1MidtermExtra Ofﬁce HoursTakeRegular Office Hours: Tuesdays 11-12Extra office hours: Wed, Feb 7 12-1pmThurs, Feb 8 11am-12Fri, Feb 9 2-4pmI WILL NOT BE HOLDING OFFICE HOURS ON TUESDAY Feb 13!!Dina, Tim, and I encourage all confused students to come to our officehours and discussion sections so we can try to help un-confuse you.No class on Tuesday Feb 13.First midterm: Thurs Feb 15 at 6pm in 155 Dwinelle (not 2050 VLSB as listedin the original schedule).Midterm will focus on material covered in lectures and will be designed tobe taken in 90 min. (We have the room till 8pm.)The GSIs will conduct a review session in our regular class period onThursday Feb 15.The extraordinary speciﬁcity ofantibodies for their antigens, and theability to generate polyclonal andmonoclonal antibodies to virtuallyanything, makes them fantasticallyuseful reagents for detecting andquantitating substances.A large number of different assays havebeen developed to detect antigens basedon antibody binding that can be used inin ﬂuids, tissues, or cells.Immunological TechniquesMonoclonal AntibodiesRadioimmune Assay (RIA)Enyzme Linked Immune Sorbant Assay (ELISA)Western blotImmunoprecipitationImmunoﬂuorescenceFlow cytometryExpression cloningRadioimmunoassay (RIA)CpmAmount unlabeled Antigen•1960 Yalow and Berson (Nobel Prize)•Very sensitive: can detect materialpresent at concentrations of <0.001micrograms/ml.•Takes advantage of protein binding toplastic of tissue culture dish.•Generate standard curve with knownamounts of unlabeled antigen•Measure unknown using standard curve.LabeledantigenUnlabeledantigenRIA to detecthepatitisdetects hepatitis virus in 1 microliterof blood.Used for screening blood fortransfusions.
2Enzyme-linked immune sorbantassay (ELISA)ColorometricassayCaptureantigen usingplate-boundantibodyAdd second speciﬁcantibody-enzymeconjugateDetection by secondary antibodies conjugated to enzymes (alkalinephosphatase, horse radish peroxidase, β-galactosidase). Break-down of substrate by enzyme produces a visible color.Variations of the ELISA methodELISA based pregnancy test
3Real life example of use of ELISA assay(from your reading assignment: “A Toll-like receptor recognizesbacterial DNA” Hemmi 2000 Nature v408 p 740 Figure 3b)Isolate macrophage from wild type and TLR9 mutant mice. Stimulate with CpG(mammalian DNA is methylated at CpG residues, and so differs fromunmethylated bacterial DNA)Peptidoglygan (PGN) and Lipopolysaccaride (LPS) trigger innate immuneresponses through other TLRs and serve as positive controls. Measure cytokineproduction (TNF-α) in culture supernatants by ELISAConcentration of the cytokine TNF-α as measured by ELISA• The ELISPOT assaycan be used todetermine thenumber of cellswithin a sample thatare secreting aparticular cytokine.Western BlotGel electrophoresis separates proteins by size, so Westernblot not only provides quantitation, but also providesinformation about the molecular weight of antigen.SDS-polyacrylamide gelelectrophoresis to separate acomplex mixture of proteinsbased on their molecular weight.Transfer proteins from gel to amembrane sheet.After transferring proteins fromgel to a membrane sheet, uselabeled antibodies to yourprotein of interest to detect therelevant band.Labeled antibodies bind to bandcontaining your protein ofinterest.Detect labeled antibody usingcolorimentic assay.
