elisa laboratory activity - images-na.ssl-images-amazon.com
TRANSCRIPT
The Biotechnology Education Company ®
EDVOTEK, Inc. • 1-800-EDVOTEK • www.edvotek.com
EVT 2011_07_25AM
EDVO-Kit #
278Quantitative ELISA Laboratory Activity
Storage: See Page 3 for specifi c 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 specifi city of antibodies, the effect of dilution on ELISA reactions, color
development and quantitation.
Updated
Revised
and
2The Biotechnology Education Company® • 1-800-EDVOTEK • www.edvotek.com
Quantitative ELISA Laboratory Activity
EVT 2011_07_25AM
All components are intended for educational research only. They are not to be used for diagnostic or drug purposes, nor administered to or con-sumed by humans or animals.
THIS EXPERIMENT DOES NOT CONTAIN HUMAN DNA. None of the experiment components are derived from human sources.
EDVOTEK and The Biotechnology Education Company are trademarks of EDVOTEK, Inc.
Page
Experiment Components 3
Experiment Requirements 3
Background Information 4
Experiment Procedures
Experiment Overview and General Instructions 6
The Enzyme Linked Immunosorbent Assay (ELISA) 8
Study Questions 14
Instructor’s Guidelines
Notes to the Instructor 15
Pre-Lab Preparations 16
Quick Reference Tables 18
Avoiding Common Pitfalls 18
Expected Results 19
Study Questions and Answers 20
Material Safety Data Sheets 21
Table of Contents
3
278278Experiment #
Quantitative ELISA Laboratory Activity
EVT 2011_07_25AM
EDVOTEK - The Biotechnology Education Company® 1-800-EDVOTEK • www.edvotek.com
FAX: (301) 340-0582 • email: [email protected]
A Antigen 1B Antigen 2 C Primary Antibody 1D Primary Antibody 2E Secondary Antibody F Gelatin (blocking agent)G Hydrogen Peroxide, stabilizedH Phosphate buffered saline concentrateI 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.
This experiment is designed for 6 groups.
Upon receipt, store the perishable components (A-I) in the refrigerator.
Experiment Components
Requirements
• Distilled or deionized water• Beakers or fl asks• 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.
Now with NEW
Substrate!
4
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
The Biotechnology Education Company® • 1-800-EDVOTEK • www.edvotek.com
Quantitative ELISA Laboratory Activity278278 Experiment
Antibodies are specifi c 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 anti-gens 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 signifi cant 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 specifi c biological antigen, it can recognize specifi c chemical charges, sequences or structural conformational elements. These structural binding char-acteristics make up the specifi c fi ngerprint for an antigen. Each antibody molecule can bind two antigen molecules. This recognition and binding is highly specifi c 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 specifi c 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 tradi-tional immunoprecipitation assay, antibodies are obtained from the serum of an animal exposed to the specifi c antigen. The serum, also known as plasma, is prepared by the removal of red blood cells. It contains the specifi c 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 purifi ed 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 anti-body 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, specifi c proteins, or a mixture of the two. There is no specifi city 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 an-tibody will bind to it and also remain attached after washing. These secondary antibod-ies are usually raised in rabbits and goats immunized with "non-self" IgG fractions. The second IgG antibodies are purifi ed and covalently cross- linked to horseradish peroxidase. This modifi cation does not signifi cantly affect the binding specifi city and affi nity of the antibody or the enzymatic activity of the peroxidase.
Background Information
5
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
The Biotechnology Education Company® • 1-800-EDVOTEK • www.edvotek.com
278278Experiment
Quantitative ELISA Laboratory Activity
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, amplifi cation 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 numer-ous 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 prepara-tions to a given antigen can have variable binding affi nities due to differences in the im-munological responses between animals. Different immunizations with the same antigen in the same animal can also produce variable binding affi nities. The use of monoclonal antibodies directed against a single epitope eliminates this variability. Western blot analysis of positive samples can be used to confi rm the presence and size of antibodies.
This ELISA experiment is designed to detect two separate circulating IgG molecules direct-ed towards two distinct antigens. Observations in this experiment include specifi city of antibodies, the effect of dilution on ELISA reactions, color development and quantitation.
