The Biotechnology Education Company. Single Antibody ELISA Diagnostics. Storage: See Page 3 for specific storage instructions EXPERIMENT OBJECTIVE:

Transcription

1 The Biotechnology Education Company ingle Antibody ELIA Diagnostics torage: ee Page 3 for specific storage instructions EXPERIMENT OBJECTIVE: This experiment introduces a rapid and sensitive one antibody procedure for Enzyme Linked Immunosorbent Assays (ELIA). AMPLE LITERATURE Please refer to included weblink for correct version. EDVOTEK, Inc EDVOTEK

2 2 xxx ingle Antibody ELIA Diagnostics Table of Contents Page Experiment Components 3 Experiment Requirements 3 Background Information 5 Experiment Procedures Experiment Overview 8 General Information and Instructions 9 Enzyme Linked Immunosorbent Assay (ELIA) 11 tudy Questions 13 Instructor's Guidelines 15 Pre-Lab Preparations 16 Quick Reference Tables 18 Experiment Results and Analysis 19 tudy Questions and Answers 20 afety Data heets can be found on our website: 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. THI EXPERIMENT DOE NOT CONTAIN HUMAN DNA. None of the experiment components are derived from human sources. EDVOTEK, The Biotechnology Education Company, and Instatain are registered trademarks of EDVOTEK, Inc.. Ready-to-Load and Ultrapec-Agarose are trademarks of EDVOTEK, Inc. EDVOTEK The Biotechnology Education Company EDVOTEK

3 ingle Antibody ELIA Diagnostics 3 Experiment Components This experiment is designed for 10 lab groups Contents A imulated Patient erum ample 1 B imulated Patient erum ample 2 C Positive Control D Hydrogen peroxide, stabilized E Peroxide co-substrate F Phosphate buffered saline concentrate Microtiter plates Transfer pipets Microtest tubes with attached caps 50 ml plastic tubes Upon receipt, refrigerate components A-F None of the kit components were prepared from human sources. Requirements Distilled or deionized water Beakers 37 C Incubation oven Disposable lab gloves afety 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. EDVOTEK The Biotechnology Education Company EDVOTEK FAX: info@edvotek.com

4 4 xxx ingle Antibody ELIA Diagnostics EDVOTEK The Biotechnology Education Company EDVOTEK

5 ingle Antibody ELIA Diagnostics 5 Introduction to ELIA Reactions ENZYME LINKED IMMUNOORBENT AAY (ELIA) Fab region Fc region Antigen binding sites Figure 1: General tructure of an Antibody All antibodies belong to a group of serum proteins known as globulins. Each antibody is made up of a heavy and light polypeptide chain. In general, antibodies are produced in response to the presence of a "non-self" antigenic response. Light chain Heavy chain Antibodies obtained from animals, such as rabbits, in response to an antigen are known as polyclonal antibodies. Polyclonal antibodies are heterogeneous in structure and vary in their ability to bind to antigens. Antibodies that have a high affinity for non-specific antigens may give unwanted cross-reactions that can result in high backgrounds. uch antibodies can also give false negative results. By contrast, antibodies with weak binding constants may not be as sensitive. ubstrate (Colorless) Product (Color) Background Information Enzyme linked immunosorbent assay (ELIA) tests were originally developed for antibody measurement but have also been adapted to successfully detect samples that contain antigens. A variety of substances can show antigenic properties. An example is a coat protein of a virus. This ELIA simulation experiment has been designed to detect an antibody directed against an antigen. Traditional ELIAs (Figure 2A) are done in microtiter plates usually made of polystyrene or polyvinyl chloride. The plates are transparent and contain many small wells, into which liquid samples are deposited. The traditional ELIA requires two antibodies, a primary and a secondary antibody to which an enzyme is covalently linked, ubstrate Covalently linked enzyme econdary Antibody Primary Antibody Antigen Figure 2A: chematic of traditional ELIA with primary and secondary antibodies.

