In Search of the "Kissing Disease"

Transcription

1 The Biotechnology Education Company 274 EDVO-Kit # In Search of the "Kissing Disease" Storage: Store entire experiment in the refrigerator. EXPERIMENT OBJECTIVES: The objective of this experiment is to understand the experimental concepts and methodology involved with enzyme-linked immunosorbent (ELISA) assays in the context of the clinical screening of serum samples for antibodies to EBV. Primary infection with EBV results in infectious mononucleosis, which is popularly known as the "Kissing Disease". 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 Background Information 4 Experiment Procedures Experiment Overview 7 Student Experimental Procedures 11 Study Questions 12 Instructor's Guidelines Notes to the Instructor 13 Pre-Lab Preparations 14 Avoiding Common Pitfalls 16 Idealized Schematic of Results 16 Study Questions and Answers 17 Material Safety Data Sheets 18

3 3 Experiment Components A B C D E F G H I EBV Antigens (simulated) Positive Control (primary antibody) Donor 1 Serum (simulated) Donor 2 Serum (simulated) Donor 3 Serum (simulated) Donor 4 Serum (simulated) Anti-IgG-peroxidase conjugate (secondary antibody) Substrate solution Phosphate buffered saline concentrate This experiment is designed for 10 groups. Store entire experiment in the refrigerator. Microtiter strips Transfer pipets Microtest tubes with attached caps 15 ml plastic tubes Requirements This experiment does not contain Epstein- Barr virus (EBV) or its components. None of the components have been prepared from human sources. Distilled or deionized water Beakers 37 C Incubation oven Disposable lab gloves Safety goggles Automatic micropipets (0-50 µl) and tips recommended Make sure glassware is clean, dry and free of soap residue. For convenience, additional disposable transfer pipets can be purchased for liquid removal and washing steps. All components are intended for educational research only. They are not to be used for diagnostic or drug purposes, nor administered to or consumed by humans or animals. EDVOTEK - The Biotechnology Education Company EDVOTEK 24-hour FAX: (301) edvotek@aol.com

4 4 Background Information Infectious mononucleosis, popularly known as the "Kissing Disease", is a fairly common illness among students on college campuses around the world. It is called the "Kissing Disease" because it is believed that the virus which causes the infection is transmitted by saliva during kissing. The culprit responsible for this disease is the Epstein-Barr virus (EBV). It is a DNA virus and is a member of the herpes virus group. EBV was first described by Epstein and coworkers. Infections due to EBV are usually of short duration, self-limiting and without long term effect, characterized by fever and sore throat. The virus is indigenous to all world populations and infects most humans at some stage of their life. Primary infections in third world countries usually occur in early childhood and are subclinical. In more developed countries primary infections usually occur at a later age and may present itself as acute infectious mononucleosis. Its presence can be easily demonstrated by immunological ELISA tests. Primary infection with EBV results in infectious mononucleosis, but the virus is also associated with numerous other diverse diseases. These include Burkitt s lymphoma and nasopharyngeal carcinoma, acquired immune deficiency syndrome (AIDS), autoimmune diseases and chronic fatigue syndrome. There is increasing evidence for their association with cellular immune defects. These illnesses are usually chronic, and correlate with elevated levels of IgG antibodies to EBV. The common denominator for all these illnesses is the presence of elevated antibody titers to EBV, the recovery of virus or identifiable EBV DNA from patients. Fatigue is a major symptom of EBV infectious mononucleosis. It is also the primary symptom for Chronic Fatigue Syndrome. Chronic Fatigue Syndrome is closely identified with EBV, and in addition to fatigue, its symptoms may include low-grade fever, headaches, sleep disorders, and depression which can last for a long time. IgG antibodies to EBV are elevated during the course of Chronic Fatigue Syndrome implicating EBV as a causative agent of the disease. However, current research indicates that patients who suffer from Chronic Fatigue Syndrome also suffer from immune deficiency problems. Patients suffering from rheumatoid arthritis tend to have higher antibody titers to EBV antigens than do normal individuals although they do not show ill effects from EBV viral infections. The T-cell dysfunction observed in patients with rheumatoid arthritis appears to be specific for this illness and is not found in other autoimmune diseases. Nasopharyngeal Carcinoma is restricted to certain geographical areas EDVOTEK and The Biotechnology Education Company are registered trademarks of EDVOTEK, Inc.

