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Immunological Techniques Radioimmunoassay (RIA) Regular Office Hours: Tuesdays 11-12 Extra office hours: Wed, Feb 7 12-1pm Thurs, Feb 8 11am-12 Midterm Fri, Feb 9 2-4pm I WILL NOT BE HOLDING OFFICE HOURS ON TUESDAY Feb 13!! Extra Office Hours Dina, Tim, and I encourage all confused students to come to our office hours and discussion sections so we can try to help un-confuse you. Take No class on Tuesday Feb 13. First midterm: Thurs Feb 15 at 6pm in 155 Dwinelle (not 2050 VLSB as listed in the original schedule). Midterm will focus on material covered in lectures and will be designed to be taken in 90 min. (We have the room till 8pm.) The GSIs will conduct a review session in our regular class period on Thursday Feb 15. The extraordinary specificity of Immunological Techniques antibodies for their antigens, and the ability to generate polyclonal and monoclonal antibodies to virtually anything, makes them fantastically Monoclonal Antibodies useful reagents for detecting and quantitating substances. Radioimmune Assay (RIA) A large number of different assays have been developed to detect antigens based Enyzme Linked Immune Sorbant Assay (ELISA) on antibody binding that can be used in in fluids, tissues, or cells. Western blot Immunoprecipitation Immunofluorescence Flow cytometry Expression cloning Radioimmunoassay (RIA) •1960 Yalow and Berson (Nobel Prize) •Very sensitive: can detect material present at concentrations of <0.001 micrograms/ml. •Takes advantage of protein binding to Cpm plastic of tissue culture dish. •Generate standard curve with known RIA to detect amounts of unlabeled antigen •Measure unknown using standard curve. hepatitis Labeled antigen detects hepatitis virus in 1 microliter Amount unlabeled Antigen Unlabeled of blood. antigen Used for screening blood for transfusions. 1 Enzyme-linked immune sorbant Variations of the ELISA method assay (ELISA) Capture Add second specific antigen using antibody-enzyme Colorometric plate-bound conjugate assay antibody Detection by secondary antibodies conjugated to enzymes (alkaline phosphatase, horse radish peroxidase, β-galactosidase). Break- down of substrate by enzyme produces a visible color. ELISA based pregnancy test 2 Real life example of use of ELISA assay (from your reading assignment: “A Toll-like receptor recognizes • The ELISPOT assay bacterial DNA” Hemmi 2000 Nature v408 p 740 Figure 3b) can be used to Isolate macrophage from wild type and TLR9 mutant mice. Stimulate with CpG (mammalian DNA is methylated at CpG residues, and so differs from determine the unmethylated bacterial DNA) Peptidoglygan (PGN) and Lipopolysaccaride (LPS) trigger innate immune number of cells responses through other TLRs and serve as positive controls. Measure cytokine within a sample that production (TNF-α) in culture supernatants by ELISA are secreting a particular cytokine. Concentration of the cytokine TNF-α as measured by ELISA Western Blot SDS-polyacrylamide gel electrophoresis to separate a complex mixture of proteins based on their molecular weight. Transfer proteins from gel to a membrane sheet. Gel electrophoresis separates proteins by size, so Western blot not only provides quantitation, but also provides information about the molecular weight of antigen. After transferring proteins from gel to a membrane sheet, use labeled antibodies to your protein of interest to detect the relevant band. Labeled antibodies bind to band containing your protein of interest. Detect labeled antibody using colorimentic assay. 3 Immunoprecipitation Immunoprecipitation: variations of the method. Anti- + antibody Protein A or Protein G Protein A and Protein G: bacterial cell wall proteins that binds to Ig. Magnetic beads coupled to antibodies can be used to isolate proteins from solution, or cells from a suspension. And check out “A Toll-like receptor recognizes bacterial DNA” Hemmi 2000 Nature v408 p 740 Figure 3 f and g For real-life examples of Immunoprecipitations and Western blots to analyze TLR signaling intermediates in wild type and TLR9- macrophage in response to different TLR agonists. Immunoaffinity Chromatography Immunofluorescence Fluorochrome-conjugated secondary-antibody (anti-Ig antibody) Fluorochrome-conjugated specific antibody Unconjugated specific antibody direct indirect 4 Immunofluorescence can provide spatial information about cells or molecules that react with antibodies. Anti-IgM stain of B cells Mixture of cells labeled with fluorescent antibodies Immunological Techniques Flow cytometry can be used to Monoclonal Antibodies determine the number of cells Radioimmune Assay (RIA) within a sample that react with a Enyzme Linked Immune Sorbant Assay (ELISA) particular Western blot antibody (or Immunoprecipitation antibodies) Flow cytometry Expression cloning 1 parameter histograms: Analysis of cells