Figure 1: ELISA “Sandwich”
Background Information
well
Anti-IgG (Rabbit)
Human Serum (IgG)
HIV Antigen
well
well
Substrate (S)(Reduced)(colorless)
Product (P)(Oxidized)
(color)
Human Serum (IgG)
HIV Antigen
Anti-IgG (Rabbit)
Human Serum (IgG)
HIV Antigen
Horseradish peroxidase
Horseradish peroxidase
2H2O2 –> H20 + 2O2
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. Copyright © 2000, 2005, 2009, 2011, EDVOTEK, Inc., all rights reserved. EVT 2011_07_25AM
The Biotechnology Education Company® • 1-800-EDVOTEK • www.edvotek.com
Quantitative ELISA Laboratory Activity278278 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 ex-periment is designed to detect circulating IgG antibodies directed towards two antigens. Observations in this experiment include specifi city 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.
LABORATORY NOTEBOOK RECORDINGS:
Address and record the following in your laboratory notebook or on a separate work-sheet.
Before starting the Experiment:
• Write a hypothesis that refl ects 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 refl ect this change.
Wear Gloves and Goggles
7
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
The Biotechnology Education Company® • 1-800-EDVOTEK • www.edvotek.com
278278Experiment
Quantitative ELISA Laboratory ActivityExp
erimen
t Proced
ure
1. Equilibrate a 37° C incubation oven before starting the experi-ment.
2. Place the microtiter plate as shown in the fi gure at right and carefully mark the plate with your initials or lab group number
3. Using a permanent marker, label the columns 1-5 across the top and the rows A-H down the side.
4. As shown in the fi gure, 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.
Microtiter plate
A
B
C
D
E
F
G
H
1 2 3 4 5
5. Label 4 large transfer pipets as follows (these are to be used for addition of reagents to the wells):
• PBS for Phosphate Buffered Saline • Ag1 for Antigen 1 • Ag2 for Antigen 2 • block for blocking agent 6. Label 2 small transfer pipets Ag1 and Ag2. These are to be used for removal of liquid from the wells.
7. Proceed to dilution of Primary Antibodies 1 and 2 on pages 8 and 9.
Experiment Overview and General Instructions
CAUTION: To avoid cross-contamination and false results, use the appropriately labelled plastic transfer pipet for liquid removals and washes as outlined in the experimental procedures.
1 Half piece of microtiter plate1 Tube labeled "Ag1"1 Tube labeled "Ag2"1 Tube labeled "block"1 Tube labeled "Ab1 - 1"1 Tube labeled "Ab2 -1"1 Tube labeled "2°Ab"1 Automatic micropipet with tips (optional)11 Transfer pipets (large)11 Transfer pipet (small)1 Beaker containing PBS1 Empty beaker labeled "waste"1 Tube labeled "Substrate" (just before the last incubation)8 Microcentrifuge tubes
STUDENT MATERIALS:Each Lab Group Should Receive the following components prior to the start of the experiment procedure.
8
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
The Biotechnology Education Company® • 1-800-EDVOTEK • www.edvotek.com
Quantitative ELISA Laboratory Activity278278 Experiment
Exp
erim
ent
Pro
ced
ure
DILUTION OF PRIMARY ANTIBODIES 1 AND 2
Primary Antibody 1
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.
If using a micropi-pet for diluting the antibodies, dilute the 1:400 antibody solu-tion 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.
2 drops(110 µl)
2 drops(110 µl)
2 drops(110 µl)
2 drops(110 µl)
6 drops(330 µl PBS)
(Obtainedfrom instructor)
6 drops(330 µl PBS)
6 drops(330 µl PBS)
6 drops(330 µl PBS)
1:400 1:1600 1:6400 1:25,600 1:102,400
Ab1 - 1 Ab1 - 2 Ab1 - 3 Ab1 - 4 Ab1 - 5
Quantitative Enzyme Linked Immunosorbent Assay (ELISA)
9
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
The Biotechnology Education Company® • 1-800-EDVOTEK • www.edvotek.com
278278Experiment
Quantitative ELISA Laboratory ActivityExp
erimen
t Proced
ure
Primary Antibody 2
1. Label four tubes as follows: “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.
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.