5 Background Information 5 which include southern China, Tunisia and Greenland. Patients have increased antibody titers to EBV, and tumor cells from these patients contain EBV. It is known that soil in these geographical areas contains high levels of nickel, nitrosoamines and diterprene esters, all of which are suspected carcinogens. These chemicals induce EBV-infected B-cell proliferation, which in turn will stimulate T-suppressor cell production. Burkitt s lymphoma is primarily associated with parts of Africa, New Guinea and Sweden. A large majority of patients with this cancer have recoverable EBV DNA from their tumors. It has been reported that individuals suffering from Burkitt s lymphoma have chromosomal translocation. Diagnosis for EBV in clinical laboratories is carried out by the patient s immune response to the virus using an enzyme-linked immunosorbent (ELISA) or immunofluorescence assay. As with many viral infections the immune response after the initial infection may be significantly delayed, especially in children and immunocompromised individuals. With Polymerase Chain Reaction (PCR), it is possible to detect viral DNA present in small quantities in blood and tissues. Description of the Immunological Screening Enzyme-linked immunosorbent assay (ELISA) tests were originally developed for antibody measurement. These immunoassays have also been adapted to successfully detect samples that contain antigens. This ELISA simulation experiment has been designed to detect a hypothetical patient's circulating IgG directed towards the viral (EBV) antigen. 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, antigen(s) are added to the wells where some are adsorbed by hydrophobic association to the walls of the well after washing away the excess. The antigens can be the whole EBV lysate, specific EBV 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 proteins, typically bovine serum albumin. Infection by EBV (positive for EBV) causes the individual to mount an antibody response which eventually results in plasma IgG molecules that bind to different EBV proteins. The EBV antibody present in the patient's plasma sample will bind to the viral proteins (antigen) in the well and remain there after washing. If the patient does not have EBV antibody in their serum, there will be no such binding. This will discontinue the ELISA steps that follow and not yield a color reaction which diagnoses the patient to be negative for the virus.

6 6 Background Information Enzyme Substrate (Colorless) If EBV antibody remains bound, then the secondary antibody will bind to it and remain attached after washing. Secondary antibodies are raised in rabbits and goats immunized with human IgG fractions. Secondary antibodies are purified and covalently cross linked to an enzyme with a high turn number, such as horseradish peroxidase. The cross-linking does not significantly affect the binding specificity and affinity of the antibody or the enzymatic activity of the peroxidase (Figure 1). After washing, a solution containing hydrogen peroxide and azino-diethylbenzthiazoline sulfonate (ABTS) is added to each well. Peroxidase possesses a high catalytic activity and can exceed turnover rates of 10 6 per second. Consequently, amplification of a positive sample can occur over several orders of magnitude. The substrate solution added is nearly colorless. Peroxidase converts the peroxide to H 2 O + O 2 using the ABTS as the hydrogen donor. The oxidized ABTS is green and can be easily observed in wells containing anti-ebv IgG (positive plasma). Product (Color) It should be noted that polyclonal antibody preparations to a given antigen can have variable binding affinities due to differences in the immunological responses between animals. Different immunizations with the same antigen in the same animal can also produce antibodies with variable binding affinities. The use of monoclonal antibodies directed against a single epitope eliminates this variability. Western blot analysis of positive ELISA samples are used to confirm infection by EBV. Secondary Antibody Primary Antibody (EBV antibody) Antigen (EBV proteins) Figure 1: Schematic for ELISA

7 Experiment Procedures 7 Experiment Overview EXPERIMENT OBJECTIVE: The objective of this experiment is to understand the experimental concepts and methodology involved with enzyme-linked immunosorbent (ELISA) assays in the context of the clinical screening of serum samples for antibodies to EBV. Primary infection with EBV results in infectious mononucleosis, which is popularly known as the "Kissing Disease". Wear gloves and safety goggles 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 heating. 3. DO NOT MOUTH PIPET REAGENTS - USE PIPET PUMPS OR BULBS. 4. Always wash hands thoroughly with soap and water after handling contaminated materials.

