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Graduate Level Immunology Workshop - Using Antibodies as Research Tools
- 1. Experimental Design Workshop #1: Antibodies October 7th, 2011
- 2. Polyclonal Antibodies or Antisera • Consists of antibodies generated in a natural immune response or after immunization in the lab – Antibodies have different specificities and affinities • Disadvantages: – Each antiserum is different – Limited volumes – Unexpected cross reactions, even after purification
- 3. Monoclonal antibodies • Unlimited supply of antibody molecules – Identical structure, antigen-binding site, and isotype. • Production utilizes tumor cells from multiple myeloma patients – Plasma cells produce antibodies – Plasma cells in these tumors proliferate indefinitely • Spleen cells from an immunized mouse are fused to mouse myeloma cells
- 4. Production of monoclonal antibodies
- 5. Affinity Chromatography to purify mAb
- 6. ELISA
- 8. Sandwich ELISA 10 OD450 1 0.1 10 100 1000 10000 Human IFNr Conc (pg/ml)
- 11. Dolence, JJ et. al EJI, 2011.
- 12. ELISPOT Capture Antibody Detection Antibody Add activated cells and target cells (if appropriate) Count spots Cytokine binds capture Ab Wash off cells
- 13. Lymphocyte Isolation – Ficoll gradient
- 14. Lymphocyte isolation by magnetic sorting Labeling methods direct indirect
- 15. SDS-PAGE & Western Blotting Gel with proteins separated by MW Nitrocellulose membrane with proteins positioned identically to those in the gel Incubation with primary antibody specific for protein of interest and secondary antibody (enzyme conjugated) Substrate added, blot developed
- 16. SDS-PAGE & Western Blotting 1. Proteins are transferred from the electrophoresis gel to the nitrocellulose membrane 2. The membrane is incubated with primary antibody 3. The membrane is incubated with secondary antibody directed against the protein of interest 4. The secondary antibody is conjugated with an enzyme to provide a detection mechanism (HRP or AP). Substrate is added to the blot and the enzyme catalyzes the conversion of substrate to product to form a colored precipitate (or chemiluminscence) at the site of the protein-antibody complex.
- 17. SDS-PAGE & Western Blotting
- 18. SDS-PAGE & Western Blotting
- 20. Flow cytometry and FACs
- 21. FSC vs SSC • A correlated measurement between FSC (size) and SSC (internal structure) can allow for differentiation of cell types in a heterogenous cell population Granulocytes Lymphocytes SSC Monocytes RBCs, Debris, Dead Cells FSC
- 22. Fluorescence Channels • As the laser interrogates the cell, fluorochromes on/in the cell (intrinsic or extrinsic) may absorb some of the light and become excited. • As fluorochromes leave their excited state, they release energy in the form of a photon with a specific wavelength, longer than the excitation wavelength. • Photons pass through the collection lens, are split and steered down specific channels with the use of filters.
- 23. Fluorescence Detectors Laser Beam FSC Detector Collection Lens Fluorescence Detector A, B, C, etc… Original from Purdue University Cytometry Laboratories
- 24. Compensation • Fluorochromes typically fluoresce over a large part of the spectrum (100nm or more) • Depending on filter arrangement, a detector may see some fluorescence from more than 1 fluorochrome. (referred to as bleed over) • You need to “compensate” for this bleed over so that 1 detector reports signal from only 1 fluorochrome
- 25. Spectra of Common Fluorochromes Laser Lines (nm) 350 457 488 514 610 632 PE-Texas Red Texas Red PI Ethidium PE FITC cis-Paranaric Acid 300 400 500 600 700 Original from Purdue University Cytometry Laboratories
- 26. Flow cytometry and FACs
- 27. Santisteban et al, Cancer Res 2009
- 28. Peptide binding to HLA-A*0201 on T2 cells Thiel U et al, Br. J. Cancer, 2011
- 29. FSC vs. SSC FACS or Flow cytometry Biexponential transformation takes cells off the axis to get clearer picture of data. DOES NOT change data—only changes the display of data!!!!!
