Biotechnology Immunology Past Paper PDF Fall 2023

Application of Immunology in Biotechnology IMMUNOLOGY | BIOT 203 FA L L 2 0 2 3 | W E E K 1 2 D R . J O N AT H A N M E M M E Fall 2023 | J.Memme IMMUNOLOGY | BIOT 203 1 Application of Immunology in Biotechnology TOPICS LEARNING OBJECTIVES Immunological Techniques Discuss immunological techni...

Application of Immunology in Biotechnology IMMUNOLOGY | BIOT 203 FA L L 2 0 2 3 | W E E K 1 2 D R . J O N AT H A N M E M M E Fall 2023 | J.Memme IMMUNOLOGY | BIOT 203 1 Application of Immunology in Biotechnology TOPICS LEARNING OBJECTIVES Immunological Techniques Discuss immunological techniques. Applications of Immunology in Treatment Paradigms Differentiate between different ELISA techniques. Analyze and interpret data collected from various immunological techniques. Fall 2023 | J.Memme IMMUNOLOGY | BIOT 203 2 Antibodies and the Counterparts Antibodies Antigens Immunoassay • Globulin proteins that are found in blood and are used by the immune system to identify and neutralize foreign objects, such as bacteria and viruses • also known as immunoglobulins abbreviated Ig) • A substance that stimulates the production of an antibody when introduced into the body • i.e. is detectable and recognized by specific antibodies • A laboratory technique that makes use of the binding between an antigen and its homologous antibody in order to identify and quantify the specific antigen or antibody in a sample Fall 2023 | J.Memme IMMUNOLOGY | BIOT 203 3 RECALL: Anatomy of an Antibody Antibody Structure: ◦ Fab: Fragment antigen-binding ◦ Fc: Fragment constant region ◦ Can be conjugated to fluorescent molecules or enzymes, that allow detection or be combined with other molecules for a variety of biotechnological applications Fab Fc HRP FITC Fall 2023 | J.Memme IMMUNOLOGY | BIOT 203 4 POLYCLONAL Ab MONOCLONAL Ab Commercial Antibodies ANTIBODY PRODUCTION https://www.ptglab.com/news/blog/polyclonal-vs-monoclonalantibodies/#:~:text=To%20answer%20different%20research%20needs,o ne%20epitope%20(Figure%201.) Fall 2023 | J.Memme https://www.researchgate.net/publication/320707210_Difference_Betwe en_Monoclonal_and_Polyclonal_Antibodies# IMMUNOLOGY | BIOT 203 https://www.infors-ht.com/en/blog/monoclonal-antibodies-from-screening-toproduction/ 5 Immunological Techniques Antigen-antibody interaction Identification and characterization of cell populations Isolation of cell populations Lymphocyte function Fall 2023 | J.Memme • High specificity of antibodies • Identify, isolate, quantify particular antigens • Immunoelectrophoresis • Mainly based on surface markers* • Immunofluorescence • Immunohistochemistry • According to surface markers • Density dependent centrifugations • Fluorescence activated cell sorting (FACS) • Antibody or cytokine production • Proliferation in response to antigen • Cytotoxicity IMMUNOLOGY | BIOT 203 6 Laser Scanning Confocal Microscopy High resolution imaging Depth sensitivity allows for optical sectioning ◦ View sections and create 3D images Want to know more? ◦ https://www.youtube.com/watch?v=QFtZFbug1SA ◦ https://www.youtube.com/watch?v=--9F1Xa2I_A By ZEISS Microscopy from Germany - ZEISS LSM 800 with Airyscan: Your Compact Confocal Power Pack, CC BY-SA 2.0, https://commons.wikimedia.org/w/index.php?curid=45625344 Fall 2023 | J.Memme IMMUNOLOGY | BIOT 203 7 Laser Scanning Confocal Microscopy Fall 2023 | J.Memme IMMUNOLOGY | BIOT 203 8 2 Denature & negatively charge protein in SDS 4 5 6 7 8 9 kDa 250 150 100 75 SDS Sodium Dodecyl Sulfate 1 2 3 Migration 1 samples er la dd Western Blot Electrophoresis SDSPAGE 50 + 37 3 Electrotransfer to nitrocellulose membrane Transfer plates Sponge Whatman Gel Nitrocellulose