.
2.
Add 6 drops (330 µl) of PBS to each tube.
3.
Obtain the “Ab1 - 1” tube from your instructor. This is your 1:400 dilution of
Primary Antibody 1.
4.
Use a fresh pipet tip or a fresh large transfer pipet to add 2 drops (110 µl) of
“Ab1 - 1” Primary Antibody to the tube labeled “Ab1 - 2”. Pipet the solution
up and down, cap the tube and mix well.
5.
Use the same large transfer pipet to add 2 drops of “Ab1 - 2” to the tube
labeled “Ab1 - 3”. Pipet the solution up and down, cap the tube and mix well.
6.
Use the same large transfer pipet to add 2 drops of “Ab1 - 3” to the tube
labeled “Ab1 - 4”. Pipet the solution up and down, cap the tube and mix well.
7.
Use the same large transfer pipet to add 2 drops of “Ab1 - 4” to the tube
labeled “Ab1 - 5”. Pipet the solution up and down, cap the tube and mix well.
See NOTE at bottom of page 9 for storage of Ab1 or just store diluted Ab1 in the
refrigerator until needed.
1:400
1:1600
2 drops
(110 µl)
Ab1 - 1
(Obtained
from instructor)
1:6400
2 drops
(110 µl)
Ab1 - 2
2 drops
(110 µl)
Ab1 - 3
6 drops
(330 µl PBS)
1:102,400
1:25,600
2 drops
(110 µl)
Ab1 - 4
6 drops
(330 µl PBS)
Ab1 - 5
6 drops
(330 µl PBS)
6 drops
(330 µl PBS)
Duplication of this document, in conjunction with use of accompanying reagents, is permitted for classroom/laboratory use only.
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Quantitative ELISA Laboratory Activity
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278
Quantitative Enzyme Linked Immunosorbent Assay (ELISA)
Primary Antibody 2
Label four tubes as follows:
2.
Add 6 drops (330 µl) of PBS to each tube.
3.
Obtain the “Ab2 - 1” tube from your instructor. This is your 1:400 dilution of Primary
Antibody 2.
4.
Use a fresh pipet tip or a fresh large transfer pipet to add 2 drops (110 µl) of “Ab2 - 1”
Primary Antibody to the tube labeled “Ab2 - 2”. Pipet the solution up and down, cap
the tube and mix well.
9.
Use the same large transfer pipet to add 2 drops of “Ab2 - 2” to the tube labeled
“Ab2 - 3”. Pipet the solution up and down, cap the tube and mix well.
6.
Use the same large transfer pipet to add 2 drops of “Ab2 - 3” to the tube labeled
“Ab2 - 4”. Pipet the solution up and down, cap the tube and mix well.
7.
Use the same large transfer pipet to add 2 drops of “Ab2 - 4” to the tube labeled
“Ab2 - 5”. Pipet the solution up and down, cap the tube and mix well.
1:400
“Ab2 - 2”. This is your 1:1600 dilution.
“Ab2 - 3”. This is your 1:6400 dilution.
“Ab2 - 4”. This is your 1:25,600 dilution.
“Ab2 - 5”. This is your 1:102,400 dilution.
1:1600
2 drops
(110 µl)
Ab2 - 1
(Obtained
from instructor)
1:6400
2 drops
(110 µl)
Ab2 - 2
1:25,600
2 drops
(110 µl)
Ab2 - 3
6 drops
(330 µl PBS)
1:102,400
2 drops
(110 µl)
Ab2 - 4
6 drops
(330 µl PBS)
Experiment Procedure
1.
Ab2 - 5
6 drops
(330 µl PBS)
6 drops
(330 µl PBS)
NOTE: Keep the Primary Antibodies 1 and 2 and their dilutions in the refrigerator
until needed.
Duplication of this document, in conjunction with use of accompanying reagents, is permitted for classroom/laboratory use only.
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Quantitative ELISA Laboratory Activity
Experiment
The Enzyme Linked Immunosorbent Assay (ELISA)
1
2
3
4
5
1.
Orient the microtiter plate so the wells labeled as columns 1-5 are on
the horizontal axis and the wells labelled as rows A-H are on the vertical axis (as shown in the figure at left).
2.
Read through the instructions and study the figure on the previous
page before beginning to add antigens to the wells. Refer to the figure often during the experimental procedure to avoid making errors.