- 30. Tetramers
- 31. Klenerman et al, Nature Rev Immuno, 2002
- 32. 1st paper to describe the use of tetramers Cell Number % Lysis Altman JD et al, Science, 1996
- 33. Before stimulation w/peptide pulsed APCs After stimulation w/peptide pulsed APCs Jiang X et al, Clin Cancer Res, 2007
- 34. Problem Set work in small groups for 30 minutes
- 35. • You are a respected cancer immunologist at the world renowned Mustard Clinic. A patient presents in your clinic with advanced ovarian cancer. This patient has ascites (fluid) accumulation in their peritoneal cavity. You hypothesize that there may be immune cells present in this area and that they may be secreting cytokines. You do not know if the immune cells in the ascites are working to eradicate the patient’s tumor or if they are being suppressed by the tumor microenvironment. • What assays or techniques could you perform to determine the types of immune cells that are present in this patient’s ascites? A • How could you determine what cytokines are present in the ascites? • After you identify the cytokines present in the ascites, how could you test whether it is the immune cells that are secreting these cytokines in response to recognizing the tumor cells? • You discover that there is a large population of CD4 T cells in the patient’s tumor microenvironment. You wish to determine if these are helper CD4 T cells that can help in the attack against the tumor cells or if they are regulatory CD4 T cells that are actually suppressing the immune response. Discuss possible assays you could use to find an answer to your question. B • You hypothesize that the cancer cells in this patient’s tumor are defective in class I antigen processing and presentation. Describe ways in which you could test for defects in this pathway. What proteins could you look at? How would you measure them? • You think you have discovered a new epitope of folate receptor alpha (a protein that is frequently found at high levels in ovarian cancer patients) which may be good at stimulating CD8+ T cells. C You’d like to determine if there is already preexisting T cell immunity against this epitope in your ovarian cancer patient and in normal patients. How could you measure the number or presence of FR-α specific T cells in the cancer patient and in a normal patient?
- 36. • You are a respected cancer immunologist at the world renowned Mustard Clinic. A patient presents in your clinic with advanced ovarian cancer. This patient has ascites (fluid) accumulation in their peritoneal cavity. You hypothesize that there may be immune cells present in this area and that they may be secreting cytokines. You do not know if the immune cells in the ascites are working to eradicate the patient’s tumor or if they are being suppressed by the tumor microenvironment. • What assays or techniques could you perform to determine the types of immune cells that are present in this patient’s ascites? – Flow cytometry • How could you determine what cytokines are present in the ascites? – ELISA, • After you identify the cytokines present in the ascites, how could you test whether it is the immune cells that are secreting these cytokines in response to recognizing the tumor cells? – ELISPOT: coat bottom of plate with an antibody for the cytokine that the T cell type usually secretes, put T cells and tumor cells in the plate, if the T cells recognize the tumor cells then they will attack them and secrete cytokine, the cytokine will bind the antibody. Wash away all unbound cells. Come in with a secondary enzyme conjugated antibody. Add substrate and quantitate the number of spots (which equals the number of T cells activated against the tumor cells).
- 37. • You discover that there is a large population of CD4 T cells in the patient’s tumor microenvironment. You wish to determine if these are helper CD4 T cells that can help in the attack against the tumor cells or if they are regulatory CD4 T cells that are actually suppressing the immune response. Discuss possible assays you could use to find an answer to your question. – Flow cytometry – look for markers of CD4 helper vs CD4 Treg. – ELISA - incubate T cells with tumor cells and then measure the cytokines present in the supernatant. Are they helper cytokines or regulatory? • You hypothesize that the cancer cells in this patient’s tumor are defective in class I antigen processing and presentation. Describe ways in which you could test for defects in this pathway. What proteins could you look at? How would you measure them? – Western Blotting – measure all of the levels of intracellular class I proteins – Flow cytometry – measure the amount of surface class I molecules
- 38. • You think you have discovered a new epitope of folate receptor alpha (a protein that is frequently found at high levels in ovarian cancer patients) which may be good at stimulating CD8+ T cells. You’d like to determine if there is already preexisting T cell immunity against this epitope in your ovarian cancer patient and in normal patients. How could you measure the number or presence of FR-a specific T cells in the cancer patient and in a normal patient? – Generate a tetramer for your new FRa epitope and whatever HLA it binds. Label this tetramer with a fluorophore and use it to stain the T cells in the patient blood (do a ficoll gradient on the patient blood to isolate the lymphocyte fraction). Do CD8 and tetramer staining. Using flow cytometry, determine the number of tetramer specific CD8 T cells in the total lymphocyte population. – Or you could isolate the CD8 T cells (using magnetic sorting) from the lymphocyte fraction, and then perform an IFN-g Elispot by incubating those CD8 T cells with folate receptor expressing cancer cells or control cells pulsed with your FRa epitope. If you detect spots then it means there are T cells present in the patient’s blood that can recognize those targets.