2 drops(110 µl)
2 drops(110 µl)
2 drops(110 µl)
2 drops(110 µl)
6 drops(330 µl PBS)
(Obtainedfrom instructor)
6 drops(330 µl PBS)
6 drops(330 µl PBS)
6 drops(330 µl PBS)
1:400 1:1600 1:6400 1:25,600 1:102,400
Ab2 - 1 Ab2 - 2 Ab2 - 3 Ab2 - 4 Ab2 - 5
Quantitative Enzyme Linked Immunosorbent Assay (ELISA)
NOTE: Keep the Primary Antibodies 1 and 2 and their dilutions in the refrigerator until needed.
10
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
The Biotechnology Education Company® • 1-800-EDVOTEK • www.edvotek.com
Quantitative ELISA Laboratory Activity278278 Experiment
Exp
erim
ent
Pro
ced
ure
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 verti-cal axis (as shown in the fi gure at left).
2. Read through the instructions and study the fi gure on the previous page before beginning to add antigens to the wells. Refer to the fi g-ure often during the experimental procedure to avoid making errors.
ADDITION OF ANTIGENS:
3. 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. 4. Incubate the plate at room temperature for 5 minutes.
The Enzyme Linked Immunosorbent Assay (ELISA)
Note:
None of the wells in column 6 will be used in this experiment.
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-contami-nation 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 micropi-pet, 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.
A
B
C
D
E
F
G
H
1 2 3 4 5
11
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
The Biotechnology Education Company® • 1-800-EDVOTEK • www.edvotek.com
278278Experiment
Quantitative ELISA Laboratory ActivityExp
erimen
t Proced
ure
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.
11. Using the small pipet labelled “Ag1”, remove the liquid from wells 1 - 5 in rows A - D.
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 fi nished.
OPTIONAL STOPPING POINT:
The experiment can be stopped after addition of PBS (step 13) and resumed during next lab period. Cover microtiter plates with parafi lm or plastic wrap and refrigerate overnight.
Use the appropri-ately labeled plastic transfer pipet for liquid removals and washes to avoid cross-contami-nation and false results.
12
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
The Biotechnology Education Company® • 1-800-EDVOTEK • www.edvotek.com
Quantitative ELISA Laboratory Activity278278 Experiment
Exp
erim
ent
Pro
ced
ure
ADDITION OF PRIMARY ANTIBODIES 1 & 2 (PREPARED ON PAGES 8 & 9)
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.
NOTE:To add primary antibody, use the same pipet going from lowest concentration to high-est concentration. (Use a fresh transfer pipet or a fresh pipet tip for addition of Antibody 1, Antibody 2, and PBS.)
Quantitative Enzyme Linked Immunosorbent Assay (ELISA)
blank
blank
blank
blank
blank
A
B
C
D
E
F
G
H
1 2 3 4 5
Ab1:1 Ab1:2 Ab1:3 Ab1:4 Ab1:5
Ab1:1 Ab1:2 Ab1:3 Ab1:4 Ab1:5
PBS PBS PBS PBS PBS
PBSPBS PBS PBS PBS
6
blank
blank
blank
Ab1:1 Ab1:2 Ab1:3 Ab1:4 Ab1:5
Ab2:1 Ab2:2 Ab2:3 Ab2:4 Ab2:5
Ab2:1 Ab2:2 Ab2:3 Ab2:4 Ab2:5
Ab2:1 Ab2:2 Ab2:3 Ab2:4 Ab2:5
Use the appropriately labeled plastic transfer pipet for liquid remov-als and washes to avoid cross-contamination and false results.
13
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
The Biotechnology Education Company® • 1-800-EDVOTEK • www.edvotek.com
278278Experiment
Quantitative ELISA Laboratory ActivityExp
erimen
t Proced
ure
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
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).
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.
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 trans-fer 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.
14
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
The Biotechnology Education Company® • 1-800-EDVOTEK • www.edvotek.com
Quantitative ELISA Laboratory Activity278278 Experiment
Exp
erim
ent
Pro
ced
ure
Study Questions
Answer the following study questions in your laboratory notebook or on a separate worksheet.
1. 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?