Pads Paper membrane 25 20 15 10 SDS Polyacrylamide Gel Denatured Proteins Negatively Charged 5 4 Protein Detection and Imaging 1 2 3 4 5 6 POI Nitocellulose Membrane Apply enzyme substrate, or expose blot to lightsensitive film or computerized imager I. Size and intensity (darkness) of a band indicates its relative abundance within the sample Fall 2023 | J.Memme Incubate: Primary antibody (1o Ab) against protein of interest kDa - 50 - Immunoblotting M ig r ati on Native Proteins + Incubate: Secondary 2o Ab against IgG of 1o Ab Nitocellulose Membrane IMMUNOLOGY | BIOT 203 9 Enzyme-linked Immunosorbent Assay ELISA Plate-based detection assay ◦ Peptides, Proteins, Antibodies, Hormones Detection and quantification ◦ Enzyme-based (colourometric) determination using spectrophotometer ◦ Fluorescence-based (fluorometric) determination using a fluorometer By Jeffrey M. Vinocur - Own work, CC BY 2.5, https://commons.wikimedia.org/w/index.php?curid=729439 Fall 2023 | J.Memme IMMUNOLOGY | BIOT 203 10 Competitive HRP: Horseradish peroxidase, enzyme that amplifies the signal in photometric assays https://youtu.be/nNjlBCnpGZ4?si=q6crohhUe7f95asj • Antigen competes for antibody binding site with an antigenic reagent • Inverse correlation between antigen concentration and substrate yield • https://youtu.be/Kb26nQVMHds?si=y4mDw p2fdwGx3fY- ELISA techniques Substrate Substrate Primary antibody Coloured product Secondary antibody Coloured product Coloured product Coloured product Substrate HRP Secondary antibody HRP HRP HRP Secondary antibody Detection primary antibody Antigen Primary antibody Antigen Substrate Antigen Capture primary antibody Primary antibody Antigen Direct Indirect Sandwich • Labeled primary antibody reacts directly with the antigen • Labeled secondary antibody is used for detection • Indirect antibody capture • Indirect antigen capture • Two antibodies sandwich antigens • Correlate antigen concentration and substrate response Fall 2023 | J.Memme IMMUNOLOGY | BIOT 203 11 ELISA Techniques https://www.cusabio.com/c-20659.html#:~:text=In%20direct%20and%20indirect%20ELISA,antibody%20is%20called%20capture%20antibody. Fall 2023 | J.Memme IMMUNOLOGY | BIOT 203 12 ELISA Data Qualitative ◦ Positive/negative results, compared to a blank well containing no antigen Quantitative ◦ Sample data interpreted in comparison to a standard curve to report concentrations ◦ Standard curve ◦ optical density vs log concentration ◦ Antigen standards of known concentrations are used to produce a standard curve, which is then use to determine the concentration of a sample Semi-quantitative ◦ Intensity of signal correlates to antigen concentration ◦ compare the relative levels of antigen in assay samples – i.e. control vs experimental groups Fall 2023 | J.Memme IMMUNOLOGY | BIOT 203 13 ELISPOT A highly sensitive immunoassay that measures the frequency of cytokine-secreting cells at the single-cell level. ◦ cells are cultured on a surface coated with a specific capture antibody in the presence or absence of stimuli. Typically used with blood cells to study various treatments for disease Technique ◦ Tiny wells are used to contain the cells, with a variety of other substances. ◦ The substances cause colored spots to form near the cells that secrete the substances in question ◦ Manual counting (microscope) ◦ Automatic counting (using specialized equipment with computer vision capabilities) Sensitive à can detect even a single substance-producing cell. Versatile à can be tailored to detect a wide variety of very specific secretions. Fall 2023 | J.Memme IMMUNOLOGY | BIOT 203 14 Image: https://www.merckmillipore.com/INTERSHOP/static/WFS/MerckSite/-/Merck/en_US/Freestyle/BI-Bioscience/Cell-Culture/elispot-learningcenter/elispot-workflow.jpg