- 39. Upcoming Sessions • Experimental Design Workshops – Workshop #2 Mouse Models: noon-2 PM on October 28th, Gugg 598 – Workshop #3 Using Ab & mouse tools: noon- 2 PM on December 2nd, Gugg 598 • Exam Review Sessions in Gugg 598: – 7-9 PM October 16th – 7-9 PM November 13th – 7-9 PM December 11th •
Editor's Notes
- Each antiserum is different from all other antisera, even if raised in a genetically identical animal by using the identical preparation of antigen. Antisera can be produced in limited volumes, so it is impossible to sue the same reagent in a long series of experiments. Antibodies generated in a natural immune response or after immunization in the laboratory are a mixture of molecules of different specificities and affinities. Ie, they are polyclonal.
- Mice are immunized intravenously with one antigen in order to produce a large population of splenocytes secreting specific antibody. Spleen cells would normally die after a few days in culture, so they must be fused with immortal myeloma cells to produce a hybridoma cell line. The myeloma cells lack the HGPRT enzyme and therefore can not survive on the HAT medium that is used to select hybrid cells. Spleen cells do have the HGPRT gene. So only hybrid cells can survive in culture. The spleen cells provide the gene necessary to survive and the myeloma cells provide the ability to continuously proliferate. Unfused myeloma cells will die because of the medium. Unfused spleen cells will die within 3 days because they cannot continuously proliferate.
- Affinity chromatography: separates molecules on the basis of their affinity for one another. Specific antibody can be isolated from an antiserum by affinity chromatography, which exploits the specific binding of antibody to antigen held on a solid matrix. -Bind antigen to beads -Load beads into a column -Pass antiserum over the column. Specific antibodies will bind. -Other proteins in the antiserum and nonspecific antibodies will pass through the column and be washed away -pH is lowered to 2.5 or raised to 11 and the specific antibodies are eluted.
- The ELISA is a fundamental immunological and biochemical technique, utilized to detect a putative antigen or antibody in a sample, based on antigen-antibody interactions. If a putative antigen (or likewise, an antibody) is detected, a signal is produced in the form of a measurable change. Steps: The unlabeled component (antigen in the scenario shown) is attached to the wells of a 96 well plate. Labeled antibody is added, unbound antibody or other proteins are washed away. Antibody binding is measured by a reaction that converts a colorless substrate into a colored reaction product. Color change happens directly in the 96 well plate and can be read on a spectrophotometer.
- To assess the presence of a particular antigen in a sample, a "Sandwich ELISA" may be performed essentially as follows. A solution of antibody, which is specific for (i.e., uniquely recognizes) the putative antigen, is immobilized on a solid surface in a well of a microtiter dish. The sample being assayed is then applied under conditions that would allow the antigen (for which the antibodies are specific) to bind to the immobilized specific antibodies. Unbound material is washed away with buffer rinses. Bound antigen is recognized once again under binding conditions by addition of a solution of a second antibody specific for the same antigen, which this time is coupled or linked to an enzyme that catalyzes the conversion of its substrate to a detectable and possibly quantifiable form. Those enzyme-linked antibodies that are bound to the immobilized antigen-antibody complex are resistant to rounds of washing, and finally the enzyme substrate is added and incubated so that the enzyme can catalyze the conversion of the substrate to signal the detection of the specific antigen. ELISAs typically employ either a chromogenic or a fluorogenic substrate, which produces a color or fluorescence change, respectively, upon conversion by the enzyme linked to the second antibody. Conversion of the enzyme substrate is conveniently observed or quantitated on a plate reader that measures optical density or fluorescence intensity. In summary, a color or fluorescence change signals the enzyme activity, which signals the presence of the second antibody, which signals the presence of the antigen sought. Sandwich ELISA schematic (Credit: biosystemdevelopment.com)
- Good link for difference between sandwich and competitive elisa http://www.komabiotech.co.kr/www/techniques/immunodection/elisaProtocol.html B. Principle of the technology IFNr ELISA is based on the principle of a solid phase enzyme-linked immunosorbent assay. The assay utilizes rabbit anti-human IFNr antibodies for immobilization on the microtiter wells and rabbit anti-human IFNr antibodies along with streptavidin conjugated to horseradish peroxidase (HRP) for detection. The test sample is allowed to react simultaneously with the two antibodies, resulting in the IFNr molecules being sandwiched between the solid phase and enzyme-linked antibodies. After incubation, the wells are washed to remove unbound-labeled antibodies. A HRP substrate, TMB, is added to result in the development of a blue color. The color development is then stopped with the addition of Stop Solution changing the color to yellow. The concentration of IFNr is directly proportional to the color intensity of the test sample. Absorbance is measured spectrophotometrically at 450 nm.