15
278278Experiment #
Quantitative ELISA Laboratory Activity
EVT 2011_07_25AM
EDVOTEK - The Biotechnology Education Company® 1-800-EDVOTEK • www.edvotek.com
FAX: (301) 340-0582 • email: [email protected]
Instructor’s Guide
Class size, length of laboratory sessions, and availability of equipment are factors which must be considered in planning and implementing this experi-ment with your students. These guidelines can be adapted to fi t your spe-cifi c set of circumstances. If you do not fi nd 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).
APPROXIMATE TIME REQUIREMENTS FOR PRE-LAB AND EXPERIMENTAL PROCEDURES
• 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.
Notes to the Instructor & Pre-Lab Preparations
Visit our web site for information about EDVOTEK's complete line of experiments for biotechnology
and biology education.
Visit the EDVOTEK web site often for continuously updated information.
Mon - Fri 9 am - 6 pm ET
(1-800-338-6835)
EDVO-TECH SERVICE
1-800-EDVOTEK
Mon - Fri9:00 am to 6:00 pm ET
FAX: (301) 340-0582Web: www.edvotek.comemail: [email protected]
Please have the following information ready:
• Experiment number and title• Kit lot number on box or tube• Literature version number (in lower right corner)• Approximate purchase date
Technical ServiceDepartment
OrderOnline
16
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
The Biotechnology Education Company® • 1-800-EDVOTEK • www.edvotek.com
278278Experiment
Inst
ruct
or’
s G
uid
eInstructor’s Guide Quantitative ELISA
Laboratory Activity
PREPARATIONS BEFORE THE LAB
Microtiter Plates
1. As shown in the fi gure 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 fi gure. 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.
PREPARATIONS ON THE DAY OF THE LAB
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. 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”.
2. 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.
Pre-Lab Preparations
A
B
C
D
E
F
G
H
1 2 3 4 5 6 7 8 9 10 11 12
NOTES:
The Phosphate Buff-ered saline may pre-cipitate 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 precipi-tate during storage. Warm @ 37° C for 5-10 minutes or until the precipitate has dissolved.
17
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
The Biotechnology Education Company® • 1-800-EDVOTEK • www.edvotek.com
278278Experiment
Quantitative ELISA Laboratory Activity
Instru
ctor’s G
uid
eInstructor’s Guide
Primary Antibodies 1 and 2 (1:400)
1. Add 0.3 ml diluted PBS to tube C. Mix well and transfer the entire con-tents 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 con-tents 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 thor-oughly 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 sec-ondary antibody are very small - the tubes should be centrifuged to collect the samples at the bottom of the tubes before they are diluted.
Pre-Lab Preparations
Quick Reference: The substrate is pre-pared for the peroxi-dase enzyme, which is attached to the anti-IgG peroxidase conju-gate (secondary anti-body). Prepare the substrate 15 - 30 min-utes before students require it for plate development (last incu-bation).
18
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
The Biotechnology Education Company® • 1-800-EDVOTEK • www.edvotek.com
278278Experiment
Inst
ruct
or’
s G
uid
eInstructor’s Guide Quantitative ELISA
Laboratory Activity
PREPARATION OF EXPERIMENT REAGENTS
1 half piece of microtiter plate1 tube labeled "Ag1"1 tube labeled "Ag2"1 tube labeled "block"1 tube labeled "Ab1 - 1"1 tube labeled "Ab2 - 1"1 tube labeled "2°Ab"1 automatic micropipet with tips (optional)11 Transfer pipets (large)11 Transfer pipet (small)1 beaker containing PBS1 empty beaker labeled "waste"1 tube labeled "Substrate" (just before the last incubation)8 microcentrifuge tubes
STUDENT MATERIALSEach Lab Group Should Receive:
Dispense for each groupLabel
A Antigen 1 Ag1 1.5 mlB Antigen 2 Ag2 1.5 mlC + PBS Antibody 1 (1:400 dilution) Ab1 - 1 0.5 mlD + PBS Antibody 2 (1:400 dilution) Ab2 - 1 0.5 mlE + PBS Secondary Antibody 2°Ab 3.0 mlG + I + PBS Peroxidase Enzyme Substrate Substrate 2.5 mlH + dH2O Phosphate buffered saline PBS 50 mlF Gelatin (Blocking Agent) block 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.