8 8 Student Experimental Procedures GENERAL INSTRUCTIONS AND PROCEDURES Experiment Procedures Remember! Equilibrate a 37 C incubation oven before starting the experiment. Labeling the Microtiter Plate: 1. Place the microtiter strip as shown in Figure Carefully mark the strip with your initials or lab group number and number the wells 1-6 down the side. 3. Do not separate the reaction wells. Labeling the Plastic Transfer pipets: 1. Use an automatic micropipet (50µl) and disposable yellow tips or the transfer pipets. 2. Label 7 transfer pipets as follows: ( - ) (negative) (+ ) (positive) DS 1 (Donor Serum 1) DS 2 (Donor Serum 2) DS 3 (Donor Serum 3) DS 4 (Donor Serum 4) PBS (Phosphate Buffered Saline) Use the appropriately labeled plastic transfer pipet for sample additions, removals, and washes as outlined in the experimental procedures. 3. Label 3 additional transfer pipets as follows: EBV 2 Ab Substrate Figure 2

9 Experiment Procedures 9 Student Experimental Procedures Useful Hint! The general procedure for adding reagents to the wells will conserve the use of a large number of pipets in this simulated classroom ELISA. If available, reagents should be dispensed with an automatic micropipet using disposable tips. INSTRUCTIONS FOR ADDING LIQUIDS AND WASHING WELLS Adding Reagents to wells: For adding reagents to the wells, use the labeled transfer pipets or use an automatic micropipet and disposable tips. Liquid Removal and Washes: When instructed in the experimental procedures, remove liquids with the appropriately labeled transfer pipet, and then wash the wells as follows: A. Use the transfer pipet labeled "PBS", to add PBS buffer to each of the wells. Add PBS buffer until each well is almost full. The capacity of each well is approximately 0.30 ml. Do not allow the liquids to spill over into adjacent wells. B. With the appropriately labeled transfer pipet, remove all the liquid (PBS buffer) from each of the wells. Dispose the liquid in the beaker labeled "waste". WEAR SAFETY GOGGLES AND GLOVES ENZYME-LINKED IMMUNOSORBENT ASSAY (ELISA) 1. To all 6 wells, add 50 µl or 3 drops of EBV (viral antigens). If using a transfer pipet, use the one labeled "EBV". 2. Incubate for 5 minutes at room temperature. 3. Remove all the liquid (viral antigens) with a transfer pipet. 4. Wash each well once with PBS buffer as described above ("Liquid Removal and Washes"). In research labs, following this step, all sites on the microtiter plate are saturated with a blocking solution consisting of a protein mixture, such as BSA. We have designed this experiment to eliminate this step to save time.