ELISpot Workflow Coat membrane with capture antibody. Add immune cells and stimulate. Fall 2023 | J.Memme Responding cells produce cytokines. Cytokine of interest binds to the capture Ab beneath the cell. Wash to remove cells. Add streptavidinAdd a second enzyme cytokineconjugate (i.e. specific Ab HRP). which binds to the cytokine-Ab complex. IMMUNOLOGY | BIOT 203 Add enzyme substrate and develop. Within a well, each responding cell will result in the development of one spot. 15 Images: https://www.sigmaaldrich.com/deepweb/assets/sigmaaldrich/marke ting/global/images/technical-documents/articles/proteinbiology/elisa/figure6-ifny-elispot-pbmc/figure6-ifny-elispot-pbmc.jpg ELISpot Example IFNγ Elispot • 384-well plates on four seeding densities of lymphocytes (T cells, B cells, NK cells) and monocytes • Well F12 magnified to demonstrate spot clarity. Results • IFNγ spot forming units (SFU) plotted against seeding density. • The assay shows a strong linear response for range of cell quantities tested. Fall 2023 | J.Memme IMMUNOLOGY | BIOT 203 16 https://www.bio-rad.com/en-ca/applicationstechnologies/multiplex-immunoassays?ID=LUSM0E8UU Multiplex Arrays 1 well Based on the principle of ELISA combined with Flow Cytometry It’s like running 10’s and 100’s of ELISAs in one go!!! Fall 2023 | J.Memme IMMUNOLOGY | BIOT 203 17 https://www.google.com/url?sa=i&url=https%3A%2F%2Fwww.bio-rad.com%2Fen-us%2Fapplications-technologies%2Fbio-plex-multipleximmunoassays%3FID%3DLUSM0ZMNI&psig=AOvVaw3I5S8iLii4edZlpdwnDPsb&ust=1700683896422000&source=images&cd=vfe&opi=89978449&ved=0CBQQjhxqFwoTCJC37q3z1YIDFQAAAAAdAAAAABAh Multiplex Assay Applications Cytokines Chemokines Infectious Diseases Autoimmunity Metabolic Diseases Fall 2023 | J.Memme IMMUNOLOGY | BIOT 203 18 Case study… Flow Cytometry Efficiently detects and quantifies multiple physical and chemical characteristics of cells within a heterogeneous suspension by optical means Applications ◦ measuring cell size, apoptosis, cell surface antigens, cytokine production, cell division, etc. VIDEO: https://www.youtube.com/watch?v=kue4zsMzJQ0 By SariSabban - Sabban, Sari (2011) Development of an in vitro model system for studying the interaction of Equus caballus IgE with its high- affinity FcεRI receptor (PhD thesis), The University of Sheffield, CC BY-SA 3.0, https://commons.wikimedia.org/w/index.php?curid=18139883 Fall 2023 | J.Memme IMMUNOLOGY | BIOT 203 19 By Kwz at Polish Wikipedia, CC BY-SA 3.0, https://commons.wikimedia.org/w/index.php?curid=9534446 By Kwz at Polish Wikipedia, CC BY-SA 3.0, https://commons.wikimedia.org/w/index.php?curid=9534450 Number of Events (counts) Flow Cytometry Data Analysis HISTOGRAM Displays frequency distribution for a single value of a data set Variable A Variable B High B SCATTERPLOT Displays values for two variables for a data set Background Signal Fall 2023 | J.Memme IMMUNOLOGY | BIOT 203 Low A&B High A&B High A Variable A 20 By Kwz at Polish Wikipedia, CC BY-SA 3.0, https://commons.wikimedia.org/w/index.php?curid=9534446 Light Scattering Side Scatter (SSC) Granularity of cells ◦ Relative granularity (shape) or cells Forward Scattering (FCS) ◦ Relative size of cells Size of cells Image: http://2010.igem.org/wiki/images/thumb/f/f4/Fsc.ssc.JPG/600px-Fsc.ssc.JPG Fall 2023 | J.Memme IMMUNOLOGY | BIOT 203 21 Light Scattering Side Scatter Projection 800 0 200 400 600 Forward Scatter Forward Scatter Projection 1000 Human white blood cells https://www.google.com/url?sa=i&url=https%3A%2F%2Fsocratic.org%2Fquestions%2Fwhat-is-the-differencebetween-monocytes-neutrophils-eosinophils-lymphocytes-and&psig=AOvVaw3kKdWOlU3WnaXmRVUPP1w&ust=1700691698597000&source=images&cd=vfe&opi=89978449&ved=0CBQQjhxqFwoTCPjLmraQ1oI