Immunology of Pregnancy Tests. Storage: Store the entire experiment in the refrigerator. EXPERIMENT OBJECTIVES:

Transcription

1 The Biotechnology Education Company 279 EDVO-Kit # Immunology of Pregnancy Tests Storage: Store the entire experiment in the refrigerator. EXPERIMENT OBJECTIVES: The objective of the simulation experiment is to introduce concepts and experimental procedures involved with enzyme linked immunosorbent assays (ELISA) in the context of testing for pregnancy. All components are intended for educational research only. They are not to be used for diagnostic or drug purposes, nor administered to or consumed by humans or animals

2 2 EDVO-Kit # 279 Table of Contents Page Experiment Components 3 Experiment Requirements 3 Background Information 4 Experiment Procedures Experiment Overview 7 Student Experimental Procedures 8 Study Questions 11 Instructor's Guidelines Notes to the Instructor 13 Pre-Lab Preparations 14 Quick Reference Tables 16 Avoiding Common Pitfalls 17 Expected Results 17 Study Questions and Answers 18 Safety Data Sheets can be found on our website:

3 EDVO-Kit # Experiment Components A B C D E F G H hcg Antibody (simulated) Positive Control Urine Sample Patient 1 (simulated) Urine Sample Patient 2 (simulated) Anti-hCG-peroxidase conjugate Hydrogen peroxide, stabilized Peroxide co-substrate Phosphate buffered saline concentrate Microtiter strips Transfer pipets Microtest tubes with attached caps 15 ml plastic tubes This experiment is designed for 10 groups. Store entire experiment in the refrigerator. Requirements Distilled or deionized water Beakers 37 C Incubation oven Disposable lab gloves Safety goggles Automatic micropipets (0-50 μl) and tips recommended Make sure glassware is clean, dry and free of soap residue. For convenience, additional disposable transfer pipets can be purchased for liquid removal and washing steps. All components are intended for educational research only. They are not to be used for diagnostic or drug purposes, nor administered to or consumed by humans or animals. EDVOTEK The Biotechnology Education Company EDVOTEK 24-hour FAX: info@edvotek.com

4 4 EDVO-Kit # 279 Background Information The female reproductive organs consist of the ovaries, the ovarian tubes, the uterus and the vagina. The principal function of the female reproductive system is to produce the ova and to provide the environment for fertilization and nurturing of the ovum to make possible normal growth to a mature fetus. The fi nal step is to deliver the offspring to the outside environment. The human female usually releases a single ovum per ovulation cycle. Upon ovulation the ovum is released into the ovarian tube. It is in the ovarian tubes that the ovum may be fertilized. It then passes into the uterus where it implants into the wall of the uterus. In the uterus, the fertilized ovum grows to become a fetus. Synchronized orchestration of a number of hormones mediates the events that are involved in ovulation. These hormones are produced in the pituitary and in the ovary itself and bring about their effects by circulating in the blood stream. During each female menstrual cycle, groups of cells (follicles) grow in the ovary. Although many follicles will grow, only one follicle will develop to maturity while the others will degenerate. The pituitary gland produces two hormones, luteinizing hormone (LH) and follicle stimulating hormone (FSH). At the beginning of the cycle, FSH stimulates growth of the follicle, which begins to secrete estrogen. The estrogen stimulates proliferation of the inner lining (endometrium) of the uterus and stimulates the synthesis of progesterone receptors in preparation for higher levels of progesterone later in the cycle. As the menstrual cycle progresses, estrogen levels rise and reach a peak approximately midway in the menstrual cycle. Decreasing levels of estrogen then bring about a surge in LH production, which induces ovulation. After ovulation, the follicle forms the corpus luteum, named after its yellow appearance, that produces high levels of progesterone. This progesterone is required for the preparation and upkeep of the endometrium that is essential for the nourishment of the implanted fetus. The corpus luteum requires LH to function. If fertilization does not occur, decreasing LH levels cause disintegration of the corpus luteum. The reduced progesterone levels then lead to breakdown of the surface of the endometrium, which is released during menstruation. After 4-5 days, menstruation stops and the cycle repeats. Human female menstrual cycles vary in length amongst individuals. Taken as an average, it is 28 days long starting from the fi rst day of vaginal bleeding. If implantation does occur, the fetal trophoblast makes human chorionic EDVOTEK and The Biotechnology Education Company are registered trademarks of EDVOTEK, Inc.