6 6 ingle Antibody ELIA Diagnostics Introduction to ELIA Reactions and a substrate that is enzymatically converted to a colored product. In a single antibody ELIA, the secondary antibody is not required. The enzyme is covalently linked to the antibody while the rest of the reaction is the same as shown in Figure 2B. Background Information The following are the basic steps of the single antibody ELIA technique. tep 1: The antigen is added to the wells where some remain adsorbed by hydrophobic association to the walls of the microtiter plate. The antigens can be a lysate, a specific protein, or a mixture. There is no specificity involved with the adsorption process, although some substances may exhibit low binding. In certain cases the antigens can be covalently cross-linked to the plastic using UV light. In this experiment, the plates have been precoated with antigen and are ready-to-use. ubstrate Antibody Antigen ubstrate (Colorless) Covalently linked enzyme Product (Color) tep 2: 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. Figure 2B: chematic of ELIA with one antibody. tep 3: A simulated serum solution, that may or may not contain the antibody, is added to the wells. If antibody is present in the solution, it will bind to the adsorbed antigen in the well and remain there after washing. tep 4: After washing the wells, substrate is added to all the wells. The enzyme attached to the antibody is a peroxidase. 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. Many hydrogen donor co-substrates can be used by peroxidase. These co-substrates include o-diansidine, aminoantipyrine, aminosalicylic acid and numerous phenolic compounds that develop color upon oxidation. ubstrate contains hydrogen peroxide and amino salicylate. The substrate solution added is nearly colorless. Peroxidase converts the peroxide to H 2 O + O 2 which oxidizes aminosalicylate. The oxidized salicylate is brown and can

7 ingle Antibody ELIA Diagnostics 7 Introduction to ELIA Reactions be easily observed in wells that contain the required reaction components. 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 animals can produce antibodies with variable binding affinities. The use of monoclonal antibodies directed against a single epitope eliminates this variability. Western blot analysis is usually used to confirm the ELIA results and to quantitate the size and amount of antigen. Western Blots and ELIA tests are used as diagnostic tools. This experiment demonstrates the use of a rapid and sensitive one antibody ELIA which has the advantage of a short assay time. The one antibody ELIA does not require a secondary antibody (which is used in the traditional ELIA) that causes a higher background and compromises the sensitivity of the ELIA. Background Information

8 8 ingle Antibody ELIA Diagnostics Experiment Overview This is a direct ELIA where one antibody is used to which the enzyme is bound. The microtiter wells are pre-treated with the antigen. After the simulated patient serum sample is added to the wells, washed, and substrate is added, the conversion of the substrate to product results in the color formation for positve samples. The Experiment LABEL MATERIAL: 1 2 PB Label wells: -, +, 1, and 2 directly on the microtiter plate, or place the plate on a labeled sheet of paper. Put your initials or group number on the plate. Label pipets: -, +, 1, 2, PB, and ub. These are designated for adding samples and removing washes - save these pipets! A B C A B C Incubate for 10 minutes. Remove all liquid using the transfer pipet designated for each row. 9 UBTRATE: C Wash each well once with PB buffer. Remove PB from each well using the transfer pipet designated for each row. Add 0.1 ml or 5 drops of the substrate to all of the wells. ub 1 2 ubstrate (Colorless) Product (Color) 11 Incubate for 10 minutes 37 C ANTIBODY CONTROL AND AMPLE: 3 4 Add 50 µl or 3 drops of PB to all the wells in Row 1. Add 50 µl or 3 drops of positive control to all the wells in Row 2. ubstrate Covalently linked enzyme Antibody ANALYI: 12 Remove the plate for analysis. 5 6 Add 50 µl or 3 drops of simulated patient serum sample 1 in all three wells in Row 3. Add 50 µl or 3 drops of simulated patient serum sample 2 in all three wells in Row 4. Antigen Enzyme Linked Immunosorbent Assay (ELIA) If color is not fully developed after 10 minutes (step 11), incubate at 37 C for a longer period of time.

9 ingle Antibody ELIA Diagnostics 9 General Information and Instructions EXPERIMENT OBJECTIVE: This experiment introduces a rapid and sensitive one antibody procedure for Enzyme Linked Immunosorbent Assays (ELIA). LABORATORY AFETY 1. Gloves and goggles should be worn routinely as good laboratory practice. 2. Exercise extreme caution when working with equipment that is used in conjunction with the heating and/or melting of reagents. 3. DO NOT MOUTH PIPET REAGENT - UE PIPET PUMP. 4. Exercise caution when using any electrical equipment in the laboratory. The Experiment 5. Always wash hands thoroughly with soap and water after handling reagents or biological materials in the laboratory. LABORATORY NOTEBOOK RECORDING: 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.