A
B
C
D
E
Experiment Procedure
F
ADDITION OF ANTIGENS:
G
3.
H
Using the appropriately labelled large transfer pipet or fresh pipet tip
for each reagent, add reagents to the wells as described below.
•
Add 50 µl or 1 drop of Antigen 1 to the wells 1 - 5 in rows A, B, C
and D.
•
Add 50 µl or 1 drop of Antigen 2 to the wells 1 - 5 in rows E, F, G
and H.
Note:
None of the wells in
column 6 will be used in
this experiment.
4.
Incubate the plate at room temperature for 5 minutes.
LIQUID REMOVAL OF ANTIGENS:
In the steps which follow, use the appropriately labelled small transfer pipets to remove
liquid from the wells. It is important to follow directions carefully to avoid cross-contamination of the wells. Save the pipets for later steps.
5.
Using the small pipet labelled "Ag1", remove the liquid from wells in rows A - D.
6.
Using the small pipet labelled "Ag 2", remove the liquid from wells in rows E - H.
PBS WASH AND LIQUID REMOVAL OF PBS
7.
Using the large transfer pipet labelled "PBS" add Phosphate Buffered Saline to wells
1 - 5 in all the rows. Fill until each well is almost full. If using an automatic micropipet, add 200 µl of PBS to each of the wells.
8.
Remove the PBS from the wells using the same procedures outlined in steps 5 and 6.
Duplication of this document, in conjunction with use of accompanying reagents, is permitted for classroom/laboratory use only.
This document, or any part, may not be reproduced or distributed for any other purpose without the written consent of EDVOTEK, Inc.
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Quantitative ELISA Laboratory Activity
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Quantitative Enzyme Linked Immunosorbent Assay (ELISA)
ADDITION OF BLOCKING AGENT
9.
Using a fresh pipet tip or the large transfer pipet labelled “block”, add 50 µl
or 1 drop of “block” (blocking agent) to each of the wells in columns 1 - 5 in
all rows (exclude wells in column 6).
10. Incubate the plate for 10 minutes at 37° C.
12. Using the small pipet labelled “Ag 2”, remove the liquid from wells 1 -5 in
rows E - H.
PBS WASH AND LIQUID REMOVAL OF BLOCKING AGENT
13. Using the large pipet labelled “PBS” add Phosphate Buffered Saline to all
the wells. Fill until each well is almost full. If using a micropipet, add 200 µl
of PBS to each of the wells.
14. Remove the PBS from the wells using the same procedures outlined in steps
11 and 12. Discard the small pipets when finished.
Experiment Procedure
11. Using the small pipet labelled “Ag1”, remove the liquid from wells 1 - 5 in
rows A - D.
Use the appropriately labeled plastic
transfer pipet for
liquid removals and
washes to avoid
cross-contamination and false
results.
OPTIONAL STOPPING POINT:
The experiment can be stopped after addition of PBS (step 13)
and resumed during next lab period. Cover microtiter plates
with parafilm or plastic wrap and refrigerate overnight.
Duplication of this document, in conjunction with use of accompanying reagents, is permitted for classroom/laboratory use only.
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Quantitative ELISA Laboratory Activity
Experiment
Quantitative Enzyme Linked Immunosorbent Assay (ELISA)
ADDITION OF PRIMARY ANTIBODIES 1 & 2
(PREPARED ON PAGES 8 & 9)
Experiment Procedure
NOTE:
To add primary antibody, use
the same pipet going from
lowest concentration to highest concentration. (Use a fresh
transfer pipet or a fresh pipet
tip for addition of Antibody 1,
Antibody 2, and PBS.)
1
2
3
4
5
6
A
Ab1:1
Ab1:2
Ab1:3
Ab1:4
Ab1:5
blank
B
Ab1:1
Ab1:2
Ab1:3
Ab1:4
Ab1:5
blank
C
PBS
PBS
PBS
PBS
PBS
blank
D
Ab2:1
Ab2:2
Ab2:3
Ab2:4
Ab2:5
blank
E
PBS
PBS
PBS
PBS
PBS
blank
F
Ab1:1
Ab1:2
Ab1:3
Ab1:4
Ab1:5
blank
G
Ab2:1
Ab2:2
Ab2:3
Ab2:4
Ab2:5
blank
H
Ab2:1
Ab2:2
Ab2:3
Ab2:4
Ab2:5
blank
Use the appropriately
labeled plastic transfer
pipet for liquid removals and washes to avoid
cross-contamination and
false results.