5 EDVO-Kit # gonadotropin (hcg), a hormone that acts like LH and prevents disintegration of the corpus luteum and thus assures a continued supply of progesterone. hcg is made throughout pregnancy by the placenta. The term pregnancy test is actually is a misnomer since most pregnancy tests do not actually determine pregnancy but rather the level of hcg. The hormone is a glycoprotein, which is made up of two subunits, the alpha subunit which has a molecular weight of 18,000 and the beta chain which has a molecular weight of 32,000. It should be noted that several glycoprotein hormones such as hcg, LH and FSH share a common alpha subunit. The pregnancy test detects only the unique beta-subunit of hcg. Since hcg is only produced in the developing embryo (except in rare cases of secretion of hcg by hydatidiform moles or choriocarcinoma), and since it is detectable within a few days of implantation, it is a very specifi c and early test for pregnancy. Originally pregnancy tests required the injection of urine from suspected pregnant women into female rabbits. The hcg in the urine brought about physiological changes in the reproductive system of the rabbit, which were detected by examination upon sacrifi cing the animal. This has long been replaced by immunological assays based on the ELISA test. Background Information ENZYME LINKED IMMUNOSORBENT ASSAY (ELISA) Enzyme linked immunosorbent assay (ELISA) tests were originally developed for antibody measurement. These immunoassays have also been adapted to successfully detect samples that contain antigens. This ELISA simulation experiment has been designed to detect a hypothetical patient's circulating level of hcg. ELISAs are done in microtiter plates or strips which are generally made of polystyrene or polyvinyl chloride. The plates or strips contain many small wells which are somewhat transparent and in which liquid samples are deposited. First, the antibody against hcg is added to the wells where some remain adsorbed by hydrophobic association to the walls after washing away the excess. There is no specifi city involved in antigen or antibody adsorption although some substances may exhibit low binding to the microwell walls. In certain cases the antigens can be covalently cross-linked to the plastic using UV light. After washing away unadsorbed material, the unoccupied sites on the walls of the plastic wells are blocked with proteins, typically gelatin or bovine serum albumin. In this simulation experiment, hcg present in the urine sample will bind to the adsorbed antibody in the well and remain there after washing.

6 6 EDVO-Kit # 279 Background Information If hcg has remained bound to the anti-hcg antibody in the well, then the secondary antibody will bind to another antigenic determinant on the hcg. This complex will remain attached after washing. The second antibody to hcg is purifi ed and covalently cross linked to an enzyme with a high turn over number such as 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. After washing the well to remove unbound secondary antibody, a solution containing hydrogen peroxide and aminosalicylate is added to each well. Peroxidase possesses a high catalytic activity and can exceed turnover rates of 10 6 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 cosubstrates include aminodiansidine, aminoantipyrine, aminosalicylic acid and numerous phenolic compounds that develop color upon oxidation. The substrate solution added is nearly colorless. Peroxidase converts the peroxide to H 2 O + O 2 using the salicylate as the hydrogen donor. Schematic for ELISA Enzyme Substrate (Colorless) Product (Color) It should be noted that polyclonal antibody preparations to a given antigen can have variable binding affi nities due to differences in the immunological 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 and makes the ELISA highly specifi c of hcg detection. In this experiment, students will use the ELISA test to determine pregnancy of four hypothetical patients. Secondary Antibody, conjugate to hcg Antigen to hcg Antibody to hcg

