Antigen-Antibody Interaction: The Ouchterlony Procedure

Transcription

1 The iotechnology Education Company 270 EDVO-Kit # Antigen-Antibody Interaction: The Ouchterlony Procedure Storage: Some components require refrigerator storage. See page 3 for storage requirements. EXPERIMENT OJECTIVES: The objective of this experiment is to introduce the principles of antigen-antibody interactions using the Ouchterlony procedure. 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.

2 2 Table of Contents Page Experiment Components 3 Experiment Requirements 3 ackground Information Antigen-Antibody Interaction 4 Experiment Procedures Experiment Overview 7 Student Experimental Procedures 8 Study Questions 12 Instructor's Guidelines Notes to the Instructor 13 Pre-Lab Preparations 14 Avoiding Common Pitfalls 16 Idealized Schematic of Results 17 Study Questions and Answers 18 Material Safety Data Sheets 19

3 3 Experiment Components This experiment is designed for 10 groups. Store components A - D in the refrigerator. A Antiserum (antibody) Refrigerator Whole serum (antigen) Refrigerator C Albumin (antigen) Refrigerator D IgG (antigen) Refrigerator E Powdered buffer Room temp. 1 Package UltraSpec-Agarose Room temp. 1 Tube practice loading solution Room temp. 40 Transfer pipets 40 Petri plates 10 Well cutters 1 Template for cutting wells 40 Microtest tubes Requirements 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 components which have been prepared from human sources. Micropipet and tips Plastic container or Pyrex baking dish Plastic wrap Distilled Water Pipets - 5 ml or 10 ml Marking pen Measuring spatula or toothpicks Heat plate, unsen burner, or microwave Paper towels Waterbath EDVOTEK - The iotechnology Education Company EDVOTEK 24-hour FAX: (301) [email protected]

4 4 The interactions of an antibody (Ab) with an antigen (Ag) is the fundamental reaction of immunology. ackground Information Supernatants Excess Ab Excess Ag Antibody Precipitated Figure 1 Figure 2 Antibody-Excess Zone Ab-Excess Zone ± Ab + Ag Ab - Ag Most antigens are proteins. The exact identity of the groups that react with the antibody are usually not known. Macromolecular antigens and antibodies form complexes that become insoluble and precipitate from solution. This property makes it possible to perform qualitative and quantitative assays on the antibody-antigen system. Equivalence Zone Equivalence Zone Antigen added Ag-Excess Zone Antigen-Excess Zone Precipitation occurs with most antigens because the antigen is multivalent, i.e., has several antigenic determinants per molecule to which antibodies can bind. Antibodies have at least two antigen binding sites, thus large aggregates or lattices of antigen and antibody are formed. Experimentally, an increasing amount of antigen is added to a constant amount of antibody in solution. Initially at low antigen concentration, all of the antigen is contained in the precipitate. This is called the antibody-excess zone. As more antigen is added, the amount of protein precipitated increases until the antigen and antibody molecules are at an optimal ratio. This is called the equivalence zone, or equivalence point, where maximum precipitation occurs. When the amount of antigen in solution exceeds the amount of antibody, the amount of precipitation will decrease. This is known as the antigen-excess zone (Figures 1 and 2). Antibody Antigen

5 ackground Information 5 Antigen 1 Antigen 2 When antibodies and antigens are inserted into different areas of an agarose gel, they diffuse toward each other and form opaque bands of precipitate at the interface of their diffusion fronts. Precipitation reactions of antibodies and antigens in agarose gels provide a method of analyzing various antibody-antigen reactions. THE OUCHTERLONY PROCEDURE Mixed Antibody 1 & 2 Figure 3 Antigen 1 Antigen 1 Antibody 1 Double diffusion in two dimensions is a simple procedure invented by and named after the Swedish scientist, Örjan Ouchterlony. Antigen and antibody solutions are placed in separate wells cut in an agarose plate. The reactants diffuse from the wells toward each other and precipitate where they meet at equivalent proportions. A single antigen will combine with its homologous antibody to form a single precipitation line. When two antigens are present, each behaves independently of each other. Thus, the number of precipitin bands indicates there are at least that many antibody-antigen pairs present (see Figure 3). Arrows indicate diffusion patterns of antigens and antibodies. Double diffusion in two dimensions is a useful technique for comparing antigens for the number of identical or cross-reacting determinants. If a solution of antigen is placed in two adjacent wells and the homologous antibody is placed in the center well, the two precipitin bands that form will join at their closest ends and fuse. This is known as a reaction of identity (Figure 4). Figure 4: Reaction of Identity Antigen 1 Antigen 2 Homologous Antigen Cross-reacting Antigen Mixture of Antibody 1 & 2 Mixture of Antibody 1 & 2 EDVOTEK and The iotechnology Education Company are registered trademarks of EDVOTEK, Inc. Figure 5 Reaction of Non-identity Figure 6 Reaction of Partial Identity The patterns shown in Figures 3-6 are the ideal representation. Under experimental conditions, the spurs are often difficult to visualize.