1.
Label 3 large transfer pipets "PBS", "Ab1", and "Ab2".
Use a fresh pipet tip or the appropriately labelled large
transfer pipet for addition of PBS and each antibody.
2.
Add 50 µl or 1 drop of PBS to the wells in rows C and E.
3.
Add 50 µl or 1 drop of “Ab1 - 5” to wells A5, B5, and F5.
4.
Add 50 µl or 1 drop of “Ab1 - 4” to wells A4, B4, and F4.
5.
Add 50 µl or 1 drop of “Ab1 - 3” to wells A3, B3, and F3.
6.
Add 50 µl or 1 drop of “Ab1 - 2” to wells A2, B2, and F2.
7.
Add 50 µl or 1 drop of “Ab1 - 1” to wells A1, B1, and F1.
8.
Add 50 µl or 1 drop of “Ab2 - 5” to wells D5, G5, and H5.
9.
Add 50 µl or 1 drop of “Ab2 - 4” to wells D4, G4, and H4.
10. Add 50 µl or 1 drop of “Ab2 - 3” to wells D3, G3, and H3.
11. Add 50 µl or 1 drop of “Ab2 - 2” to wells D2, G2, and H2.
12. Add 50 µl or 1 drop of “Ab2 - 1” to wells D1, G1, and H1.
13. Incubate the plate at 37° C for 30 minutes.
LIQUID REMOVAL OF ANTIBODIES:
14. Label 4 small transfer pipets “AB”, “CD”, “EF”, and “GH”.
15. Starting with the most dilute antibody (column 5) in rows
A and B, use the pipet labelled “AB” to remove the liquid
from the wells moving from right to left (i.e. remove in
order A5, B5, A4, B4, A3, B3, A2, B2, A1, B1).
16. Repeat the procedure with the other rows (C-H) using the
appropriately labelled transfer pipets.
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Quantitative ELISA Laboratory Activity
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Quantitative Enzyme Linked Immunosorbent Assay (ELISA)
PBS WASH AND LIQUID REMOVAL OF PBS
17. Using the large pipet labelled “PBS” add Phosphate Buffered Saline to all the wells.
Fill until each well is almost full. If using a micropipet, add 200 µl to each well.
18. Remove the PBS from the wells using the same procedures outlined in steps 2 and 3.
Discard transfer pipets.
ADDITION OF SECONDARY ANTIBODY
20. Incubate the plate at 37° C for 15 minutes.
LIQUID REMOVAL OF SECONDARY ANTIBODIES
21. Label 4 small transfer pipets “AB”, “CD”, “EF”, and “GH”.
22. Starting with the most dilute antibody (column 5) in rows A and B, use the pipet
labelled “AB” to remove the liquid from the wells moving from right to left (i.e.
remove, in order A5, B5, A4, B4, A3, B3, A2, B2, A1, B1).
23. Repeat the procedure with the other rows (C-H) using the appropriately labeled
transfer pipets.
Experiment Procedure
19. Use a fresh pipet tip or a large transfer pipet to add 50 µl or 1 drop of Secondary
Antibody to each of the wells in rows A-H (all 40 wells).
PBS WASH AND LIQUID REMOVAL OF PBS
24. Using the large pipet labelled “PBS” add Phosphate Buffered Saline to all the wells.
Fill until each well is almost full. If using a micropipet, add 200 µl to each well.
25. Remove the PBS from the wells using the same procedures outlined in steps 22 and
23.
ADDITION OF SUBSTRATE
26. Start with column 5 and move to the left - use a fresh pipet tip or a fresh large transfer pipet to add 50 µl or 1 drop of Substrate to 40 wells (exclude wells in column 6).
27. Incubate at room temperature for 5 minutes.
28. Periodically observe the plate for color development.
29. If color is not fully developed after 5 minutes, incubate for a longer period of time.
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Quantitative ELISA Laboratory Activity
Experiment
Study Questions
Answer the following study questions in your laboratory notebook or on a separate
worksheet.
To what do antibodies respond?
2.
Can an antibody act as an antigen?
3.
Describe the ELISA antigen antibody reactions.
4.
Is a positive result always visualized as a brown color in the ELISA assay?
Experiment Procedure
1.
Duplication of this document, in conjunction with use of accompanying reagents, is permitted for classroom/laboratory use only.