7 EDVO-Kit # Experiment Overview EXPERIMENT OBJECTIVE: The objective of this simulation experiment is to introduce concepts and experimental procedures involved with enzyme linked immunosorbent assays (ELISA) in the context of testing for pregnancy. Wear gloves and safety goggles LABORATORY SAFETY Gloves and goggles should be worn routinely as good laboratory practice. Experiment Procedures

8 8 EDVO-Kit # 279 Student Experimental Procedures GENERAL INSTRUCTIONS AND PROCEDURES Experiment Procedures Equilibrate a 37 C incubation oven before starting the experiment. Labeling the Microtiter Strip: Place the microtiter strip as shown in Figure 1. Carefully mark the strip with your initials or lab group number and number the wells 1-4 down the side. Do not separate the reaction wells. Labeling the Plastic Transfer pipets: Label 5 transfer pipets as follows: ( - ) (negative) (+ ) (positive) USP 1 (Urine Sample Patient 1) USP 2 (Urine Sample Patient 2) PBS (Phosphate Buffered Saline) Figure 1 Use the appropriately labeled plastic transfer pipet for sample additions, removals, and washes as outlined in the experimental procedures. If using transfer pipets to add reagents to the wells, label 3 additional pipets "hcg Ab", "hcg Ab2" (hcgab-hrp conjugate), and "substrate". 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 200 μl. Do not allow the liquids to spill over into adjacent wells. B. With the appropriately labeled transfer pipet, remove all the liquid (PBS buffer) from each of the wells. Dispose the liquid in the beaker labeled "waste".

9 EDVO-Kit # Student Experimental 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 USP 1 Well 3 Transfer pipet USP 2 Well 4 Dispose the liquid into a beaker labeled "waste". Washes: ENZYME LINKED IMMUNOSORBENT ASSAY (ELISA) 1. To all 4 wells, add 50 μl or 3 drops of hcg Ab (human chorionic gonadotropin antibody). If using a transfer pipet, use the one labeled "hcg Ab". 2. Incubate for 5 minutes at room temperature. 3. Remove all the liquid (hcg antibody) with a transfer pipet. 4. Wash each well once with PBS buffer as described above ("Liquid Removal and Washes"). In research labs, following this step, all sites on the microtiter strip are saturated with a blocking solution consisting of a protein mixture, such as BSA. We have designed this experiment to eliminate this step to save time. 5. To Test Patient 1, add reagents as outlined below: Remember to use the appropriately labeled transfer pipets provided with this experiment for adding a new reagent. If you are using automatic micropipets, use a clean micropipet tip for each reagent. Add 50 μl or 3 drops of PBS Buffer to the fi rst well. (This is the negative control.) Experiment Procedures For all wells, use the transfer pipet labeled "PBS" to add PBS until each well is almost full. (Steps 4, 8, & 12) Quick Reference: The positive control contains hcg. Negative urine samples will not contain hcg. Urine samples obtained from a pregnant patient will contain hcg and thus have a positive result. Add 50 μl or 3 drops of "+" (positive) to the second well. (This is the positive control.) Add 50 μl or 3 drops Urine Sample from Patient 1 USP 1 to the third well. Add 50 μl or 3 drops Urine Sample from Patient 2 USP 2 to the fourth well. 6. Incubate at 37 C for 15 minutes. 7. Remove all the liquid from each well with the appropriately labeled transfer pipet. 8. Wash each well once with PBS buffer (as described under "Instructions for Adding Liquids and Washing Wells" on page 8). Optional Stopping Point: The experiment can be stopped after step 8, but requires that PBS be added to all the wells for overnight storage at room temperature. The experiment can be resumed during the next lab period. Remove the PBS and continue with step 9.

