Immunology MBioS 440
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This 25 page Class Notes was uploaded by Carol McDermott on Thursday September 17, 2015. The Class Notes belongs to MBioS 440 at Washington State University taught by Staff in Fall. Since its upload, it has received 45 views. For similar materials see /class/205940/mbios-440-washington-state-university in Molecular Biosciences at Washington State University.
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Date Created: 09/17/15
More interesting points about antibodies Production of secretory lgA luminal surface abluminal surface Stroma Apical I L RER endosome L j g g N Basolater al quot endosome mmlnl 2 gt J chain I 212 transmembrane SC pig receptnr I 3 free or bound SC Production of monoclonal antibodies What are monoclonal antibodies Production of monoclonal antibodies Production of monoclonal antibodies Epllapcs Plasma cells Myclom cells Hybridornas Clones Monoclonal antibodies Polyclonul zlmiwulm How are purines made De novo synthesis J Purine nucleoside monophosphosphate Purine nucleoside HGPRT Purine base J J Uric acid How does HAT work Myeloma fusion partner is HGPRT deficient hypoxanthineguanine phosphoribosyl transferase HGPRT is a purine salvage enzyme HGPRT uses hypoxanthine or guanine to make purines Hypoxanthine Aminopterine Thymidine How does HAT work Myeloma Plasma cell 1 Eternal life 1 Finite life 2 Makes no Ab 2 Makes desired Ab 3 Has HGPRT 3 Has no HGPRT AntibodyAntigen Interactions ANTIGEN ANTI BODY N H CHZ OH 0 c 7CH27CH17 042 Hz mu 0 c CHZ CHZ CH CHa CI CH3 CHrCH CH2 7 IH CH3 CH3 vCIH o CHz C HgNiCHzi Kuby 0 chapter 6 Interaction of Ab with Ab results in Precipitation Agglutination Fixation of complement AgAb complexes monovalent antigen AgA b complexes divalent antigen AgA b complexes Equivalence Antigen EXCESS excess quota Q 9 as 3quot E 52 i 39 n f gt a 1quot 5 3 gm in I 39 g 1 a E hi Amddnt df antibddy precipitated Amudtunt m antigen added Precipi39l39in RXN What happens when you add a soluble antigen 1390 an agar gel A FDiffusion 1 What happens when you add a soluble antigen To an agar gel Molecules begin to diffuse from well into gel Double Immunodiffusion aka aso known as Ouch relony double gel diffusion Agar matrix PreCIpItate Kuby chapter 6 A more realistic cartoon of what you see Anfiqen Antibody As reactants diffuse precipitation occurs where equivalence is reached A side view of the precipitin band formation in Ouchterlony double gel diffusion Ab 1 Ag Ab Ag Ab A9 Conc Conc Conc Cone Distance Distance Does molecular size and concentration affect the diffusion rate In a limited time concentrated In a limited time smaller molecules move farther than molecules move more rapidly less concentrated molecules than larger ones Let39s talk about these antibodyantigen interactions can be used in immunological assays 12 Double gel diffusion aka also known as The Ouch rer lony Assay emu12 Single radial immunodiffusion QC internal quality control Uses measurement of proteins in serum amniotic fluid cerebrospinal uid saliva Acutephase proteins transport proteins coagulation proteins and tumor markers Comenualion 0 protein gll Actually a that be added here is bugging One of The widely used assays reciprocal sarle dllmian h 391 n a 5 at v w ms 91 9quot WM teslsera Dl r IO 0 00 I l u This is a cross secfion of whaf is happening in The well of an ELISA Add antIA antibody covalently linked to enzyme mple 2 mple 1 a amigen A antigen 3 Enzymesubstrate pairs useful for ELISA Horseradish peroxidase TMB blue tetramethylbenizidine Chloronapthal blue DAB brown diaminobenzene Alkaline phosphatase NBT nitroblue tetrazolium chloride Ano iher widely used Technique in medicine and research is immunofluorescence How do you do immunofluorescence Several ways l39his 7 77 can be done llumescemamd amibmlv Mummy anuhody 53933 A wash wash I wash 1 add39mavg geinmed addcomplemem s A 1T 5 4 add rluoresceinaxeo39 armc antibu y Fluorochromes used for immunofluorescence FITC fluorescein isothiocyanate Texas red CyT39V39S Confocal microscopy And you can use immunofluorescence to determine where proteins exist in the cell during cell cycle as shown here Green indicates where the Pim1 kinase exists and the red indicates Where the DNA Observing the distribution of proteins during myeloid cell differentiation M M Cells stained wuh Fluorescence can also be used in another x widely used technique called flow cytometry A a nm A B39cells Flow cytometry can be used To identify cells ngm A24 Immunnhialagy Ema Garland Science Inns Markers common To certain leukemias used in flow cytometry analysis An ALL ofthe pre B lineage the most commonly occurring ALL CD10 a metalloproteinase CD19 quotNv B H r t a ce co ecep or markemf hematopoietic precursors Markers common To certain leukemias used in flow cytometry analysis ALL ofthe T lineage CDS CD4 coreceptor coreceptor fOTMHCl forMHCII an MHC classl like molecule CD2 an adhesion 7 molecule CD5 a Tcel marker Markers common To certain leukemias used in flow cytometry analysis A B lineage CLL MHCH CD44 adhesion molecule CD23 lowaffinity lgE receptor CD19 c020 CD5 Bcell marker 20 How analysis of a blood lymphocyte sample would be done Green photomulllpller 0 tube PMT stream at uid 3 containing anlibody 1 labeled cell Red PMT Side scatter Laser Forward scatter What you see in the analysis of cells stained with antibodies to two different surface antibodies igM Dot plots 10W 5 ndard 1W quot in I i i 01 i i i I 1 ll 1W I lll39l 01 l 0 HID 15W 19 10W 1 Colordensxly mu W l y I D 9 M 1 I 10 mo woo 01 1 10 woo woo Flgure AIS pm 1 a 1 lmmunablalogh a lo inland same was 21 This might be considered to be the most widely used immunotechnique Immunoblotting also known as Western blotting l sepantiun gel anngen samples nevus and fl aulorsdiograph antigen blinds visuallia hlolling xank immunoslaining ol blot lransli r DEDlI ES lo numceuuiuse sheel b all aulovadiogranhy Immunoblotting AKA Western blotting O W 22 By treating cells with tanic acid one can attach other proteins to the surface of the RBC Prescription drugs lattice WQ es sera red cell H amigens l sensiti ng an gen an hady i reciprocal serum dilution k n v 3 3951 a 19 1 a 6 199 new ImanDU Where the mat settles one can assume there was an antibodyantigen interaction that allowed a lattice to form Kuby chapter6 23 Immunoelectmphonesis was one of the first tiltnimin it n h luhulnia m 1 g nigh J lligrntnni diminu How immunoelec rrophoresis is used today clinically Whyis this nding important What does it tell us Normal Myeloma Deniitometric trace A dz i z Y Aa1u2 1 2Mband Protein elec trophoresis A01 12 ml Y A l11t12 1 2 Mband 25
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