6 6 ackground Information When unrelated antigens are placed in adjacent wells and the center well is filled with antibodies for each antigen, the precipitin bands will form independently of each other and will cross. This is known as a reaction of non-identity (Figure 5). If two purified antigens cross-react, then placing them in adjacent peripheral wells with antibody to one in the central well will give a single band with the homologous and cross-reacting antigen. Since the crossreacting antigen lacks some of the antigenic determinants present in the homologous antigen, it is not able to precipitate all of the antibody. The remaining antibody will diffuse beyond the line of cross-reacting precipitate to react with the homologous antigen to produce a spur. The spur that forms projects toward the antigen with the fewer determinants, i.e., the cross-reacting antigen. This is called a reaction of partial identity. Since these non-cross-reacting antibodies often are only a fraction of the total antibody involved in the homologous precipitin reaction, the spur is usually less dense (often difficult to visualize) than the precipitin band from which it projects (See Figure 7). Homologous Antigen Cross-reacting Antigen Antiserum to Homologous Antigen Figure 7

7 Experiment Procedures 7 Experiment Overview EXPERIMENT OJECTIVE: The objective of this experiment is to introduce the principles of antigenantibody interactions using the Ouchterlony procedure. LAORATORY SAFETY Wear gloves and safety goggles Gloves and goggles should be worn routinely as good laboratory practice.

8 8 Student Experimental Procedures A. PREPARATION OF AGAROSE AND POURING OF OUCHTERLONY PLATES Experiment Procedures 1. Each group requires 4 plates: 1 practice loading plate and 3 experimental plates. Using a 5 ml or a 10 ml pipet, carefully pipet 5 ml of the cooled agarose (55 C) into each plate, rotating the plate to cover the bottom with agarose. Repeat with the remaining plates. 2. If the molten agarose contains bubbles, gently swirl to remove the bubbles. 55 C 3. Allow the agarose to solidify. This will take approximately minutes, at which time the gel will appear slightly opaque. 4. If the plates are not to be used that day, the plates can be wrapped with plastic wrap and stored inverted in the refrigerator for two weeks.. PRACTICE WELL LOADING (OPTIONAL) This experiment contains practice loading solution. This solution is included to allow instructors and students to practice loading the sample wells before performing the actual experiment. Use a micropipetting device, or one of the plastic transfer pipets included in your experiment kit to practice loading the sample wells with the practice loading solution. 1. One practice plate should be prepared for each group. Enough reagents have been provided for this purpose. 2. Using the well cutters provided, cut several rows of wells as shown in the diagram at left. 3. Practice loading the sample wells with the plastic, disposable transfer pipets. (See "Sample Loading of Wells with Transfer Pipets"). 4. If you are using an automatic micropipetting device, the amount of sample that should be loaded is 30 microliters.