- The ELISPOT detecting interferon (IFNγ) has become widely used as a means of quantization of T cell-mediated immune responses (CD4+ or CD8+) and is becoming the assay of choice for clinical trials for detecting T cell responses to gene therapy product and candidate vaccines. It is relatively rapid; sensitive, and allows the screening of multiple samples on a single day. The ELISPOT assay is based on the principle of the ELISA detecting antigen-induced secretion of cytokines trapped by an immobilized antibody and visualized by an enzyme-coupled second antibody.
- Lymphocytes are analyzed by density centrifugation over a step gradient consisting of the carbohydrate polymer Ficoll Hypaque. RBCs and polymorphonuclear leukocytes or granulocytes are denser and travel through the Ficoll layer, while mononuclear cells consisting of lymphocytes together with some monocytes will form a band over the ficoll and can be recovered at the interface.
- Negative selection Positive selection Also, you can stain cells first with CD4 PE (as you would for flow) then use anti PE beads to sort them. Cells in a single-cell suspension are magnetically labeled with MACS® MicroBeads. The sample is applied to a MACS Column placed in a MACS Separator. The unlabeled cells pass through while the magnetically labeled cells are retained within the column. The flow-through can be collected as the unlabeled cell fraction. After a short washing step, the column is removed from the separator, and the magnetically labeled cells are eluted from the column. Thus, with MACS Technology both labeled and unlabeled cells can easily be isolated with high purity and recovery. MACS MicroBeads are 50-nm superparamagnetic particles that are conjugated to highly specific antibodies against a particular antigen on the cell surface. Due to the small size, they do not activate cells and they will not saturate cell surface epitopes. Unlike larger beads, MACS MicroBeads do not have to be removed for any downstream application. MicroBeads are non-toxic and biodegradable
- Western blotting is used to identify the presence of a given protein in a cell lysate. Discuss SDS-PAGE vs Native
- Proteins are transferred from the electrophoresis gel to the nitrocellulose membrane The membrane is incubated with primary antibody The membrane is incubated with secondary antibody directed against the protein of interest The secondary antibody is conjugated with an enzyme to provide a detection mechanism (HRP or AP). Substrate is added to the blot and the enzyme catalyzes the conversion of substrate to product to form a colored precipitate (or chemiluminscence) at the site of the protein-antibody complex.
- This technique is used to determine whether a give protein interacts physically with another given protein. Cell extracts containing the presumed interaction complex are first immunoprecipitated with antibody against one of the proteins. The immune complexes are then recovered by incubation with Protein A agarose beads or Protein G agarose beads. The immune complexes are then washed and dissociated in SDS sample buffer. The samples are then analyzed by SDS-PAGE and Western blotting. The material identified by this means is tested for the presence of the other protein by immunoblotting with a specific antibody. Goal of this experiment was to see if the receptor tyrosine kinase called Flt3 activates the JAK/STAT pathway in Baf3/FLT3 cells (which stably express the Flt3 receptor). Figure 1. FL induces tyrosine phosphorylation of Stat5 in Baf3/Flt3 cells. (A) Growth factor starved Baf3/Flt3 cells were stimulated with Flt3 ligand (FL) (100 ng/ml) or IL-3 (10 ng/ml) for 5 min. Stat5 and Stat3 were immunoprecipitated from cell lysates and immunoblotted with antiphosphotyrosine antibody.
- Cyto = cells Metry = measure Flow = cells in motion Flow cytometry = measuring the properties of cells while they are in a fluid stream Light traveling in the forward direction is in the forward scatter channel (FSC) and is attributed to cell size and refractive index (membrane permeability). Laser light that is scattered at 90 degrees to the axis of the laser path is detected in the side scatter channel (SSC). The intensity of this signal is proportional to the amount of cytosolic structure in the cell (eg. granules, cell inclusions, etc.)
- CD24-/lo and CD44+ phenotype is consistent with BCSCs.
- EZH2 and CHM1 are Ewing tumor associated antigens
- Flow jo website: When data is properly compensated, it is common that a large number of cells are displayed squished against the axis. The cells become piled up in the first channel (against the axis) because the fluorescence parameters are displayed on a log scale where it is not possible to display "zero" or negative values. These negative values occur as a result of measurement error (not as a result of compensation). The spreading of a population into the negative is the result of measurement error that is inherent in the data we collect on flow cytometers. Even though the measurement error is the same in uncompensated samples, the error becomes obvious in the low regions of the log scale because log scales expand the view of data in the lower regions (frist decade) and compress the view of data in the upper regions (fourth decade).