Pre-Lab Preparations
AVOIDING COMMON PITFALLS
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 la-beled pipets and avoid contaminating adjacent wells.
3. Do not attempt to empty the micro-titer wells by shaking it out. This will not work - it will result in contaminat-ing adjacent wells.
4. Wash the wells gently and slowly, without force.
19
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
The Biotechnology Education Company® • 1-800-EDVOTEK • www.edvotek.com
278278Experiment
Quantitative ELISA Laboratory Activity
Instru
ctor’s G
uid
eInstructor’s Guide
Expected Results
1:400 1:1600 1:6400 1:25,600
A
B
CD
E
F
G
H
1 2 3 4 5
1:102,400
The idealized schematic at right shows representative results. Actual results may vary.
21
278278Experiment
Material Safety Data SheetsFull-size (8.5 x 11”) pdf copy of MSDS is available at www. edvotek.com or by request.
Mat
eria
l Saf
ety
Dat
a S
hee
tM
ay b
e us
ed to
com
ply
with
OS
HA
's H
azar
d C
omm
unic
atio
nS
tand
ard.
29
CF
R 1
910.
1200
Sta
ndar
d m
ust b
e co
nsul
ted
for
spec
ific
requ
irem
ents
.
IDE
NT
ITY
(A
s U
sed
on L
abel
and
Lis
t)N
ote:
Bla
nk s
pace
s ar
e no
t per
mitt
ed.
If an
y ite
m is
not
ap
plic
able
, or
no in
form
atio
n is
ava
ilabl
e, th
e sp
ace
mus
t be
mar
ked
to in
dica
te th
at.
Sec
tio
n I
Man
ufac
ture
r's N
ame
Sec
tio
n II
- H
azar
do
us
Ing
red
ien
ts/Id
enti
fy In
form
atio
n
Em
erge
ncy
Tele
phon
e N
umbe
r
Tele
phon
e N
umbe
r fo
r in
form
atio
n
Dat
e P
repa
red
Sig
natu
re o
f Pre
pare
r (o
ptio
nal)
Add
ress
(N
umbe
r, S
tree
t, C
ity, S
tate
, Z
ip C
ode)
ED
VO
TE
K, I
nc.
1467
6 R
oth
geb
Dri
veR
ock
ville
, MD
208
50
Haz
ardo
us C
ompo
nent
s [S
peci
fic
Che
mic
al Id
entit
y; C
omm
on N
ame(
s)]
O
SH
A P
EL
AC
GIH
TLV
Oth
er L
imits
R
ecom
men
ded
% (
Opt
iona
l)
(301
) 25
1-59
90
(301
) 25
1-59
90
Boi
ling
Poi
nt
Sec
tio
n II
I - P
hys
ical
/Ch
emic
al C
har
acte
rist
ics
Unu
sual
Fire
and
Exp
losi
on H
azar
ds
Spe
cial
Fire
Fig
htin
g P
roce
dure
s
Vap
or P
ress
ure
(mm
Hg.
)
Vap
or D
ensi
ty (
AIR
= 1
)
Sol
ubili
ty in
Wat
er
App
eara
nce
and
Odo
r
Sec
tio
n IV
- P
hys
ical
/Ch
emic
al C
har
acte
rist
ics
Fla
sh P
oint
(M
etho
d U
sed)
Ext
ingu
ishi
ng M
edia
Fla
mm
able
Lim
itsU
EL
LEL
Mel
ting
Poi
nt
Eva
pora
tion
Rat
e(B
utyl
Ace
tate
= 1
)
Spe
cific
Gra
vity
(H
0 =
1)
2
EDVOTEK
®
A -
E 2
78
CA
S# 1
39-3
3-3
CA
S# 2
6628
-22-
8
V
ery
dilu
te
No
data
No
data
No
data
No
data
No
data
No
data
Solu
ble
Cle
ar li
quid
, no
odor
No
data
N.D
. = N
o da
ta
Dry
che
mic
al, c
arbo
n di
oxid
e, h
alon
. wat
er s
pray
or
stan
dard
foa
m
Mor
e co
ntai
ner
from
fir
e ar
ea if
pos
sibl
e. D
ike
fire
con
trol
w
ater
for
late
r di
spos
al
The
rmal
dec
ompo
sitio
n pr
oduc
ts m
ay in
clud
e to
xic
and
haza
rdou
sox
ides
of
carb
on, n
itrog
en, a
nd s
odiu
m
07/2
7/09
Sta
bilit
y
Sec
tio
n V
- R
eact
ivit
y D
ata
Uns
tabl
e
Sec
tio
n V
I - H
ealt
h H
azar
d D
ata
Inco
mpa
tibili
ty
Con
ditio
ns to
Avo
id
Rou
te(s
) of
Ent
ry:
Inha
latio
n?In
gest
ion?