9 Experiment Procedures 9 Student Experimental Procedures Sample Loading of Wells With Transfer Pipets 1. Squeeze the pipet stem, not the bulb, to slowly draw a portion of the sample up into the pipet. The sample should remain in the lower portion of the pipet. If the sample is overdrawn and becomes lodged in the bulb or on the walls, tap until the sample moves down into the lower stem of the pipet. Eject it back into the tube. Try step 1 again. 2. While holding the pipet tip above the tube, slowly squeeze the pipet stem until the sample is nearly at the opening of the pipet tip. 3. Place the pipet tip just over, not inside, the sample well. Maintain steady pressure on the pipet stem to prevent sample from being drawn back up into the pipet. 4. Slowly squeeze the pipet bulb to eject two (2) drops of sample. The well should appear full, but be careful not to overfill the wells and cause spillage on the agarose surface. Put any remaining sample in the pipet back into the tube. C. PREPARATION OF SAMPLE WELLS Top 1. Make several copies of the template (at left) for your lab group. 2. Place the template under one of the plates so that the pattern is in the center of the plate. The distances between the wells is important. Try to follow the template as accurately as possible. 3. Cut the five wells using the well cutter (provided in the kit) in a gentle punching motion. Remove the agarose plugs with a flat-edged toothpick or spatula. Template 4. If well placement is not accurate, there should be enough room on the plate to re-cut the wells using the template. 5. Repeat steps 2 and 3 with the remaining two plates.

10 10 Student Experimental Procedures D. LOADING THE SAMPLES Experiment Procedures WEAR SAFETY GOGGLES AND GLOVES A 1. Orient your lab number or group designation at the top before loading samples. 2. Using the same pipet, fill the center wells of all three plates with 30 microliters (2 drops with a transfer pipet) of antiserum (antibody) from Tube A. Wells should appear full, but be careful not to overfill the wells and cause spillage on the agarose surface. This may affect your results. 3. Fill the outer wells with 30 microliters of antigen using a clean pipet tip for each antigen as follows: Plate 1 Center well: antiserum to the fluid containing antibodies (Tube A) Left upper well: Whole serum (Tube ) Right upper well: Whole serum (Tube ) Left lower well: Whole serum (Tube ) Right lower well: Whole serum (Tube ) C A C Plate 2 Center well: antiserum to the fluid containing antibodies (Tube A) Left upper well: Whole serum (Tube ) Right upper well: albumin (Tube C) Left lower well: albumin (Tube C) Right lower well: Whole serum (Tube ) D C Plate 3 Center well: antiserum to the fluid containing antibodies (Tube A) Left upper well: IgG (Tube D) Right upper well: albumin (Tube C) Left lower well: albumin (Tube C) Right lower well: IgG (Tube D) C A D

11 Experiment Procedures 11 Student Experimental Procedures E. INCUATION Replace lids onto plates. Carefully place the covered plates in the incubation chamber on top of the wet paper towel layer. Do not invert the plates. Cover the chamber with plastic wrap and let incubate at room temperature hours to allow precipitin lines to form or the chamber can be placed in a 37 C incubation oven. F. READING THE RESULTS The precipitin lines will be visible in hours. Carefully hold a plate up so that the overhead room lights shine through it. You should be able to see opaque white arcs in each side of the plate where the antibody and antigen precipitated. A drawing of the results should be made.

12 12 Experiment Results and Study Questions LAORATORY NOTEOOK RECORDINGS: Experiment Procedures Address and record the following in your laboratory notebook or on a separate worksheet. efore 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. Explain how qualitative observations can be performed on the antigen-antibody system. 2. What is the equivalence zone or equivalence point? 3. When would you observe the antigen-excess zone? What effect does this have on the amount of precipitation? 4. What would cause two or more precipitin bands to form in an antigen-antibody experiment?

13 13 Notes to the Instructor 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 EDVOTEK ( ). APPROXIMATE TIME REQUIREMENTS Your individual schedule and time requirements will determine when the Ouchterlony plates should be prepared. It takes approximately 20 to 30 minutes to prepare the plates (generally 10 minutes of this time is required for solidification). Students can prepare the plates, if time allows. Online Ordering now available Instructor's Guide Visit our web site for information about EDVOTEK 's complete line of experiments for biotechnology and biology education. Technical Service Department M o n EDVO-TECH - Fri 9 SERVICE EDVOTEK ( ) am - 6 E T pm Mon - Fri 9:00 am to 6:00 pm ET FAX: (301) web: [email protected] Please have the following information: The experiment number and title Kit Lot number on box or tube The literature version number (in lower right corner) Approximate purchase date