4Immunoprecipitation+Protein AorProtein GAnti-antibodyProtein A and Protein G: bacterial cell wall proteins that binds to Ig.Immunoprecipitation: variations of the method.Magnetic beads coupled to antibodies can beused to isolate proteins from solution, or cellsfrom a suspension. And check out “A Toll-like receptor recognizesbacterial DNA” Hemmi 2000 Nature v408 p 740Figure 3 f and gFor real-life examples of Immunoprecipitations andWestern blots to analyze TLR signaling intermediatesin wild type and TLR9- macrophage in response todifferent TLR agonists.Immunoafﬁnity Chromatography Immunoﬂuorescencedirect indirectFluorochrome-conjugatedspeciﬁc antibodyUnconjugatedspeciﬁc antibodyFluorochrome-conjugatedsecondary-antibody(anti-Ig antibody)
5Immunoﬂuorescence can provide spatial informationabout cells or molecules that react with antibodies.Anti-IgM stain of B cellsImmunological TechniquesMonoclonal AntibodiesRadioimmune Assay (RIA)Enyzme Linked Immune Sorbant Assay (ELISA)Western blotImmunoprecipitationFlow cytometryExpression cloningMixture of cells labeled with ﬂuorescent antibodiesFlow cytometrycan be used todetermine thenumber of cellswithin a samplethat react with aparticularantibody (orantibodies)Fluorescence intensity (red)CellnumberFluorescence intensityCellnumberUnstained cells(negative control)Stainedcells30%positive< 1 %positiveFluorescence intensity (green)Cellnumber Stainedcells40%positiveFlow cytometricanalysisof cells stained with 2different labeledantibodiesFluorescence intensity (red)Fluorescenceintensity(green)2 parameter dot plot:1 parameter histograms:20%“doublePositive”20%Greenonly10%redonly50%“doublenegative”Analysis of cells stained with labeled antibodiesDot plots Contour plotshistogramsAmt. IgDNumberofcellsNumberofcells
6The Power of Flow CytometryQuantitative:Accurately determine relative ﬂuorescent levels (proteinslevels) on individual cells.Accurately determine the number of ﬂuorescent cells withina population.Sensitive:Analysis can be performed with <104 cells.Flexible:Fluorescent labeled antibodies speciﬁc for many cellsurface proteins are readily available. Can simultaneouslystain for >4 markers.Real life example of use of ﬂow cytometry(from your reading assignment: “A Toll-like receptor recognizesbacterial DNA” Hemmi 2000 Nature v408 p 740 Figure 3d)Fluorescence intensityWild typeTLR9-/-CD40 CD80 CD86 MHC-IIIsolate macrophage from wild type and TLR9 mutant mice.Stimulate with CpG (or LPS as positive control). Examineexpression of activation markers by ﬂow cytometry.Note: CD40, CD90, CD86, and MHC-II are all important for T cell activationby macrophage (innate immunity stimulating adaptive immunity).NumberofcellsThe generation of monoclonal antibodiesspeciﬁc for cell surface proteins, coupledwith ﬂow cytometry, provides a powerfultool for identifying different lymphocytepopulations.Flow cytometry can be used toidentify different kinds of leukemia.(“CD” nomenclature)Fluorescenceactivated Cell-sorting(FACS)using the ﬂowcytometerUltrasonic nozzle vibratorCell suspensionSheath ﬂuidDrop-chargingsignalCell collectorCell collectorFlask for undeﬂected dropletsCell-sortingusing theﬂowcytometerlaser
7Magnetic beads coupled to antibodies can alsobe used to purify cells (an alternative to FACS,that is especially useful for processing largernumbers of cells.)Flow cytometry is used here to monitor the compositionof the cell sample before and after separation.Expression cloning genes in E.coliCell expressingProtein of interestPuriﬁedmRNA cDNA cDNA library inBacterial expressionvectorSpeciﬁcantibodyBacterialColonies,EachExpressingOnecDNABacteriaexpressingcDNA ofinterestExpressionCloning•Make antiserum againstgene-product of interest•Make cDNA expressionlibrary from cell lineexpressing gene•Transfect library into cellline which does not normallyexpress gene-product•Select cells expressingproduct by antibody-binding•Recover cDNA expressionvector•Repeat. Recover. Sequence.Victory!A a result of V(D)J recombination every mature B cell expresses a unique antibody.Encounter with an antigen leads to clonal expansion of B cells with a particularspeciﬁcity.Where we have been and where we are goingInnate ImmunityAntibodies and antigens I (emphasis on antibody structure)Antibodies and antigens II(emphasis onantigen-antibodybinding interactions)Techniques based onantibodiesV(D)J recombinationB cell development andfunctionV(D)J RecombinationDiscovery of Ig gene rearrangementsStructure of antibody genes (RSS)Role of RAG proteins and DNA repair machinery