This document, or any part, may not be reproduced or distributed for any other purpose without the written consent of EDVOTEK, Inc.
Copyright © 2000, 2005, 2009, 2011, EDVOTEK, Inc., all rights reserved.
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Quantitative ELISA
Laboratory Activity
278
Experiment #
Instructor’s
Guide
Notes to the Instructor & Pre-Lab Preparations
Class size, length of laboratory sessions, and availability of equipment are
factors which must be considered in planning and implementing this experiment with your students. These guidelines can be adapted to fit your specific set of circumstances. 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. 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).
Order
Online
APPROXIMATE TIME REQUIREMENTS FOR PRE-LAB AND
EXPERIMENTAL PROCEDURES
Visit our web site for information
about EDVOTEK's complete line
of experiments for biotechnology
and biology education.
E
O
DV
-TE
C H S E RV I C E
•
Pre-lab preparation of biologicals and reagents takes approximately
one and one-half hours.
•
The student experimental activity requires approximately 2 hours with
optional stopping points available.
Technical Service
Department
Mon - Fri
9:00 am to 6:00 pm ET
1-800-EDVOTEK
ET
(1-800-338-6835)
Mo
FAX: (301) 340-0582
Web: www.edvotek.com
email: info@edvotek.com
m
6p
n - Fri 9 am
Please have the following
information ready:
• Experiment number and title
• Kit lot number on box or tube
• Literature version number
Visit the EDVOTEK web site often for
continuously updated information.
(in lower right corner)
• Approximate purchase date
EDVOTEK - The Biotechnology Education Company®
1-800-EDVOTEK • www.edvotek.com
FAX: (301) 340-0582 • email: info@edvotek.com
EVT 2011_07_25AM
15
278
Instructor’s Guide
Quantitative ELISA
Laboratory Activity
Experiment
Pre-Lab Preparations
PREPARATIONS BEFORE THE LAB
Microtiter Plates
1.
As shown in the figure at right, orient the microtiter
plates so that the numbers 1-12 are at the top and the
letters A-H are on the left.
2.
Cut each plate along the solid lines as
shown in the figure. Each piece will be 6
wells on one axis and 8 wells on the other
axis. Each lab group will receive one piece
containing 48 wells.
Instructor’s Guide
1
2
3
4
5
6
7
8
9 10 11 12
A
B
C
D
E
F
G
PREPARATIONS ON THE DAY OF THE
LAB
H
Preparation of Phosphate Buffered Saline
1.
Add all of the Phosphate Buffered Saline concentrate
(comp. H) to 360 ml of distilled water. Mix.
2.
Label this diluted Phosphate Buffered saline as “PBS”.
3.
Dispense 50 ml into small beakers for each of the 6 lab
groups.
Antigens 1 and 2
1.
2.
Label 6 tubes “Ag1” and “Ag2” and dispense 1.5 ml
Antigen 1 (comp. A) into the tubes labeled “Ag1” and
1.5 ml Antigen 2 (comp. B) into the tubes labeled “Ag2”.
Distribute one tube of “Ag1” and “Ag2” each per group.
Blocking Agent
1.
If the Blocking agent has gelled, place the bottle in a
37° C waterbath or oven to melt the gelatin.
2.
Label 6 larger test tubes “block” and dispense 3.0 ml
Gelatin Blocking Agent (comp. F) into the tubes.
3.
Distribute one tube of Blocking agent per group.
NOTES:
The Phosphate Buffered saline may precipitate or crystalize.
Warm the buffer in
a 37° C water bath
before using and
check that there is
no precipitate.
The blocking buffer
will likely precipitate during storage.
Warm @ 37° C
for 5-10 minutes or
until the precipitate
has dissolved.
Duplication of this document, in conjunction with use of accompanying reagents, is permitted for classroom/laboratory use only.
This document, or any part, may not be reproduced or distributed for any other purpose without the written consent of EDVOTEK, Inc.
Copyright © 2000, 2005, 2009, 2011, EDVOTEK, Inc., all rights reserved.
EVT 2011_07_25AM
16
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Quantitative ELISA
Laboratory Activity
Instructor’s Guide
278
Experiment
Pre-Lab Preparations
Primary Antibodies 1 and 2 (1:400)
Add 0.3 ml diluted PBS to tube C. Mix well and transfer the entire contents to a larger tube containing 5.7 ml diluted PBS. This is 1:400 Primary
Antibody 1. Dispense 0.5 ml diluted Primary Antibody 1 (comp. C) into 6
tubes labeled "Ab1 - 1".