14 14 PreLab Preparations Instructor's Guide A. PREPARATION OF AGAROSE AND POURING OF OUCHTERLONY PLATES 1. In a 500 ml Erlenmeyer flask or beaker, add the entire contents of powdered buffer package (Component E) to 225 ml of distilled water. Swirl the flask or beaker to dissolve the powder. 2. Add the entire contents of the agarose package to the flask or beaker. Swirl to disperse large clumps. With a marking pen, indicate the level of solution volume on the outside of the flask or beaker. 3. You must boil the solution to dissolve the agarose. This can be accomplished with a hot plate or unsen burner. Cover the beaker with foil. Heat the mixture to boiling over a burner with occasional swirling. Wear safety goggles and a hot glove. oil the mixture until all the gelatinous agarose is dissolved. During heating, occasionally remove the beaker from the heat and check to see that there are no small, clear particles of agarose. Continue heating with occasional swirling. The final solution should be clear. A microwave can also be used to melt the agarose (no foil cover) on high in 30 sec. pulses for 2 minutes if required. Swirl the flask in between pulses and microwave for an additional 1-2 minutes. Check to see that there are no small clear particles of agarose. The final solution should be clear. 4. If detectable evaporation has occurred, add distilled water to bring the solution up to the original volume as marked on the flask or beaker in step 2. Total volume should be a minimum of 230 ml. 55 C 5. Cool the agarose solution to 55 o C in a waterbath. Swirl to promote even dissipation of heat. 6. Aliquot 25 ml of the agarose solution (55 C) for each of the ten groups.

15 15 Pre-Lab Preparations. PREPARATION OF INCUATION CHAMER (Prepare day of laboratory) Line the bottom of a plastic container or glass container (such as a pyrex dish) with several paper towels. Soak the paper towels with distilled water. There should not be any layer of liquid above the paper towels. All liquid should be absorbed into the paper toweling. Cover the entire chamber with plastic wrap. C. PREPARATION OF ANTIODY AND ANTIGENS Antibody (Ab) and the three antigens (Ag) have been supplied in bulk. It is recommended that the Ab and Ag are aliquoted for each group. Instructor's Guide 1. Label 10 microtest tubes "A". 2. Label 10 microtest tubes "". 3. Label 10 microtest tubes "C". 4. Label 10 microtest tubes "D". 5. Aliquot 100 µl of Sample A into each tube "A". 6. Aliquot 200 µl of Sample into each tube "". 7. Aliquot 150 µl of Sample C into each tube "C". 8. Aliquot 80 µl of Sample D into each tube "D". 9. Each group requires one each of tube A,, C, and D. 10. Aliquot 10 microtest tubes 100 µl of practice solution per group.

16 16 Avoiding Common Pitfalls Instructor's Guide 1. Follow instructions carefully when preparing the gel for the plates. Make sure the agarose is completely dissolved. 2. Make neat, clean wells with the well cutters. Take measures to ensure that the wells are properly spaced according to the template on page Add samples to the wells carefully and precisely. Avoid overfilling the wells. 4. Do not tip or invert plates when transferring to the humidity chamber. 5. Placing the humidity chamber in a 37 C incubation oven will expedite the formation of precipitin arcs. 6. Absence of precipitin lines is usually due to disproportionate pipetting between Ag and Ab wells, or distance between Ag and Ab wells. 7. Spurs may be faint. IgG and albumin are immunologically nonidentical components of whole serum. They are both recognized by different antibodies in the antiserum made against whole rabbit serum.

17 17 Idealized Schematic of Results A Plate 1: Reaction of Identity Note the series of precipitation lines indicating many Ab-Ag systems may be visible. Instructor's Guide Plate 2: Reaction of Partial Identity C A C Spurs may be faint. Albumin is recognized by the antiserum in purified form or as a serum component. The antiserum also recognizes many other serum components. Plate 3: Reaction of Non-Identity D C A C D Spurs may be faint. IgG and albumin are immunologically nonidentical components of whole serum. They are both recognized by different antibodies in the antiserum made against whole rabbit serum. Technically, this is not a reaction of partial identity. It is a reaction of non-identity since albumin is in the serum too. The spur is all the other proteins reacting with the antiserum that antigenically have nothing in common with albumin.