stained with labeled antibodies Flow cytometric Dot plots Contour plots Cell < 1 % Unstained cells analysis number positive (negative control) of cells stained with 2 different labeled Number Fluorescence intensity of antibodies cells Cell 30% ) 2 parameter dot plot: number positive n Stained e e r cells g ( 20% histograms y 20% t i Green s “double n only e Positive” Fluorescence intensity (red) t n i e c n e c Number s e of Cell 40% r 10% o 50% cells number positive u Stained l “double red F cells negative” only Fluorescence intensity (red) Fluorescence intensity (green) Amt. IgD 5 The Power of Flow Cytometry Real life example of use of flow cytometry Quantitative: (from your reading assignment: “A Toll-like receptor recognizes Accurately determine relative fluorescent levels (proteins bacterial DNA” Hemmi 2000 Nature v408 p 740 Figure 3d) levels) on individual cells. Isolate macrophage from wild type and TLR9 mutant mice. Accurately determine the number of fluorescent cells within Stimulate with CpG (or LPS as positive control). Examine expression of activation markers by flow cytometry. a population. CD40 CD80 CD86 MHC-II Sensitive: Wild type Analysis can be performed with <104 cells. Flexible: TLR9-/- Number of cells Fluorescent labeled antibodies specific for many cell surface proteins are readily available. Can simultaneously Fluorescence intensity Note: CD40, CD90, CD86, and MHC-II are all important for T cell activation stain for >4 markers. by macrophage (innate immunity stimulating adaptive immunity). The generation of monoclonal antibodies Flow cytometry can be used to identify different kinds of leukemia. specific for cell surface proteins, coupled (“CD” nomenclature) with flow cytometry, provides a powerful tool for identifying different lymphocyte populations. Ultrasonic nozzle vibrator Fluorescence Cell suspension Sheath fluid Cell- activated Cell- sorting sorting Drop-charging (FACS) signal using the using the flow laser flow cytometer cytometer Cell collector Cell collector Flask for undeflected droplets 6 Magnetic beads coupled to antibodies can also Expression cloning genes in E. be used to purify cells (an alternative to FACS, that is especially useful for processing larger coli numbers of cells.) Cell expressing Purified cDNA cDNA library in Protein of interest mRNA Bacterial expression vector Bacterial Colonies, Each Bacteria Expressing expressing cDNA of Flow cytometry is used here to monitor the composition One Specific cDNA interest of the cell sample before and after separation. antibody A a result of V(D)J recombination every mature B cell expresses a unique antibody. Expression Encounter with an antigen leads to clonal expansion of B cells with a particular Cloning specificity. •Make antiserum against gene-product of interest •Make cDNA expression library from cell line expressing gene •Transfect library into cell line which does not normally express gene-product •Select cells expressing product by antibody-binding •Recover cDNA expression vector •Repeat. Recover. Sequence. Victory! Where we have been and where we are going Innate Immunity V(D)J Recombination Antibodies and antigens I (emphasis on antibody structure) Discovery of Ig gene rearrangements Antibodies and antigens II (emphasis on antigen-antibody binding interactions) Structure of antibody genes (RSS) Techniques based on antibodies Role of RAG proteins and DNA repair machinery V(D)J recombination B cell development and function 7 HV3 HV1 HV2 The puzzle of antibody diversity • Limitless array of Ig sequences (too large to be encoded in genome) • Variation limited to V domain. • Identical V segment could be associated with two different C regions (myeloma Variability protein with γ and µ chains) of Ig • Germ-line vs somatic variation models • Dreyer and Bennett (1965) the 2 gene model; a violation of the “one gene, one Sequences polypeptide” rule • 1976: the emerging tools of molecular biology open the way for the breakthrough. Review of Southern blot method The breakthrough paper Restriction endonucleases cleave at specific sequences in DNA and can be used to generate a physical map of DNA. (e.g. EcoRI cleaves at the sequence: 5’-GAATTC-3’) Surprising Southern blot Surprising Southern blot 3 3 a a 1 2 1 2 m m a a a a o o l l m m m m e e n n o o o o y y l l l l e e r r e e e e m m e n e n e e l i l i y y y y v v p k p k i i l m m s s l m m s s Jκ1Jκ2Jκ3Jκ4 Cκ Vκ1 Vκ 2 Cκ probe 8 Detecting Ig gene rearrangement using Southern blot V(D)J Recombination Discovery of Ig gene rearrangements Structure of antibody genes (RSS) Evidence for role of RAG proteins and DNA repair machinery Multigene organization of Ig genes: Multigene organization of Ig genes: light chain genes heavy chain genes VK gene segments JK gene segments VH gene segments DH gene segments JH gene segments
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