DFQAAAAAdAAAAABAe Fall 2023 | J.Memme 0 IMMUNOLOGY | BIOT 203 200 400 600 Side Scatter 800 1000 22 Fluorescence intensity is proportional to the number of binding sites Fluorescence ◦ More binding sites = more intense Fluoresce labeling ◦ fluorescent labeled antibodies recognize target (antigen) on cell surface Relative fluorescence intensity ◦ Markers (i.e.. CD#) Fall 2023 | J.Memme IMMUNOLOGY | BIOT 203 23 XF Analysis Extracellular Flux Analyzer LIVE cell measurements in real time ◦ oxygen consumption rate (OCR) ◦ extracellular acidification rate (ECAR) Immune cell activation ◦ Metabolic pathways and immune response ◦ T-cell, macrophage, neutrophil activation https://www.agilent.com/en/products/cell-analysis/seahorseanalyzers/cell-analysis-measuring-cell-metabolism-transientmicro-chamber Fall 2023 | J.Memme IMMUNOLOGY | BIOT 203 24 Announcements IMPORTANT Preparation Introduction to the Case Study will be available on Blackboard Case Study Fall 2023 | J.Memme Review this material before the beginning of the next class. IMMUNOLOGY | BIOT 203 Group Project 25 Case Study IMMUNOLOGY | BIOT 203 Fall 2023 | J.Memme IMMUNOLOGY | BIOT 203 26 Case Study IMPORTANT PREPERATION Introduction to the Case Study is available in the Unit Learning Material Read the introduction before the beginning of the next class. Too Many B Cells: Chronic Lymphocytic Leukemia and the Role of Flow Cytometry Objectives ◦ Explain the principle of flow cytometry and how it is applied to leukemia diagnosis ◦ Interpret flow cytometry data ◦ Distinguish basic immune cell types based on size and granularity Format The case study has been modified from the original source and used according to “fair use” guidelines: Debby Walser-Kuntz (2011). National Center for Case Study Teaching in Science, University at Buffalo, State University of New York. Fall 2023 | J.Memme ◦ Case study file posted in the Assessments folder on Blackboard ◦ Review the supplied case study ◦ Submit responses on Blackboard (9 questions) using the template provided (save as Word or PDF format) IMMUNOLOGY | BIOT 203 27 Figure 2 Consider the following… - B cells: CD20, CD19 - T cells: CD3, CD4 or CD8 - Consider other markers, relevant to identification and diagnosis. Fall 2023 | J.Memme IMMUNOLOGY | BIOT 203 28 Figure 3 Positive population Consider the following… Negative population Fall 2023 | J.Memme IMMUNOLOGY | BIOT 203 29 Figure 4 🗸 CD8 X CD4 Consider the following… 🗸 CD8 🗸 CD4 X CD8 X CD4 X CD8 🗸 CD4 Cells expressing CD4 Fall 2023 | J.Memme IMMUNOLOGY | BIOT 203 30 Granularity of cells Figure 5 Consider the following… Size of cells Consider size and granularity of: • Lymphocytes • Neutrophils • Monocytes Fall 2023 | J.Memme IMMUNOLOGY | BIOT 203 31 Progress Tracking Assessment #10 Available on Blackboard in Unit Learning Materials ◦ You are in encouraged to collaborate with your peers but must submit your own individual product. nucleic ◦ Submit via Blackboard using theacidassignment submission link before the end of the day based testing serologybased testing Fall 2023 | J.Memme IMMUNOLOGY | BIOT 203 COVID19 testing & screening antigenbased testing 33 PTA#10 Research the three listed methods for COVID-19 Testing & Screening Prepare an organized table that provides the following information for each of the three methods: • Identify the routine (typical) use • Describe the test/technology use to confirm the presence of nucleic acid, antigen, or antibody in the sample COVID-19 Testing & Screening Te Antigenbased testing Serologybased testing Routine Use Test Technology Fall 2023 | J.Memme IMMUNOLOGY | BIOT 203 34