10 10 Student Experimental Procedures Experiment Procedures REMINDERS: Adding Reagents: Be sure to use a fresh tip for each reagent. (Steps 1, 5, 9, & 13). Alternatively, use the appropriately labeled transfer pipet for each reagent. Liquid Removals: Use the appropriately labeled transfer pipet to remove all liquid from each of the wells (steps 3, 7, & 11) and after washes (steps 4, 8 & 12) Transfer pipet (-) Well 1 Transfer pipet (+) Well 2 Transfer pipet DS 1 Well 3 Transfer pipet DS 2 Well 4 Transfer pipet DS 3 Well 5 Transfer pipet DS 4 Well 6 ELISA, CONTINUED 5. Add reagents as outlined below: Remember to use the appropriately labeled transfer pipet for adding a new reagent. If you are using automatic micropipets, use a clean micropipet tip for each reagent. Add 50 µl or 3 drops of PBS Buffer to the first well. (This is the negative control.) Add 50 µl or 3 drops of "+" (positive) to the second well. (This is the positive control.) Add 50 µl or 3 drops of Donor Serum DS1 to the third well. Add 50 µl or 3 drops of Donor Serum DS2 to the fourth well. Add 50 µl or 3 drops of Donor Serum DS3 to the fifth well. Add 50 µl or 3 drops of Donor Serum DS4 to the sixth well. Dispose the liquid in a beaker labeled "waste". Washes: For all wells, use the transfer pipet labeled "PBS" to add PBS until each well is almost full. (Steps 4, 8, & 12) Quick Reference: The positive control, which contains IgG directed against EBV antigens, is the primary antibody. Positive serum samples will also contain anti-ebv IgG, while negative serum samples will not contain anti-ebv IgG. 6. Incubate at 37 C for 15 minutes. 7. Remove all the liquid from each well with the appropriately labeled transfer pipet. 8. Wash each well once with PBS buffer (as described under "Liquid Removal and Washes"). Optional Stopping Point: The experiment can be stopped after step 8, but requires that PBS be added to all the wells for overnight storage at room temperature. The experiment can be resumed during the next lab period. Remove the PBS and continue with step 9.

11 Experiment Procedures 11 Student Experimental Procedures ELISA, CONTINUED 9. Add 50 µl or 3 drops of the anti-igg peroxidase conjugate (2 Ab) to all 6 wells. If using a transfer pipet, use the one labeled "2 Ab". 10. Incubate at 37 C for 15 minutes. At this time you can obtain the substrate to be used in step Remove all the liquid from each well with the appropriately labeled transfer pipet. 12. Wash each well once with PBS buffer (as described under "Liquid Removal and Washes"). 13. Add 50 µl or 3 drops of the substrate to all 6 wells. If using a transfer pipet, use the one labeled "substrate". 14. Incubate at 37 C for 5 minutes. 15. Remove the wells for analysis. 16. If color is not fully developed after 5 minutes, incubate at 37 C for a longer period of time.

12 12 Experiment Results and Study Questions LABORATORY NOTEBOOK RECORDINGS: Experiment Procedures 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. STUDY QUESTIONS Answer the following study questions in your laboratory notebook or on a separate worksheet. 1. Describe the reaction that generates the color in positive wells in the ELISA reaction. 2. How is the EBV infection transmitted through kissing? 3. Why was EBV suspected to be the cause of chronic fatigue? 4. (OPTIONAL) What is PCR and how can it be used to detect EBV virus?

13 13 Notes to the Instructor APPROXIMATE TIME REQUIREMENTS FOR PRE-LAB AND EXPERIMENTAL PROCEDURES 1. Pre-lab preparation of biologicals and reagents takes approximately one and one-half hours. 2. The student experiment requires approximately 60 minutes. Class size, length of laboratory sessions, and availability of equipment are factors which must be considered in the planning and the implementation of this experiment with your students. These guidelines can be adapted to fit your specific set of circumstances. Online Ordering now available Visit our web site for information about EDVOTEK 's complete line of experiments for biotechnology and biology education. 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 ( ). Technical Service Department Instructor's Guide M o n EDVO-TECH EDVOTEK - ( ) Fri 9 SERVICE am - 6 E T pm Mon - Fri 9:00 am to 6:00 pm ET FAX: (301) web: edvotek@aol.com 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 PREPARATIONS BEFORE THE LAB Microtiter Wells 1. Carefully divide each microtiter plate into 8-well strips. Each student group requires one 8-well strip. Dispensing Components A through F: 2. Use a clean pipet for dispensing each of the components A-F directly from the component tubes provided in this experiment kit. Label microtest tubes and dispense volumes as outlined in the chart "Preparation of Experiment Reagents" below. Preparation of Experiment reagents Dispense for Component Label each group A* EBV Antigens EBV 0.5 ml B* Positive control + 75 µl C* Donor Serum # 1 DS 1 75 µl D* Donor Serum # 2 DS 2 75 µl E* Donor Serum # 3 DS 3 75 µl F* Donor Serum # 4 DS 4 75 µl G + PBS Anti-IgG-peroxidase-conjugate 2 Ab 0.5 ml H Substrate Solution Substrate 0.4 ml I + water Phosphate Buffered Saline PBS 10 ml * Components A - F can be dispensed before the actual day of the lab and stored in the refrigerator. If these components are dispensed on the day of the lab, leave at room temperature.