2.
Distribute one tube of “Ab1 - 1” per group.
3.
Add 0.3 ml diluted PBS to tube D. Mix well and transfer the entire contents to a larger tube containing 5.7 ml diluted PBS. This is 1:400 Primary
Antibody 2. Dispense 0.5 ml diluted Primary Antibody 2 (comp. D) into 6
tubes labeled "Ab2 - 1".
4.
Distribute one tube of “Ab2 - 1” per group.
Preparation of Secondary Antibody
1.
Add 0.3 ml diluted PBS to the tube containing Secondary Antibody (comp.
E). Add 18 ml diluted PBS into the 50 ml conical tube provided and transfer
the entire contents of tube E into the PBS. Cap the tube and mix. Dispense
3.0 ml diluted secondary antibody into 6 tubes labeled "2°Ab".
2.
Distribute one tube of “2°Ab” per group.
Preparation of Peroxidase Substrate During the Lab Experiment
Prepare 15 - 30 minutes before the last incubation:
1.
Dispense 16 ml of diluted Phosphate buffered saline (PBS) to the second 50
ml tube provided.
2.
Add all of the Aminosalicylic acid (I) to the 16 ml of PBS. Cap and mix thoroughly by shaking and/or vortexing. There is usually undissolved material
remaining.
3.
Then add 1.8 ml of Hydrogen peroxide (G). Cap and mix.
4.
Dispense 2.5 ml of the peroxidase substrate for each group.
5.
Keep refrigerated in the dark until use.
NOTE:
The sample volumes
of primary antibodies
1 and 2 and the secondary antibody are
very small - the tubes
should be centrifuged
to collect the samples
at the bottom of the
tubes before they are
diluted.
Quick Reference:
The substrate is prepared for the peroxidase enzyme, which is
attached to the antiIgG peroxidase conjugate (secondary antibody). Prepare the
substrate 15 - 30 minutes before students
require it for plate
development (last incubation).
Instructor’s Guide
1.
Duplication of this document, in conjunction with use of accompanying reagents, is permitted for classroom/laboratory use only.
This document, or any part, may not be reproduced or distributed for any other purpose without the written consent of EDVOTEK, Inc.
Copyright © 2000, 2005, 2009, 2011, EDVOTEK, Inc., all rights reserved.
EVT 2011_07_25AM
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17
278
Quantitative ELISA
Laboratory Activity
Instructor’s Guide
Experiment
Pre-Lab Preparations
PREPARATION OF EXPERIMENT REAGENTS
Label
Instructor’s Guide
A
B
C + PBS
D + PBS
E + PBS
G + I + PBS
H + dH2O
F
Antigen 1
Antigen 2
Antibody 1 (1:400 dilution)
Antibody 2 (1:400 dilution)
Secondary Antibody
Peroxidase Enzyme Substrate
Phosphate buffered saline
Gelatin (Blocking Agent)
Dispense for
each group
Ag1
Ag2
Ab1 - 1
Ab2 - 1
2°Ab
Substrate
PBS
block
1.5 ml
1.5 ml
0.5 ml
0.5 ml
3.0 ml
2.5 ml
50 ml
3.0 ml
* Components A, B, C, D, E, F can be dispensed before the actual day
of the lab and stored in the refrigerator.
STUDENT MATERIALS
AVOIDING COMMON PITFALLS
Each Lab Group Should Receive:
1
1
1
1
1
1
1
1
11
11
1
1
1
8
half piece of microtiter plate
tube labeled "Ag1"
tube labeled "Ag2"
tube labeled "block"
tube labeled "Ab1 - 1"
tube labeled "Ab2 - 1"
tube labeled "2°Ab"
automatic micropipet with tips (optional)
Transfer pipets (large)
Transfer pipet (small)
beaker containing PBS
empty beaker labeled "waste"
tube labeled "Substrate"
(just before the last incubation)
microcentrifuge tubes
1.
Students should be advised to be very
careful when transferring solutions
into and out of the microliter plate
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,
without force.
Duplication of this document, in conjunction with use of accompanying reagents, is permitted for classroom/laboratory use only.
This document, or any part, may not be reproduced or distributed for any other purpose without the written consent of EDVOTEK, Inc.
Copyright © 2000, 2005, 2009, 2011, EDVOTEK, Inc., all rights reserved.