18 Please refer to the kit insert for the Answers to Study Questions

19 Material Safety Data Sheet May be used to comply with OSHA's Hazard Communication Standard. 29 CFR Standard must be consulted for specific requirements. Section V - Reactivity Data Stability Unstable Stable Incompatibility available X Conditions to Avoid IDENTITY (As Used on Label and List) Agarose Section I Manufacturer's Name EDVOTEK, Inc. Address (Number, Street, City, State, Zip Code) Rothgeb Drive Rockville, MD Note: lank spaces are not permitted. If any item is not applicable, or no information is available, the space must be marked to indicate that. Emergency Telephone Number (301) Telephone Number for information (301) Date Prepared Signature of Preparer (optional) Section II - Hazardous Ingredients/Identify Information Hazardous Components [Specific Other Limits Chemical Identity; Common Name(s)] OSHA PEL ACGIH TLV Recommended % (Optional) This product contains no hazardous materials as defined by the OSHA Hazard Communication Standard. CAS # Section III - Physical/Chemical Characteristics oiling Point For 1% solution 194 F Specific Gravity (H 0 = 1) 2 Vapor Pressure (mm Hg.) Melting Point Evaporation Rate Vapor Density (AIR = 1) (utyl Acetate = 1) Solubility in Water Insoluble - cold Appearance and Odor White powder, no odor Section IV - Physical/Chemical Characteristics N.D. = Flash Point (Method Used) Flammable Limits LEL N.D. UEL N.D. Extinguishing Media Water spray, dry chemical, carbon dioxide, halon or standard foam Special Fire Fighting Procedures Possible fire hazard when exposed to heat or flame Hazardous Decomposition or yproducts Hazardous May Occur Conditions to Avoid Polymerization Will Not Occur X Section VI - Health Hazard Data Route(s) of Entry: Inhalation? Yes Skin? Yes Ingestion? Yes Health Hazards (Acute and Chronic) Inhalation: available Ingestion: Large amounts may cause diarrhea Carcinogenicity: NTP? IARC Monographs? OSHA Regulation? Signs and Symptoms of Exposure available Medical Conditions Generally Aggravated by Exposure available Emergency First Aid Procedures Treat symptomatically and supportively Section VII - Precautions for Safe Handling and Use Steps to be Taken in case Material is Released for Spilled Waste Disposal Method Precautions to be Taken in Handling and Storing Other Precautions Section VIII - Control Measures Respiratory Protection (Specify Type) Ventilation Local Exhaust Special Protective Gloves Yes Sweep up and place in suitable container for disposal Normal solid waste disposal Chemical cartridge respirator with full facepiece. Mechanical (General)Gen. dilution ventilationother Eye Protection Splash proof goggles Unusual Fire and Explosion Hazards Other Protective Clothing or Equipment Work/Hygienic Practices Impervious clothing to prevent skin contact Material Safety Data Sheet May be used to comply with OSHA's Hazard Communication Standard. 29 CFR Standard must be consulted for specific requirements. Section V - Reactivity Data Stability Unstable Stable Incompatibility X Conditions to Avoid IDENTITY (As Used on Label and List) Note: lank spaces are not permitted. If any item is not applicable, or no information is available, the space must Practice Gel Loading Solution be marked to indicate that. Section I Manufacturer's Name Emergency Telephone Number (301) EDVOTEK, Inc. Telephone Number for information Address (Number, Street, City, State, Zip Code) (301) Rothgeb Drive Rockville, MD Date Prepared Signature of Preparer (optional) Section II - Hazardous Ingredients/Identify Information Hazardous Components [Specific Chemical Identity; Common Name(s)] OSHA PEL ACGIH TLV Section III - Physical/Chemical Characteristics oiling Point Vapor Pressure (mm Hg.) Vapor Density (AIR = 1) Solubility in Water Soluble Specific Gravity (H 0 = 1) 2 Melting Point Evaporation Rate (utyl Acetate = 1) Other Limits Recommended This product contains no hazardous materials as defined by the OSHA Hazard Communication Standard. % (Optional) Hazardous Decomposition or yproducts Hazardous May Occur Conditions to Avoid Polymerization Will Not Occur X Section VI - Health Hazard Data Route(s) of Entry: Inhalation? Yes Skin? Yes Ingestion? Yes Health Hazards (Acute and Chronic) Acute eye contact: May cause irritation. available for other routes. Carcinogenicity: NTP? IARC Monographs? available OSHA Regulation? Signs and Symptoms of Exposure May cause skin or eye irritation Medical Conditions Generally Aggravated by Exposure reported Emergency First Aid Procedures Treat symptomatically and supportively. Rinse contacted area with copious amounts of water. Section VII - Precautions for Safe Handling and Use Steps to be Taken in case Material is Released for Spilled Wear eye and skin protection and mop spill area. Rinse with water. Waste Disposal Method Observe all federal, state, and local regulations. Precautions to be Taken in Handling and Storing Avoid eye and skin contact. Sulfur oxides, and bromides Appearance and Odor lue liquid, no odor Section IV - Physical/Chemical Characteristics Flash Point (Method Used) Flammable Limits LEL UEL Extinguishing Media Dry chemical, carbon dioxide, water spray or foam Special Fire Fighting Procedures Use agents suitable for type of surrounding fire. Keep upwind, avoid breathing hazardous sulfur oxides and bromides. Wear SCA. Unusual Fire and Explosion Hazards Unknown Other Precautions Section VIII - Control Measures Respiratory Protection (Specify Type) Ventilation Local Exhaust Yes Special Mechanical (General) Yes Other Protective Gloves Yes Eye Protection Other Protective Clothing or Equipment required Work/Hygienic Practices Avoid eye and skin contact Splash proof goggles