Ski
n?
Oth
er
Sta
ble
Haz
ardo
us
Pol
ymer
izat
ion
May
Occ
urC
ondi
tions
to A
void
Will
Not
Occ
ur
Hea
lth H
azar
ds (
Acu
te a
nd C
hron
ic)
Car
cino
geni
city
:N
TP
?O
SH
A R
egul
atio
n?IA
RC
Mon
ogra
phs?
Sig
ns a
nd S
ympt
oms
of E
xpos
ure
Med
ical
Con
ditio
ns G
ener
ally
Agg
rava
ted
by E
xpos
ure
Em
erge
ncy
Firs
t Aid
Pro
cedu
res
Sec
tio
n V
II -
Pre
cau
tio
ns
for
Saf
e H
and
ling
an
d U
seS
teps
to b
e Ta
ken
in c
ase
Mat
eria
l is
Rel
ease
d fo
r S
pille
d
Was
te D
ispo
sal M
etho
d
Pre
caut
ions
to b
e Ta
ken
in H
andl
ing
and
Sto
ring
Oth
er P
reca
utio
ns
Sec
tio
n V
III -
Co
ntr
ol M
easu
res
Ven
tilat
ion
Loca
l Exh
aust
Spe
cial
Mec
hani
cal (
Gen
eral
)
Res
pira
tory
Pro
tect
ion
(Spe
cify
Typ
e)
Pro
tect
ive
Glo
ves
Oth
er P
rote
ctiv
e C
loth
ing
or E
quip
men
t
Wor
k/H
ygie
nic
Pra
ctic
es
Eye
Pro
tect
ion
Haz
ardo
us D
ecom
posi
tion
or B
ypro
duct
s
X
Exc
essi
ve h
eat,
spar
ks o
r op
en f
lam
e, p
rote
in d
enat
uran
ts
Aci
ds, a
lum
inum
, met
als,
oxi
dize
rs (
stro
ng)
The
rmal
dec
ompo
sitio
n pr
oduc
ts o
f tox
ic &
haz
ardo
us o
xide
sof
car
bon
and
nitr
ogen
X Yes
Yes
Yes
M
oder
atel
y to
xic
by in
gest
ion.
Sys
tem
atic
toxi
city
may
res
ult.
May
ch
elat
e le
ad, m
agne
sium
, zin
c, tr
ace
met
als
if p
rese
nt in
inte
stin
e. S
ensi
tivity
rea
ctio
ns -
ana
phyl
actic
sho
ck
Non
e
No
data
No
data
No
data
Muc
ous
mem
bran
e ir
rita
tion,
eye
/ski
n ir
rita
tion,
irri
tatin
g to
ga
stro
inte
stin
al s
yste
m
Ren
al o
r he
art d
isea
se, p
otas
sium
def
icie
ncy.
Ins
ulin
-dep
ende
nt d
iabe
tes,
seiz
ures
or
intr
acra
nial
lesi
ons
Tre
at s
ympt
omat
ical
ly a
nd s
uppo
rtiv
ely
Mop
up
with
abs
orpt
ive
mat
eria
l. C
onta
iner
ize
to d
ispo
se o
f pr
oper
ly
Obs
erve
fed
eral
, sta
te, a
nd lo
cal l
aws.