EVT 2011_07_25AM
18
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Quantitative ELISA
Laboratory Activity
Instructor’s Guide
278
Experiment
Expected Results
The idealized schematic at right
shows representative results. Actual
results may vary.
1
2
3 4
5
A
B
C
D
E
F
H
1:400
1:1600 1:6400 1:25,600 1:102,400
Instructor’s Guide
G
Duplication of this document, in conjunction with use of accompanying reagents, is permitted for classroom/laboratory use only.
This document, or any part, may not be reproduced or distributed for any other purpose without the written consent of EDVOTEK, Inc.
Copyright © 2000, 2005, 2009, 2011, EDVOTEK, Inc., all rights reserved.
EVT 2011_07_25AM
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19
Please refer to the kit
insert for the Answers to
Study Questions
1121 5th Street NW
Washington DC 20001
08-15-11
®
Material Safety Data Sheet
10/10/06
OSHA PEL
soluble
No data
No data
100C
Evaporation Rate
(Butyl Acetate = 1)
Melting Point
Flammable Limits
LEL
UEL
No data
No data
1.017
% (Optional)
Unstable
Stable
Yes
Inhalation?
Skin?
Yes
Yes
Ingestion?
NTP?
IARC Monographs?
OSHA Regulation?
Yes
Local Exhaust
Mechanical (General)
Work/Hygienic Practices
Other
Special
Eye Protection
N/A
N/A
Do not ingest. Avoid contact with skin, eyes and clothing.
Wash thoroughly after handling.
Other Protective Clothing or Equipment
Protective Gloves
Ventilation
Respiratory Protection (Specify Type) NIOSH/MSHA approved respirator
Section VIII - Control Measures
N/A
N/A
Yes
®
A - E 278
Very dilute
OSHA PEL
Clear liquid, no odor
Unstable
Route(s) of Entry:
X
Conditions to Avoid
Excessive heat, sparks or open flame, protein denaturants
Inhalation?
Skin?
Yes
Conditions to Avoid
Ingestion? Yes
NTP?
IARC Monographs?
OSHA Regulation?
No data
No data
No data
Mucous membrane irritation, eye/skin irritation, irritating to
gastrointestinal system
Treat symptomatically and supportively
Observe federal, state, and local laws.
None
Store away from strong oxidizers or heat. Avoid eye/skin contact
Work/Hygienic Practices
Other Protective Clothing or Equipment
Yes
Splash proof goggles
Emergency eye wash should be available
Impervious clothing to prevent contact
Eye Protection
Chemical cartridge respirator with full facepiece and organic vapor cartridge
Special
Local Exhaust
No
None
Dilution vent sys Other
None
Mechanical (General)
Protective Gloves
Ventilation
Respiratory Protection (Specify Type)
Section VIII - Control Measures
Other Precautions
Precautions to be Taken in Handling and Storing
Waste Disposal Method
Mop up with absorptive material. Containerize to dispose of properly
Steps to be Taken in case Material is Released for Spilled
Section VII - Precautions for Safe Handling and Use
Emergency First Aid Procedures
Renal or heart disease, potassium deficiency. Insulin-dependent diabetes,seizures or intracranial lesions
Medical Conditions Generally Aggravated by Exposure
Signs and Symptoms of Exposure
None
Carcinogenicity:
Moderately toxic by ingestion. Systematic toxicity may result. May
chelate lead, magnesium, zinc, trace metals if present in intestine. Sensitivity reactions - anaphylactic shock
Yes
X
Thermal decomposition products of toxic & hazardous oxides
of carbon and nitrogen
Will Not Occur
May Occur
Health Hazards (Acute and Chronic)
UEL
Thermal decomposition products may include toxic and hazardous
oxides of carbon, nitrogen, and sodium
Section VI - Health Hazard Data
Hazardous
Polymerization
LEL
More container from fire area if possible. Dike fire control
water for later disposal
Acids, aluminum, metals, oxidizers (strong)
Stable
Hazardous Decomposition or Byproducts
Incompatibility
Stability
Section V - Reactivity Data
Flammable Limits
No data
No data
No data
% (Optional)
Dry chemical, carbon dioxide, halon. water spray or standard foam
No data
Unusual Fire and Explosion Hazards
Special Fire Fighting Procedures
Extinguishing Media
Flash Point (Method Used)
Section IV - Physical/Chemical Characteristics
Appearance and Odor
Solubility in Water
07/27/09
N.D. = No data
Evaporation Rate
(Butyl Acetate = 1)
No data
Soluble
Melting Point
No data
Vapor Density (AIR = 1)
Specific Gravity (H 0 = 1)
2
No data
Vapor Pressure (mm Hg.)