8Variabilityof IgSequencesHV1 HV2HV3 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 (myelomaprotein with γ and µ chains)• Germ-line vs somatic variation models• Dreyer and Bennett (1965) the 2 gene model; a violation of the “one gene, onepolypeptide” rule• 1976: the emerging tools of molecular biology open the way for the breakthrough. . .The breakthrough paperReview of Southern blot methodRestriction endonucleases cleave at speciﬁc sequences inDNA and can be used to generate a physical map of DNA.(e.g. EcoRI cleaves at the sequence: 5’-GAATTC-3’)Surprising Southern blotliverskinspleenmyeloma1myeloma2myeloma3Surprising Southern blotliverskinspleenmyeloma1myeloma2myeloma3probeVκ1Vκ 2 CκJκ1Jκ2Jκ3Jκ4 Cκ
9Detecting Ig gene rearrangement using Southern blotV(D)J RecombinationDiscovery of Ig gene rearrangementsStructure of antibody genes (RSS)Evidence for role of RAG proteins and DNArepair machineryVK gene segments JK gene segmentsCK exonsLight chains encoded by 2 gene loci: kappa and lambdaEach light chain encoded by 3 kinds of gene segments:V (variable), J (joining), C (constant)A V and J segment are brought together by somatic DNA rearrangementprocess: “V(D)J recombination”Multigene organization of Ig genes:light chain genesVH gene segmentsFirst 96 aa’s of Ig HCDH gene segments3-6 aa’s HCJH gene segments10-12 aa’s HC CH exonsMultigene organization of Ig genes:heavy chain genesHeavy chains encoded by a single gene locus.Each heavy chain encoded by 4 kinds of gene segments:V(variable), D (diversity), J (joining), C (constant)V, D, and J segments are brought together by somatic DNArearrangement process: “V(D)J recombination”Multigene organization of Ig genesV(D)J Rearrangement: light chainGenerearrangementTranscriptionVK gene segments JK gene segmentsCK exonsRNAsplicing mRNAV and J gene segments brought together in DNA beforetranscription. (RNA slicing removes introns.)
10V(D)J Rearrangement: heavy chainVH gene segmentsFirst 96 aa’s of Ig HCDH gene segments3-6 aa’s HCJH gene segments10-12 aa’s HC CH exonsGenerearrangementTranscriptionRNAsplicing mRNAV, D, and J gene segments brought together in DNA beforetranscription. (RNA slicing removes introns.)V(D)J RearrangementVH gene segmentsFirst 96 aa’s of Ig HCDH gene segments3-6 aa’s HCJH gene segments10-12 aa’s HC CH exonsGenerearrangementVariable domainexonConstant domainexonsCombinatorial Diversity in humansJunctional diversity (ﬂexible joining of segments, P and Nregion additions at junctions) also contributes substantially tothe total diversity of antibodies.Gene rearrangement juxtaposespromoter and enhancersPromoters: relatively short nucleotide sequences within ~200 bp oftranscriptional start site that initiate transcription in a certain direction.Enhancers: nucleotide sequences located up-stream or down-stream ofa gene that activated the promoter in an orientation independentmanner.
11Ig promoters are actively transcribed when theyare brought close to enhancers due to generearrangement.Allelic exclusionof Ig genes:ensures that most B cellwill express a singleantibody speciﬁcity(allele: two or morealternative forms of agene.)Rearranging gene segments areﬂanked by a conserved“rearrangement signal sequence”Gene-segment7-mer 9-mer12 or 23ntheptamer spacer nonamerRSSAlso known as “one turn RSS” and “two turn RSS”.Rearranging gene segments are ﬂanked by aconserved “rearrangement signal sequence”The 12/23 RuleOnly gene segments ﬂanked by RSSs withdissimilar spacers can undergo V(D)Jrecombination with one another.Ensures that V segments don’t join withother Vs, that VH don’t join with JH, etc.V(D)J Recombination:Reactants & Products
12Flexibility in joining ofgene segmentscontributes to junctionaldiversity.Note mostrearrangements are non-productive!(Only 1/3rearrangementspreserves the correctreading frame of the Jsegment.)P nucleotides are generated by resolution of hairpin structures. Nnucleotide are added by an enzyme called terminal deoxynucleotidetransferase (TdT).P and N nucleotide addition also contribute tojunctional diversity.