20 IDENTITY (As Used on Label and List) A,,C and D/270 Section I Manufacturer's Name EDVOTEK, Inc. Address (Number, Street, City, State, Zip Code) Rothgeb Drive Rockville, MD Material Safety Data Sheet May be used to comply with OSHA's Hazard Communication Standard. 29 CFR Standard must be consulted for specific requirements. Note: lank spaces are not permitted. If any item is not applicable, or no information is available, the space must be marked to indicate that. Emergency Telephone Number (301) Telephone Number for information (301) Date Prepared Signature of Preparer (optional) Section II - Hazardous Ingredients/Identify Information Hazardous Components [Specific Other Limits Chemical Identity; Common Name(s)] OSHA PEL ACGIH TLV Recommended Ethylendiaminetetraacetic acid C10-H14-08-N2.2Na CAS # Section III - Physical/Chemical Characteristics oiling Point Specific Gravity (H 0 = 1) 2 Vapor Pressure (mm Hg.) Melting Point Vapor Density (AIR = 1) Evaporation Rate (utyl Acetate = 1) Solubility in Water Soluble Appearance and Odor Clear liquid, no odor Section IV - Physical/Chemical Characteristics Flash Point (Method Used) Flammable Limits LEL Extinguishing Media Dry chemical, carbon dioxide, halon, water spray or standard foam Special Fire Fighting Procedures % (Optional) Unusual Fire and Explosion Hazards Thermal decomposition products may include toxic and hazardous oxides of carbon, nitrogen, and sodium. IDENTITY (As Used on Label and List) Powdered uffer Section I Manufacturer's Name EDVOTEK, Inc. UEL Move container from fire area if possible. Dike fire control water for later disposal Address (Number, Street, City, State, Zip Code) Rothgeb Drive Rockville, MD Material Safety Data Sheet May be used to comply with OSHA's Hazard Communication Standard. 29 CFR Standard must be consulted for specific requirements. Section II - Hazardous Ingredients/Identify Information Hazardous Components [Specific Chemical Identity; Common Name(s)] OSHA PEL ACGIH TLV Special Fire Fighting Procedures Note: lank spaces are not permitted. If any item is not applicable, or no information is available, the space must be marked to indicate that. Emergency Telephone Number (301) Telephone Number for information (301) Date Prepared Signature of Preparer (optional) Other Limits Recommended Section III - Physical/Chemical Characteristics oiling Point 100C Specific Gravity (H 0 = 1) 2 Vapor Pressure (mm Hg.) Melting Point Vapor Density (AIR = 1) Evaporation Rate (utyl Acetate = 1) Solubility in Water soluble Appearance and Odor solid Section IV - Physical/Chemical Characteristics Flash Point (Method Used) Flammable Limits LEL Noncombustible Extinguishing Media Use extinquishing media appropriate to surrounding fire % (Optional) Wear SCA and protective clothing to prevent contact with skin and eyes Unusual Fire and Explosion Hazards Emits toxic fumes under fire conditions UEL Section V - Reactivity Data Stability Unstable Conditions to Avoid Stable X Excessive heat, sparks or open flame, protein denaturants Incompatibility Acids, aluminum, metals, oxidizers (strong) Hazardous Decomposition or yproducts Thermal decomposition products of toxic & hazardous oxides of Carbon and nitrogen Hazardous May Occur Conditions to Avoid Polymerization Will Not Occur X Section