ACGIH TLV
Boiling Point
Section III - Physical/Chemical Characteristics
CAS# 139-33-3
CAS# 26628-22-8
Hazardous Components [Specific
Chemical Identity; Common Name(s)]
(301) 251-5990
(301) 251-5990
Other Limits
Recommended
Signature of Preparer (optional)
Date Prepared
Telephone Number for information
Emergency Telephone Number
Note: Blank spaces are not permitted. If any item is not
applicable, or no information is available, the space must
be marked to indicate that.
Section II - Hazardous Ingredients/Identify Information
14676 Rothgeb Drive
Rockville, MD 20850
Material Safety Data Sheet
May be used to comply with OSHA's Hazard Communication
Standard. 29 CFR 1910.1200 Standard must be consulted for
specific requirements.
Address (Number, Street, City, State, Zip Code)
EDVOTEK, Inc.
Manufacturer's Name
Section I
IDENTITY (As Used on Label and List)
EDVOTEK
Material Safety Data Sheets
Other Precautions
Wear appropriate NIOSH/MSHA approved respirator, chemical resistant gloves, safety goggles
safety shower and eye bath.
Precautions to be Taken in Handling and Storing
For small quantities - cautiosly add to a large stirred excess of water. Adjust pH to neutral
Waste Disposal Method
Wear respirator, chemical safety goggles,
rubber boots and heavy rubber gloves, sweep up, place in a bag and hold for waste disposal.
Steps to be Taken in case Material is Released for Spilled
Section VII - Precautions for Safe Handling and Use
Swallowed - wash out mouth with water provided person is conscious.
Skin/eye contact - flush with water Inhalation - remove to fresh air
Emergency First Aid Procedures
Medical Conditions Generally Aggravated by Exposure
Signs and Symptoms of Exposure
Carcinogenicity:
& upper respiratory tract. Toxocological properties have not been thoroughly investigated.
Health Hazards (Acute and Chronic) Cause eye & skin irritation, irritating to mucous membranes
Route(s) of Entry:
Conditions to Avoid
Nature of decomposition products not known
Will Not Occur
May Occur
Conditions to Avoid
Strong acids
Section VI - Health Hazard Data
Hazardous
Polymerization
Hazardous Decomposition or Byproducts
Incompatibility
Stability
Section V - Reactivity Data
Emits toxic fumes under fire conditions
Unusual Fire and Explosion Hazards
Wear SCBA and protective clothing to prevent contact with skin and eyes
Special Fire Fighting Procedures
Use extinquishing media appropriate to surrounding fire
Extinguishing Media
Noncombustible
Flash Point (Method Used)
ACGIH TLV
Specific Gravity (H 0 = 1)
2
solid
Section IV - Physical/Chemical Characteristics
Appearance and Odor
Solubility in Water
Vapor Density (AIR = 1)
Vapor Pressure (mm Hg.)
Boiling Point
Section III - Physical/Chemical Characteristics
Hazardous Components [Specific
Chemical Identity; Common Name(s)]
(301) 251-5990
Other Limits
Recommended
Signature of Preparer (optional)
Date Prepared
Section II - Hazardous Ingredients/Identify Information
14676 Rothgeb Drive
Rockville, MD 20850
(301) 251-5990
Telephone Number for information
Emergency Telephone Number
Note: Blank spaces are not permitted. If any item is not
applicable, or no information is available, the space must
be marked to indicate that.
May be used to comply with OSHA's Hazard Communication
Standard. 29 CFR 1910.1200 Standard must be consulted for
specific requirements.
Address (Number, Street, City, State, Zip Code)
EDVOTEK, Inc.
Manufacturer's Name
Section I
PBS
IDENTITY (As Used on Label and List)
EDVOTEK
Full-size (8.5 x 11”) pdf copy of MSDS is available at www. edvotek.com or by request.
278
Experiment
21
278
1121 5th Street NW
Washington DC 20001
08-15-11
Experiment
22
Material Safety Data Sheets
Full-size (8.5 x 11”) pdf copy of MSDS is available at www. edvotek.com or by request.