10 10 ingle Antibody ELIA Diagnostics General Information and Instructions INTRUCTION FOR ADDING LIQUID AND WAHING WELL Adding Reagents to wells: The Experiment For adding reagents to the wells, use the appropriately labeled transfer pipets or use an automatic micropipet and disposable tips. If using automatic micropipets, use a fresh tip for every step. Liquid Removal and Washes: When instructed to remove liquid reagents, use the appropriately labeled transfer pipet designated for each row. Alternatively, use an automatic micropipet and disposable tips. To wash the wells, do the following: A. Use the transfer pipet labeled "PB", to add PB buffer to the wells. Add PB buffer until each well is almost full. The capacity of each well is approximately 0.2 ml. Do not allow the liquids to spill over into adjacent wells. B. Remove all the PB from each of the wells with the transfer pipet designated for each row. REMINDER FOR AVOIDING COMMON PITFALL 1. Be very careful when transferring solutions into and out of the microtiter plate wells. 2. Do not attempt to empty the microtiter wells by shaking them out. This will not work - it will result in contaminating adjacent wells. 3. Use only clean or appropriately labeled pipets and avoid contaminating adjacent wells. 4. Wash the wells gently and slowly, without force.

11 ingle Antibody ELIA Diagnostics 11 Enzyme Linked Immunosorbent Assay (ELIA) In this direct ELIA one antibody is used to which the enzyme is bound. The microtiter wells are pre-treated with the antigen. Equilibrate a 37 C incubation oven before starting the experiment. LABEL THE MICROTITER PLATE: 1. Orient the microtiter plate as shown in figure 3. Carefully mark the microtiter plate with your initials or lab group number. If your microtiter plate is pre-labeled by the manufacturer, mark out the letters or numbers and re-label the plate as follows: Label the microtiter plate A, B and C across the top A B C Figure 3 Label the rows of wells consecutively, +, 1 and 2 down the left side of the microtiter plate. The Experiment The use of automatic micropipets and disposable tips is recommended to minimize cross-contamination and maximize the best results. However, if automatic micropipets are unavailable, use transfer pipets included in the experiment kit. LABEL THE PLATIC TRANFER PIPET: 2. Label 6 transfer pipets as follows: PB Phosphate Buffered aline ub ubstrate Use the appropriately labeled plastic transfer pipet for sample additions, removals, and washes as outlined in the experimental procedures. PB ub Figure

12 12 ingle Antibody ELIA Diagnostics Enzyme Linked Immunosorbent Assay (ELIA) ANTIGEN The microtiter wells are pre-treated with antigen. The Experiment Reminders: ADDING REAGENT: Be sure to use a fresh tip for the addition of each reagent. Alternatively, use the appropriately labeled transfer pipet for each reagent. LIQUID REMOVAL: Use the appropriately labeled transfer pipet to remove all liquid from each of the wells and after washes. Dispose the liquid into a beaker labeled "waste". WAHE: For all wells, use the transfer pipet labeled "PB" to add PB until each well is almost full. ANTIBODY CONTROL AND AMPLE: 3. Add 50 µl or 3 drops of PB to all the wells in Row Add 50 µl or 3 drops of positive control to all the wells in Row Add 50 µl or 3 drops of simulated patient serum sample 1 in all three wells in Row 3. UBTRATE: Figure Add 0.1 ml or 5 drops of the substrate to all of the wells (Figure 6). 11. Incubate for 10 minutes at 37 C. 12. Remove the plate for analysis A B C Negative control Positive control imulated erum ample 1 imulated erum ample 2 6. Add 50 µl or 3 drops of simulated patient serum sample 2 in all three wells in Row Incubate for 10 minutes at 37 C. 8. Remove all liquid using the transfer pipet designated for each row. 9. Wash each well once with PB buffer. Remove PB from each well using the transfer pipet designated for each row. A B C If color is not fully developed after 10 minutes, incubate at 37 C for a longer period of time. Figure 6

13 ingle Antibody ELIA Diagnostics 13 tudy Questions Answer the following study questions in your laboratory notebook or on a separate worksheet. 1. Why is there a 37 C incubation step after the addition of the substrate? 2. What would be the effect of not including the antigen or the antibody in the ELIA reaction? 3. Why is it important to wash all the wells between the additions of the various components? 4. Can nucleic acids be detected by the ELIA format? The Experiment