15 15 Pre-Lab Preparations PREPARATIONS ON THE DAY OF THE LAB Preparation of Phosphate Buffered Saline 1. Add all of the Phosphate Buffered Saline concentrate (I) to 135 ml of distilled water. Mix. 2. Label this diluted Phosphate Buffered saline as PBS. 3. Dispense 10 ml into small beakers for each of the 10 lab groups. Preparation of Anti-IgG Peroxidase Conjugate (Secondary Antibody) Instructor's Guide Useful Hint! The sample volume of the secondary antibody is very small - the tube can be centrifuged to collect the sample at the bottom. Note: Prepare on same day as needed for the experiment. 4. Add 0.3 ml of diluted Phosphate Buffered Saline (PBS) to the concentrated Anti-IgG peroxidase conjugate (G). Mix thoroughly by tapping and inverting the tube. 5. Transfer 6 ml of diluted Phosphate Buffered Saline (PBS) to one of the 15 ml plastic tubes provided. 6. Transfer the entire contents of tube G prepared in step 4 to the 15 ml tube containing 6 ml of PBS prepared in step 5. Label the tube "2 Ab" (Secondary Antibody). Mix. STUDENT MATERIALS Each Lab Group Should Receive: 1 piece of microtiter strip 1 tube labeled "EBV" 1 tube labeled "+" 1 tube labeled "DS 1" 1 tube labeled "DS 2" 1 tube labeled "DS 3" 1 tube labeled "DS 4" 1 tube labeled "2 Ab" 1 automatic micropipet with tips (optional) 1 pipet pump 10 Transfer pipets 1 beaker or tube containing PBS 1 empty beaker labeled "waste" 1 tube labeled "Substrate" 7. Dispense 0.5 ml of the diluted Anti-IgG peroxidase conjugate for each group. Substrate Solution 8. Dispense 0.4 ml of the substrate solution for each of the 10 groups.

16 16 Avoiding Common Pitfalls Instructor's Guide 1. Students should be advised to be very careful when transferring solutions into and out of the microtiter strips. 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. Idealized Schematic of Results Donors 2 and 3 should show positive for EBV as represented in the figure below. Actual liquid amounts in the wells are not drawn to scale, but the color of the positive donors should look similar to the positive control, which is in the second well Experiment Results 1. Negative Control 2. Positive Control 3. Donor 1 (Negative) 4. Donor 2 (Positive) 5. Donor 3 (Positive) 6. Donor 4 (Negative) Donors