Stor
e aw
ay f
rom
str
ong
oxid
izer
s or
hea
t. A
void
eye
/ski
n co
ntac
t
Non
e
No
Non
e
Dilu
tion
vent
sys
Non
e
Yes
Spla
sh p
roof
gog
gles
Impe
rvio
us c
loth
ing
to p
reve
nt c
onta
ct
Em
erge
ncy
eye
was
h sh
ould
be
avai
labl
e
Che
mic
al c
artr
idge
res
pira
tor
with
ful
l fac
epie
ce a
nd o
rgan
ic v
apor
car
trid
ge
1121
5th
Str
eet
NW
Was
hin
gto
n D
C 2
0001
08-1
5-11
Mat
eria
l Saf
ety
Dat
a Sh
eet
May
be
use
d t
o c
om
ply
wit
h O
SHA
's H
azar
d C
om
mu
nic
atio
nSt
and
ard
. 29
CFR
191
0.12
00 S
tan
dar
d m
ust
be
con
sult
ed f
or
spec
ific
req
uir
emen
ts.
IDEN
TITY
(A
s U
sed
on
Lab
el a
nd
Lis
t)N
ote
: B
lan
k sp
aces
are
no
t p
erm
itte
d.
If a
ny
item
is n
ot
app
licab
le, o
r n
o in
form
atio
n is
ava
ilab
le, t
he
spac
e m
ust
b
e m
arke
d t
o in
dic
ate
that
.
Sect
ion
IM
anu
fact
ure
r's
Nam
e
Sect
ion
II -
Haz
ard
ou
s In
gre
die
nts
/Id
enti
fy In
form
atio
n
Emer
gen
cy T
elep
ho
ne
Nu
mb
er
Tele
ph
on
e N
um
ber
fo
r in
form
atio
n
Dat
e Pr
epar
ed
Sig
nat
ure
of
Prep
arer
(o
pti
on
al)
Ad
dre
ss (
Nu
mb
er, S
tree
t, C
ity,
Sta
te,
Zip
Co
de)
EDV
OTE
K, I
nc.
1467
6 R
oth
geb
Dri
veR
ock
ville
, MD
208
50
Haz
ard
ou
s C
om
po
nen
ts [
Spec
ific
C
hem
ical
Iden
tity
; C
om
mo
n N
ame(
s)]
O
SHA
PEL
AC
GIH
TLV
Oth
er L
imit
s R
eco
mm
end
ed%
(O
pti
on
al)
(301
) 25
1-59
90
(301
) 25
1-59
90
Bo
ilin
g P
oin
t
Sect
ion
III -
Ph
ysic
al/C
hem
ical
Ch
arac
teri
stic
s
Un
usu
al F
ire
and
Exp
losi
on
Haz
ard
s
Spec
ial F
ire
Fig
hti
ng
Pro
ced
ure
s
Vap
or
Pres
sure
(m
m H
g.)
Vap
or
Den
sity
(A
IR =
1)
Solu
bili
ty in
Wat
er
Ap
pea
ran
ce a
nd
Od
or
Sect
ion
IV -
Ph
ysic
al/C
hem
ical
Ch
arac
teri
stic
sFl
ash
Po
int
(Met
ho
d U
sed
)
Exti
ng
uis
hin
g M
edia
Flam
mab
le L
imit
sU
ELLE
L
Mel
tin
g P
oin
t
Evap
ora
tio
n R
ate
(Bu
tyl A
ceta
te =
1)
Spec
ific
Gra
vity
(H
0 =
1)
2
PBS
10/1
0/06
100C
No
dat
a
No
dat
a
1.01
7
No
dat
a
No
dat
a
solu
ble
solid
No
nco
mb
ust
ible
Use
ext
inq
uis
hin
g m
edia
ap
pro
pri
ate
to s
urr
ou
nd
ing
fir
e
Wea
r SC
BA
an
d p
rote
ctiv
e cl
oth
ing
to
pre
ven
t co
nta
ct w
ith
ski
n a
nd
eye
s
Emit
s to
xic
fum
es u
nd
er f
ire
con
dit
ion
s
Stab
ility
Sect
ion
V -
Rea
ctiv
ity
Dat
aU
nst
able
Sect
ion
VI -
Hea
lth
Haz
ard
Dat
a
Inco
mp
atib
ility
Co
nd
itio
ns
to A
void
Ro
ute
(s)
of
Entr
y:In
hal
atio
n?