14 14 ingle Antibody ELIA Diagnostics Experiment Notes The Experiment

15 ingle Antibody ELIA Diagnostics 15 Instructor s Guide 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. 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 ervice is available from 9:00 am to 6:00 pm, Eastern time zone. Call for help from our knowledgeable technical staff at EDVOTEK ( ). ABOUT THI EXPERIMENT In this simulation experiment, the microtier wells are pre-treated with the antigen. This is a direct ELIA where one antibody is used to which the enzyme is bound. After the simulated patient serum sample is added to the wells, washed, and substrate is added, the conversion of the substrate to product results in the color formation for the positve samples. APPROXIMATE TIME REQUIREMENT FOR PRE-LAB AND EXPERIMENTAL PROCEDURE 1. Pre-lab preparation of biologicals and reagents takes approximately one and one-half hours. 2. The student experimental activity requires approximately 60 minutes. EDVOTEK The Biotechnology Education Company EDVOTEK FAX: info@edvotek.com

16 16 ingle Antibody ELIA Diagnostics Pre-Lab Preparations PREPARATION BEFORE THE LAB Microtiter Plates Instructor s Guide The microtier wells are pre-treated with the antigen. 1. As shown in the figure 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 figure. Each piece will be 3 wells on one axis and 4 wells on the other axis. Each lab group will receive one piece. Row 1 A Row 2 Row 3 Row 4 Row 1 Row 2 Row 3 Row 4 B C D E F G H Cutting lines depicted by dashed lines PREPARATION OF REAGENT ON THE DAY OF THE LAB Preparation of Phosphate Buffered aline 1. Add all of the Phosphate Buffered aline concentrate (F) to 135 ml of distilled water. Mix. 2. Label this diluted Phosphate Buffered saline as PB. 3. Dispense 12 ml into 10 small beakers or tubes for each lab group.

17 ingle Antibody ELIA Diagnostics 17 Pre-Lab Preparations PREPARATION OF PATIENT AND CONTROL AMPLE Note: The concentrated patient and control samples are very small and some of the liquid may get caught in the cap. Use caution to avoid losing the samples. Component Tube Volume to Label Dispense A + PB imulated Patient P ml erum ample 1 B + PB imulated Patient P ml erum ample 2 C + PB Positive Control ml F + water Phosphate buffered PB 8.0 ml aline PB + D + E Peroxidase-enzyme substrate 1.5 ml 1. Add 3 ml of diluted Phosphate Buffered aline (PB) to the imulated Patient erum ample 1 concentrate (A). Cap the bottle and mix thoroughly by tapping and inverting. 2. Dispense 0.25 ml of the diluted imulated Patient erum ample 1 for each group, 3. Using a clean pipet, repeat steps 1 and 2 for the imulated Patient erum ample 2 concentrate (B) and Positive Control concentrate (C). 4. Label tubes and dispense the reagents into the corresponding tubes. Instructor s Guide Prepare the substrate minutes before students require it for plate development (last incubation). PEROXIDAE UBTRATE (Prepare During the lab experiment) Prepare minutes before the last incubation: 1. Dispense 14 ml of diluted Phosphate buffered saline (PB) to a 50 ml tube. 2. Add Peroxide co-substrate (E) to the 14 ml of PB. Cap and mix thoroughly by shaking and/or vortexing. There is usually undissolved material remaining. 3. Add 1.5 ml of Hydrogen peroxide (D). Cap and mix. 4. Dispense 1.5 ml peroxidase substrate () for each of the 10 groups.

18 18 ingle Antibody ELIA Diagnostics Quick Reference Tables Preparation of Experiment reagents Components Description Label Dispense for each group Instructor s Guide A Patient erum ample 1 P ml B Patient erum ample 2 P ml C Positive Control ml F + water Phosphate Buffered aline PB 12.0 ml PB + D + E Peroxidase-enzyme substrate** 1.5 ml ** Peroxidase-enzyme substrate should be prepared minutes before the last incubation. Each Lab Group hould Receive: 1 microtiter section (pre-treated with antigen) 1 tube labeled "+" for positive control 1 tube labeled "imulated patient serum sample 1" 1 tube labeled "imulated patient serum sample 2" 1 automatic micropipet with tips (optional) 6 Transfer pipets 1 beaker or tube containing PB 1 empty beaker labeled "waste" 1 tube labeled "" (just before the last incubation)

19 ingle Antibody ELIA Diagnostics 19 Experiment Results and Analysis Color should appear only in Rows 2 and 4. Row 1 is a negative control. Row 2 is a positive control. Row 3 is a negative patient sample. Row 4 is a positive patient sample A B C Instructor s Guide

20 Please refer to the kit insert for the Answers to tudy Questions