17 17 Study Questions and Answers 1. Describe the reaction that generates the color in positive wells in the ELISA reaction. The antigen (protein) is adsorbed to the plastic well by non-covalent forces. The antibody is then bound to the antigen at the first step. The secondary antibody, to which an enzyme is covalently attached, is then bound to the antigen-antibody complex. Upon introduction of the substrate, the enzyme will convert the substrate to product resulting in a color change. 2. How is the EBV infection transmitted through kissing? The main mode of transmission for the EBV virus is through the saliva to which an individual may be exposed during kissing. Epithelail cells in the throat of an individual become infected by the EBV virus. This is followed by virus replication and lysis of cells which in turn become readily available in the saliva. Instructor's Guide 3. Why was EBV suspected to be the cause of chronic fatigue? Current research indicates that individuals who suffer from chronic fatigue suffer from immune deficiency. EBV infection is not a causative agent, but often is an opportunistic infection. EBV, however, is directly implicated in infectious mononucleosis. 4. (OPTIONAL) What is PCR and how can it be used to detect EBV virus? Polymerase Chain Reaction (PCR) is a method used to amplify the DNA virus to make it s detection possible. For example, in the case of the EBV virus, the amount present in the saliva of a newly infected person is not detectable. Detection is usually determined by blood tests that check for antibody levels. Using PCR, which requires two primers (one complementary to each of the two DNA strands), a thermally stable DNA polymerase, and the four dideoxy-triphosphates, copies of a unique part of the virus, is amplified. The amplified viral DNA can be detected and identified by standard molecular biology techniques, such as agarose gels.

18 IDENTITY (As Used on Label and List) A,B,D / 274 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. Section II - Hazardous Ingredients/Identify Information Hazardous Components [Specific Chemical Identity; Common Name(s)] OSHA PEL ACGIH TLV CAS # CAS # Very dilute 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. Emergency Telephone Number (301) Telephone Number for information (301) Date Prepared Signature of Preparer (optional) Other Limits Recommended Section III - Physical/Chemical Characteristics Boiling Point Specific Gravity (H 0 = 1) 2 Vapor Pressure (mm Hg.) Melting Point Vapor Density (AIR = 1) Evaporation Rate (Butyl 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 % (Optional) Special Fire Fighting Procedures Move container from fire area if possible. Dike fire control water for later disposal Unusual Fire and Explosion Hazards Thermal decomposition products may include toxic and hazardous oxides of carbon, nitrogen, and sodium. 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 Byproducts 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? None Signs and Symptoms of Exposure Mucous membrane irritation, eye/skin irritation, irritating to gastrointestinal system. Medical Renal Conditions or heart disease, Generally potassium Aggravated deficiency, by Exposure 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 NONE Precautions Section VIII - Control Measures Respiratory Protection (Specify Type) Chemical cartridge respirator with full facepiece and organic vapor cartridge Ventilation Local Exhaust No Special None Mechanical (General) Gen. dilution vent sys. Other None 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 IDENTITY (As Used on Label and List) C and H/274 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. Section II - Hazardous Ingredients/Identify Information Hazardous Components [Specific Chemical Identity; Common Name(s)] OSHA PEL ACGIH TLV 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. Emergency Telephone Number (301) Telephone Number for information (301) Date Prepared 02/17/00 Signature of Preparer (optional) Polyoxyethylenesorbitan monolaurate CAS # Other Limits Recommended % (Optional) Section III - Physical/Chemical Characteristics Boiling Point Specific Gravity (H 0 = 1) 2 Vapor Pressure (mm Hg.) Melting Point Vapor Density (AIR = 1) Evaporation Rate (Butyl 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 UEL Extinguishing Media Water spray, carbon dioxide, dry chemical powder, alcohol or polymer foam Special Fire Fighting Procedures Wear SCBA and protective clothing to prevent contact w/ skin and eyes. Unusual Fire and Explosion Hazards Emits toxic fumes under fire conditions Section V - Reactivity Data Stability Unstable Stable Incompatibility Strong oxidizers, acids Conditions to Avoid Hazardous Decomposition or Byproducts Carbon dioxide and carbon monoxide may form when heated to decomposition 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) Unknown Carcinogenicity: NTP? IARC Monographs? OSHA Regulation? None Signs and Symptoms of Exposure May cause irritation Medical Conditions Generally Aggravated by Exposure No information Emergency First Aid Procedures Ingestion: give several glasses of water to drink to dilute Skin: Flush with plenty of soap and water for 15 minutes. Eyes: Flush with plenty of water for 15 min. Section VII - Precautions for Safe Handling and Use Steps to be Taken in case Material is Released for Spilled Ventilate area of leak or spill. Contain and recover liquid when possible. Collect liquid in appropriate container or absorb with inert material and place in a chemical waste container. Waste Disposal Method Observe all federal, state and local regulations Precautions to be Taken in Handling and Storing Keep in tightly closed container, stored in cool, dry, ventilated area. Other Precautions NONE X Section VIII - Control Measures Respiratory Protection (Specify Type) Ventilation Local Exhaust Special None Mechanical (General) Other None 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