- Staining specific T cells with their ligand of soluble peptide:MHC complexes has failed because there is a fast dissociation rate from the T cell antigen receptor. Multimeric pMHC complexes can bind more than one TCR on a specific T cell and have slower dissociation rates, making the complexes more suitable for use as immunological stains. Tetramers are produced by mixing the biotinylated pMHC complex with PE labeled avidin.
- Staining specific T cells with their ligand of soluble peptide:MHC complexes has failed because there is a fast dissociation rate from the T cell antigen receptor. Multimeric pMHC complexes can bind more than one TCR on a specific T cell and have slower dissociation rates, making the complexes more suitable for use as immunological stains. Tetramers are produced by mixing the biotinylated pMHC complex with PE labeled avidin. The basic construction of MHC class I tetramers has not evolved significantly since its first description in 1996 (Ref. 5), although factors that affect the interaction with T-cell receptor (TCR)–CD8 have been explored94, 95, 96. Although some interaction of T cells with MHC dimers can be detected, reproducible staining is best obtained with tetramers. These tetramers must be formed in solution before the addition of T cells — adding fluorescent streptavidin after the incubation of cells with monomers does not lead to cell labelling. Internalization of tetramers has been observed and might contribute to the stability of binding. The tetramers themselves might bind together to form higher-order oligomers. The tetramer remains essentially a tool for flow cytometric studies — its use in histological sections has been limited to studies by only a few groups97. The advantages of tetramer assays are that they are specific and sensitive, allow for surface and intracellular phenotyping, and can be combined readily with functional assays. Very small samples can be evaluated in detail (for example, organ infiltrates) and cryopreserved samples are satisfactory; indeed, in longitudinal studies, they are even preferable. Sorting and cloning from low specific T-cell frequencies (for example, human influenza-specific T-cell populations) have been successful; these experiments were important initially to confirm that populations of tetramer-positive cells were in fact antigen-specific28. The main disadvantage of tetramers is that only single specificities can be analysed. This is not a problem in inbred mouse strains or human infections, such as with Epstein–Barr virus, for which immunodominant peptides exist, but is a problem for infection with HIV and hepatitis C virus, in which a complex set of epitopes might be targeted by T cells. Tracking T cells with tetramers: new tales from new tools Paul Klenerman, Vincenzo Cerundolo & P. Rod Dunbar Nature Reviews Immunology 2 , 263-272 (April 2002)
- Staining specific T cells with their ligand of soluble peptide:MHC complexes has failed because there is a fast dissociation rate from the T cell antigen receptor. Multimeric pMHC complexes can bind more than one TCR on a specific T cell and have slower dissociation rates, making the complexes more suitable for use as immunological stains. Tetramers are produced by mixing the biotinylated pMHC complex with PE labeled avidin. Fig. 1. Staining by MHC-peptide tetramers correlates with peptide-dependent cytotoxicity. Flow cytometric analysis of CD8+ T cells from (A) clone 20 stained with A2-Pol (solid line) and A2-Gag (dotted line) tetramers, (B) clone 20 showed specific killing of autologous Epstein-Barr virus- transformed B cells pulsed with Pol peptide (closed 4C squares) but not target cells without added peptide (closed circles).
- Fig. 1. Identification of the HLA-A2/pIL-13Rα2345-354-aAPCs–induced specific CTLs by HLA-A2/peptide tetramer staining. Frequency of HLA-A2/peptide tetramer–binding CD8+ T lymphocytes before stimulation and after four rounds of stimulation with HLA-A2/pIL-13Rα2345-354-aAPCs. The HLA-A2/pIL-13Rα2345-354 tetramer and the control HLA-A2/pHIV tetramer were used. A, the frequency of HLA-A2/pHIV tetramer–binding CD8+ T lymphocytes before stimulation was 0.09%. B, the frequency of HLA-A2/pIL-13Rα2345-354 tetramer–binding CD8+ T lymphocytes before stimulation was 0.11%. C, the frequency of HLA-A2/pHIV tetramer–binding CD8+ T lymphocytes after stimulation was 0.06%. D, the frequency of HLA-A2/pIL-13Rα2345-354 tetramer–binding CD8+ T lymphocytes after stimulation was 14.7%. Representative results of many experiments, showing that the coculture of HLA-A2/pIL-13Rα2345-354-aAPCs and HLA-A2+ PBMCs can expand the pIL-13Rα2345-354–specific CTLs. HLA Tetramer–Based Artificial Antigen-Presenting Cells Efficiently Stimulate CTLs Specific for Malignant Glioma