10 10 EDVO-Kit # 279 Student Experimental Procedures ENZYME LINKED IMMUNOSORBENT ASSAY, CONTINUED Experiment Procedures 9. Add 50 μl or 3 drops of the anti-hcg peroxidase conjugate (hcg Ab2) to all 4 wells of each strip. If using a transfer pipet, use the one labeled "2 Ab". 10. Incubate at 37 C for 15 minutes. At this time you can obtain the substrate to be used in step 13. Since the substrate must be prepared just prior to use, your instructor will prepare it towards the end of the incubation in step Remove all the liquid from each well with the appropriately labeled transfer pipet. 12. Wash each well once with PBS buffer (as described under "Liquid Removal and Washes"). 13. Add 100 μl or 5 drops of the substrate to all 4 wells. If using a transfer pipet, use the one labeled "substrate". 14. Incubate at 37 C for 5 minutes. 15. Remove the strip for analysis. 16. If color is not fully developed after 5 minutes, incubate at 37 C for a longer period of time.

11 EDVO-Kit # Experiment Results and Study Questions LABORATORY NOTEBOOK RECORDINGS: Address and record the following in your laboratory notebook or on a separate worksheet. Before starting the experiment: Write a hypothesis that 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. STUDY QUESTIONS Experiment Procedures Answer the following study questions in your laboratory notebook or on a separate worksheet. 1. Why is the ELISA reaction so sensitive? 2. Why is it important to have a positive control? 3. What is being detected in the standard home pregnancy test? 4. What is the difference between poyclonal and monoclonal antibodies?

12 12 EDVO-Kit # 279 Notes: Experiment Procedures

13 EDVO-Kit # 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 experimental activity requires approximately 60 minutes. 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. 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 ( ). Instructor's Guide Safety Data Sheets can be found on our website:

14 14 EDVO-Kit # 279 PreLab Preparations Instructor's Guide PREPARATIONS BEFORE THE LAB Microtiter Plates 1. As shown in the fi gure below, orient the microtiter plates so that the numbers 1-12 are at the top and the letters A - H are on your left. 2. Cut each plate on the dotted lines as shown in the fi gure. Each piece will be two rows of four wells. Each lab group will receive one piece and use one row. Row 1 Row 2 Row 3 Row 4 Row 5 Row 6 Row 7 Row 8 A B C D E F G H Cutting guide depicted by dashed lines Dispensing Components A through D: Use a clean pipet for dispensing each of the components A-D directly from the component tubes provided in this experiment kit. Label microtest tubes and dispense volumes as outlined in the chart "Quick Reference: Preparation of Experiment Reagents". Note: This experiment is ideal for problem-based experimentation. Instructors may wish to dispense the patient samples so the various student groups get different results.

15 EDVO-Kit # Pre-Lab Preparations PREPARATIONS ON THE DAY OF THE LAB Preparation of Phosphate Buffered Saline 1. Add all of the Phosphate Buffered Saline concentrate (Component H) to 135 ml of distilled water. Mix. 2. Label this diluted Phosphate Buffered saline as PBS. 3. Dispense 10 ml into small beakers or tubes for each of the 10 lab groups. Preparation of Anti-hCG Peroxidase Conjugate (Secondary Antibody) Instructor's Guide Note: Prepare on same day as needed for the experiment. The sample volume of the secondary antibody is very small - the tube can be centrifuged to collect the sample at the bottom. 4. Add 0.3 ml of diluted Phosphate Buffered Saline (PBS) to the tube of concentrated Anti-hCG peroxidase conjugate (Component E). Mix thoroughly by tapping and inverting the tube. 5. Transfer 6 ml of diluted Phosphate Buffered Saline (PBS) to one of the 15 ml plastic tube provided. 6. Transfer the entire contents of tube E prepared in step 4 to the 15 ml tube containing 6 ml of PBS. Label the tube "2 Ab" (Secondary Antibody). Mix. 7. Dispense 0.5 ml of the diluted Anti-hCG peroxidase conjugate for each of the ten lab groups. Quick Reference: The substrate is prepared for the peroxidase enzyme, which is attached to the anti-hcg peroxidase conjugate (secondary antibody). Prepare the substrate minutes before students require it for plate development (last incubation). PREPARATION OF PEROXIDASE SUBSTRATE DURING THE LAB EXPERIMENT Prepare minutes before the last incubation: 1. Dispense 9 ml of diluted Phosphate buffered saline (PBS) to the second 15 ml tube provided. 2. Add Peroxide co-substrate (Component G) to the 9 ml of PBS. Cap and mix thoroughly by shaking and/or vortexing. There is usually undissolved material remaining. 3. Then add 1 ml of Hydrogen peroxide (Component F). Cap and mix. 4. Dispense 0.8 ml of the peroxidase substrate for each of the 10 groups.