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 Conditions to Avoid Stable Incompatibles Incompatibility Strong oxidizing agents, strong acids IDENTITY (As Used on Label and List) ABTS Substrate Section I Manufacturer's Name EDVOTEK, Inc. Address (Number, Street, City, State, Zip Code) Rothgeb Drive Rockville, MD Section II - Hazardous Ingredients/Identify Information Hazardous Components [Specific Chemical Identity; Common Name(s)] OSHA PEL ACGIH TLV 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. Emergency Telephone Number (301) Telephone Number for information (301) Date Prepared Signature of Preparer (optional) 03/14/00 Other Limits Recommended 2,2'-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) Diammonium salt CAS# % (Optional) Section III - Physical/Chemical Characteristics Boiling Point Specific Gravity (H 0 = 1) 2 Vapor Pressure (mm Hg.) Melting Point >300 C Vapor Density (AIR = 1) Evaporation Rate (Butyl Acetate = 1) Solubility in Water Appearance and Odor Soluble colorless, odorless liquid Section IV - Physical/Chemical Characteristics N.D. = Flash Point (Method Used) Flammable Limits LEL UEL N.D. N.D. Extinguishing Media Water spray. Carbon dioxide powder or appropriate foam Special Fire Fighting Procedures Wear self-contained breathing apparatus and protective clothing to prevent contact with skin and eyes. Unusual Fire and Explosion Hazards Hazardous Decomposition or Byproducts Toxic fumes of carbon monoxide, carbon dioxide, nitrogen oxides sulfure oxides. Hazardous May Occur Conditions to Avoid Polymerization Will Not Occur No Data Section VI - Health Hazard Data Route(s) of Entry: Inhalation? Yes Skin? Yes Ingestion? Yes Health Hazards (Acute and Chronic) Irritant Carcinogenicity: NTP? IARC Monographs? OSHA Regulation? Signs and Symptoms of Exposure Eye, skin and respiratory system irritation Medical Conditions Generally Aggravated by Exposure Emergency First Aid Procedures Eyes/Skin: Flush with copious amounts if water. If swallowed, wash out mouth with water, call a physician. 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 Avoid contact. Wear self-contained breathing apparatus, rubber boots and rubber gloves. Other Precautions Section VIII - Control Measures Respiratory Protection (Specify Type) Ventilation Local Exhaust No Special None Mechanical (General) Yes Other None Protective Gloves Chemical resistant Eye Protection Chemical safety goggles Other Protective Clothing or Equipment Work/Hygienic Practices Wear suitable protective clothing and mop up Dissolve or mix the material with a combustible solvent and burn in a chemical incinerator equipped with an afterburrner and scrubber. Observe federal, state, and local laws. None Lab coat, NIOSH/MSHA approved respirator Avoid contact IDENTITY (As Used on Label and List) PBS 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. Section II - Hazardous Ingredients/Identify Information Hazardous Components [Specific Chemical Identity; Common Name(s)] OSHA PEL ACGIH TLV Special Fire Fighting Procedures 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. Emergency Telephone Number (301) Telephone Number for information (301) Date Prepared 03/14/05 Signature of Preparer (optional) Other Limits Recommended Section III - Physical/Chemical Characteristics Boiling Point 100C Specific Gravity (H 0 = 1) 2 Vapor Pressure (mm Hg.) Melting Point Vapor Density (AIR = 1) Evaporation Rate (Butyl 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 SCBA 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 Incompatibility Strong acids Hazardous Decomposition or Byproducts 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? Ye 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.