VI - Health Hazard Data Route(s) of Entry: Inhalation? Skin? Ingestion? Yes Yes Yes Health Hazards (Acute and Chronic) Moderately toxic by ingestion. Systematic toxicity may result. May chelate lead, magnesium, zinc, trace metals if present in intestine. Sensitivity reactions-anaphylactic shock Carcinogenicity: NTP? IARC Monographs? OSHA Regulation? Signs and Symptoms of Exposure Mucous membrane irritation, eye/skin irritation, irritating to gastrointestinal system. Medical Conditions Generally Aggravated by Exposure Renal or heart disease, potassium deficiency, insulin-dependent, diabetes, seizures or intracranial lesions Emergency First Aid Procedures Treat symptomatically and supportively Section VII - Precautions for Safe Handling and Use Steps to be Taken in case Material is Released for Spilled Mop up with absorptive material. Containerize to dispose of properly. Waste Disposal Method Observe all federal, state and local regulations Precautions to be Taken in Handling and Storing Store away from strong oxidizers or heat. Avoid eye/skin contact. Other Precautions NONE Section VIII - Control Measures Respiratory Protection (Specify Type) Chemical cartridge respirator with full facepiece and organic vapor cartridge Ventilation Local Exhaust No Special Mechanical (General) Gen. dilution vent sys. Other Protective Gloves Yes Eye Protection Splash proof goggles Other Protective Clothing or Equipment Impervious clothing to prevent contact. Work/Hygienic Practices Emergency eye wash should be available Section V - Reactivity Data Stability Unstable Conditions to Avoid Stable Incompatibility Strong acids Hazardous Decomposition or yproducts Nature of decomposition products not known Hazardous May Occur Conditions to Avoid Polymerization Will Not Occur Section VI - Health Hazard Data Route(s) of Entry: Inhalation? Skin? Ingestion? Yes Yes Yes Health Hazards (Acute and Chronic) Cause eye & skin irritation, material is irritating to mucous membranes and upper respiratory tract. The toxocological properties have not been thoroughly investigated. Carcinogenicity: NTP? IARC Monographs? OSHA Regulation? Signs and Symptoms of Exposure Medical Conditions Generally Aggravated by Exposure Emergency First Aid Procedures Swallowed - wash out mouth with water provided person is conscious. Skin/eye contact - flush with water Inhalation - remove to fresh air Section VII - Precautions for Safe Handling and Use Steps to be Taken in case Material is Released for Spilled Wear respirator, chemical safety goggles, rubber boots and heavy rubber gloves, sweep up, place in a bag and hold for waste disposal. Waste Disposal Method For small quantities - cautiosly add to a large stirred excess of water. Adjust ph to neutral Precautions to be Taken in Handling and Storing Wear appropriate NIOSH/MSHA approved respirator, chemical resistant gloves, safety goggles safety shower and eye bath. Other Precautions Section VIII - Control Measures Respiratory Protection (Specify Type) Ventilation Local Exhaust N/A Special Mechanical (General) N/A Other Protective Gloves Yes Eye Protection Other Protective Clothing or Equipment Work/Hygienic Practices NIOSH/MSHA approved respirator N/A N/A Yes Do not ingest. Avoid contact with skin, eyes and clothing. Wash thoroughly after handling.