16 16 EDVO-Kit # 279 Quick Reference Tables Instructor's Guide PREPARATION OF EXPERIMENT REAGENTS Label Dispense for each group A* hcg Antibody hcg Ab 0.5 ml B* Positive control + 75 μl C* Urine Sample Patient 1 USP 1 75 μl D* Urine Sample Patient 2 USP 2 75 μl E + PBS Anti-hCG-peroxidase-conjugate 2 Ab 0.5 ml PBS + G + F Peroxidase-enzyme substrate** Substrate 0.8 ml H + water Phosphate Buffered Saline PBS 10 ml * Components A - D can be dispensed before the actual day of the lab and stored in the refrigerator. If these components are dispensed on the day of the lab, leave at room temperature. ** Peroxidase-enzyme substrate should be prepared minutes before the last incubation. STUDENT MATERIALS Each Lab Group Should Receive: 1 Piece of microtiter plate 1 Tube labeled "hcg Ab" 1 Tube labeled "+" 1 Tube labeled "USP 1" 1 Tube labeled "USP 2" 1 Tube labeled "2 Ab" 1 Automatic micropipet with tips (optional) 10 Transfer pipets 1 Beaker or tube containing PBS 1 Empty beaker labeled "waste" 1 Tube labeled "Substrate" (just before the last incubation)

17 EDVO-Kit # Avoiding Common Pitfalls 1. Students should be advised to be very careful when transferring solutions into and out of the microtiter strip wells. 2. Use only clean or appropriately labeled pipets and avoid contaminating adjacent wells. 3. Do not attempt to empty the microtiter wells by shaking it out. This will not work - it will result in contaminating adjacent wells. 4. Wash the wells gently and slowly. Instructor's Guide Expected Results Pregnancy tests together with positive and negative controls as represented in the fi gure below. Actual liquid amounts in the wells are not drawn to scale, but the color of the positive patients should look similar to the positive control, which is in the second well. ( - ) ( + ) USP 1 USP 2 Problem-based Individualized Experiment Students may be given combinations of positive and negative samples so the various student groups obtain different results. Instructors can code samples to represent various numbers of samples.

18 18 EDVO-Kit # 279 Study Questions and Answers 1. Why is the ELISA reaction so sensitive? Instructor's Guide The sensitivity of the ELISA is due to the high catalytic turnover rates of the enzyme linked conjugate. One conjugate can generate millions of product molecules in a few minutes. 2. Why is it important to have a positive control? A positive control assures that the reagents and plates are working optimally. 3. What is being detected in the standard home pregnancy test? The pregnancy test detects ( hcg) human Chorionic Gonadotropin in maternal urine. The hormone hcg is expressed shortly after the zygote is embeded in the uterine lining. hcg is a glycoprotein that is made of two subunits α and β. The two subunits individually are physiologically inactive. The β subunit has unique polypeptide sequences against which antibodies can be generated and which can function as the primary antibody for early pregnancy tests. 4. What is the difference between poyclonal and monoclonal antibodies? When a foreign substance such as a protein (antigen) is introduced into an animal, the animal immune system responds by making polyclonal antibody against the foreign protein. As an example, a rabbit will generate polyclonal antibodies against hcg if injected with the hormone. Polyclonal antibodies are a mixture of antibodies against different regions of the polypeptide. Unlike polyclonal antibodies, monoclonal antibodies are directed against specifi c antigenic determinants within a polypeptide. Thus a specifi c monoclonal antibody can be generated against a specifi c antigenic determinant in the β subunit of hcg.