Ing
esti
on
?Sk
in?
Oth
er
Stab
le
Haz
ard
ou
s Po
lym
eriz
atio
nM
ay O
ccu
rC
on
dit
ion
s to
Avo
id
Will
No
t O
ccu
r
Hea
lth
Haz
ard
s (A
cute
an
d C
hro
nic
)
Car
cin
og
enic
ity:
NTP
?O
SHA
Reg
ula
tio
n?
IAR
C M
on
og
rap
hs?
Sig
ns
and
Sym
pto
ms
of
Exp
osu
re
Med
ical
Co
nd
itio
ns
Gen
eral
ly A
gg
rava
ted
by
Exp
osu
re
Emer
gen
cy F
irst
Aid
Pro
ced
ure
s
Sect
ion
VII
- Pr
ecau
tio
ns
for
Safe
Han
dlin
g a
nd
Use
Step
s to
be
Take
n in
cas
e M
ater
ial i
s R
elea
sed
fo
r Sp
illed
Was
te D
isp
osa
l Met
ho
d
Prec
auti
on
s to
be
Take
n in
Han
dlin
g a
nd
Sto
rin
g
Oth
er P
reca
uti
on
s
Sect
ion
VIII
- C
on
tro
l Mea
sure
s
Ven
tila
tio
nLo
cal E
xhau
stSp
ecia
l
Mec
han
ical
(G
ener
al)
Res
pir
ato
ry P
rote
ctio
n (
Spec
ify
Typ
e)
Pro
tect
ive
Glo
ves
Oth
er P
rote
ctiv
e C
loth
ing
or
Equ
ipm
ent
Wo
rk/H
ygie
nic
Pra
ctic
es
Eye
Pro
tect
ion
Haz
ard
ou
s D
eco
mp
osi
tio
n o
r B
ypro
du
cts C
ause
eye
& s
kin
irri
tati
on
, irr
itat
ing
to
mu
cou
s m
emb
ran
es
& u
pp
er r
esp
irat
ory
tra
ct.
Toxo
colo
gic
al p
rop
erti
es h
ave
no
t b
een
th
oro
ug
hly
inve
stig
ated
.
Swal
low
ed -
was
h o
ut
mo
uth
wit
h w
ater
pro
vid
ed p
erso
n is
co
nsc
iou
s.
Skin
/eye
co
nta
ct -
flu
sh w
ith
wat
er
Inh
alat
ion
- r
emo
ve t
o f
resh
air
Yes
Y
es
Y
es
W
ear
resp
irat
or,
chem
ical
saf
ety
go
gg
les,
ru
bb
er b
oo
ts a
nd
hea
vy r
ub
ber
glo
ves,
sw
eep
up
, pla
ce in
a b
ag a
nd
ho
ld f
or
was
te d
isp
osa
l.
NIO
SH/M
SHA
ap
pro
ved
res
pir
ato
r
N/A
N/A
N/A
N/A
Yes
Yes
Do
no
t in
ges
t. A
void
co
nta
ct w
ith
ski
n, e
yes
and
clo
thin
g.
Was
h t
ho
rou
gh
ly a
fter
han
dlin
g.
Stro
ng
aci
ds
Nat
ure
of
dec
om
po
siti
on
pro
du
cts
no
t kn
ow
n
For
smal
l qu
anti
ties
- c
auti
osl
y ad
d t
o a
larg
e st
irre
d e
xces
s o
f w
ater
. A
dju
st p
H t
o n
eutr
al
Wea
r ap
pro
pri
ate
NIO
SH/M
SHA
ap
pro
ved
res
pir
ato
r, ch
emic
al r
esis
tan
t g
love
s, s
afet
y g
og
gle
ssa
fety
sh
ow
er a
nd
eye
bat
h.
ED
VO
TE
K®
Material Safety Data SheetsFull-size (8.5 x 11”) pdf copy of MSDS is available at www. edvotek.com or by request.278278
Experiment
22
1121
5th
Str
eet
NW
Was
hin
gto
n D
C 2
0001
08-1
5-11