Interleukin-2 ELISA Assay Enzyme-Linked Immunosorbent Assays (ELISAs) areenzyme immunoassays widely used for detecting specific antibodiesbut can be used to detect antigens also. This procedure employs thespecificity of both Ab and enzymes to achieve a highly sensitive andprecise test. It is one of the standard procedures used for thedetection of Ab to the HIV virus. The ELISA takes advantage of the fact that most proteins willbind firmly to the surface of several different kinds of plastic, usuallyby hydrophobic interactions between the nonpolar residues of theprotein and the naturally hydrophobic components of the plastic.ELISAs are normally performed on a solid phase. Most often the solidphase consists of the surfaces of individual wells in multi-well plasticplates (96-well plates are commonly used). In the assays, a series ofantibody-antigen or antibody-antibody interactions are used to bindenzyme molecules to the bottom of a 96-well plate in such a way thatthe amount of enzyme is proportional to the amount of antibody/antigen in the system. The amount of enzyme activity is thenmeasured using a color producing substrate. From this the amount ofantibody/antigen can be calculated. The indirect ELISA is used to measure antibody. In this type ofELISA (figure 1), the antigen (peptide or protein) is bound to thepolystyrene microtiter plate first. Proteins like to stick irreversibly tovirgin plastic (passive absorption) and most of the plastic surface willbecome coated with the antigen.Figure 1: The indirect ELISA.
Any "unused" plastic surface is then "blocked" with an irrelevantprotein to prevent nonspecific binding of any of the subsequentadditives. Serum or some other sample containing the primaryantibody is then added to the well and allowed to bind. Next, samplesare removed and the wells are "washed" several times with anappropriate buffer solution. Finally, a second antibody, specific for thefirst antibody and labeled for detection, is added to the well andallowed to bind; the second antibody usually has an enzymeconjugated to it (e.g. horseradish peroxidase). This enzyme catalyzesthe formation of a colored substance from the substrate which is thenquantified and the amount of antibody/antigen present can becalculated. The ELISA procedure as outlined above can be modified inseveral ways. One important way is to convert it to detect andquantify antigen (sandwich ELISA). This is accomplished by firstlayering an Ab specific for the Ag in question on the plastic wells.After "blocking" you add the sample to be analyzed. If it contains thatantigen it will bind to the "solid phase" Ab-specific preparation. Youthen again add a preparation of antibodies which are specific to theantigen, this time with an enzyme coupled to them. Cleavage ofsubsequently added substrate is an indication that antigen is present,causing the enzyme labeled second antibody to bind. Another variation for measuring amounts of antigen is thecompetitive ELISA. In this technique, samples containing antigenare added to a pre-coated antibody plate. Addition of an enzyme-conjugated secondary antibody specific for the isotype of the primaryantibody is also added. The antigen and the enzyme-conjugatedsecondary antibody “compete” for binding sites with the primaryantibody that is coating the wells. After washing the wells and addinga color-producing substrate, the intensity of the color (the absorbancevalue) is inversely proportional to the amount of antigen containedwithin the sample.Cytokines and Interleukins Effective immune responses require communication betweenseveral cell types, namely lymphoid cells, inflammatory cells, andhematopoeitic stems cells. Chemical messengers called cytokinesare used to mediate such interactions between these cells.Cytokines are proteins that are secreted by leukocytes and various
other cells within the body in response to a number of stimuli.Cytokine proteins assist in regulating the development of immuneeffector cells, and some cytokines possess effector functions of theirown. Interleukins are a discrete group of cytokine messengermolecules that help coordinate intercommunication betweenleukocytes (hence the name “interleukins”). Arguably, the mostimportant protein of the interleukin family is Interleukin-2 (IL-2)because of its instrumental role in the bodys natural response tomicrobial infection and in discriminating between foreign (non-self)and self. The primary function of IL-2 is to act as a facilitator,mediator, and regulator of T cell activation and proliferation.However, in addition to regulating T cell proliferation, IL-2 canregulate Natural Killer cell activation and proliferation, B cellproliferation, and can be used to facilitate the manufacturing ofimmunoglobulins originating from B cells. In this experiment, you will perform a sandwich ELISA todetermine the amount of IL-2 in an unknown sample. To do this, youwill generate a standard curve from which you will be able tocalculate the quantity of your unknown. OBJECTIVES: Understand how the ELISA method works. Be able to compare and contrast the different ELISA methods. Understand the role of cytokines and interleukins, specifically Interleukin 2.Materials:Mouse Interleukin-2 ELISA kit purchased from eBioscience thatincludes the following: • 96-well microtiter plate. • Capture antibody: 500µL • Detection antibody: 500µL • Mouse Interleukin-2 standard: 20µL
• Substrate: 100mL. Stabilized 3,3’, 5,5’ Tetramethylbenzidine plus hydrogen peroxide. Light sensitive. • 5X Assay Diluent: 150mL. Diluted 5 fold with deionized water. • Enzyme (Avidin-HRP): 500µL1X PBS with 0.05% Tween 20 wash buffer2N H2SO4 stop solutionELISA plate readerProcedure:Day 1: Plate the standards and the samples*You will be working in tables for this experiment 1. Add 100µL per well of capture antibody in coating buffer to the appropriate wells of your table’s 96 well microtiter plate. Seal the plate and incubate overnight at 40C (T.A. will do this). 2. Aspirate wells and wash three times with 200µL per well of 1X PBS with 0.05% Tween 20 wash buffer using a multi-channel pipettor (T.A. will do this). 3. Block wells with 200µL per well of 1X Assay Diluent. Incubate at room temperature for 1 hour (T.A. will do this). 4. Aspirate wells and wash three times with 200µL per well of 1X PBS with 0.05% Tween 20 wash buffer using a multi-channel pipettor. 5. Label seven 15mL conical tubes for performing a serial dilution of the top standard. Perform 2-fold serial dilution of the top standard using 1X Assay Diluent to make the standard curve. 6. Add 100µL per well of standard to the appropriate wells. 7. Add 100µL per well of an unknown sample to the appropriate wells. T.A. will provide you with an unknown sample.
8. Once the standards and the unknown sample have been added to the appropriate wells, seal the plate and incubate for 48hrs at 40C.Day 2: Reading the IL-2 ELISA assay 1. Aspirate wells and wash three times with 200µL per well of 1X PBS with 0.05% Tween 20 wash buffer using a multi-channel pipettor (T.A. will do this). 2. Add 100µL per well of detection antibody diluted in 1X Assay Diluent. Seal the plate and incubate at room temperature for 1 hour (T.A. will do this). 3. Aspirate wells and wash three times with 200µL per well of 1X PBS with 0.05% Tween 20 wash buffer using a multi-channel pipettor. 4. Add 100µL per well of Enzyme (Avidin-HRP) diluted in 1X Assay Diluent. Seal the plate and incubate at room temperature for 30 minutes. 5. Aspirate wells and wash three times with 200µL per well of 1X PBS with 0.05% Tween 20 wash buffer using a multi-channel pipettor 6. Add 100µL of substrate solution to each well. Seal the plate and incubate for 15 minutes at room temperature. 7. Add 50µL of stop solution to each well. 8. Read plate at 450nm. 9. Average the absorbance values of the triplicates for the standards and use these averages to create a standard curve with the concentration of IL-2 in pg/ml on the X axis and the average absorbance value on the Y axis. Include Y error bars.
10. Using the y equation from your standard curve, determine the concentration of IL-2 (pg/ml) in your unknown sample.