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Immunology Field Trip BTCI (parent company Promega) – Madison November, 9th 2010 Cost: $40.00 ea (class 24) approximate cost Two Labs + Tour (Immunology & Transformation) Classroom 6:50am Leave:7:00:01.0am SHARP 9:00am in lab 12:00pm Lunch – bring lunch 2:45pm leave Madison Home 4:45pm ± 30min. BW’s? +$7 - $15.00 Home 5:30pm ± 45min. (as late as 6:15pm) Purpose Describe antibodies (ab) and antigens (ag) and how they work. Purify IgY antibodies from egg yolk and identify using the dot-blot method of immunodetection. Describe immune response and relate to vaccination. Explain how principles of immunology are used in clinical diagnostics. Background to Immunology Antigen not recognized as self Invasive/foreign to body Virus, Bacteria, protein, foreign material (allergies) Antibody Protein Produced by B-cells Act against antigen Immunology Macrophages, B-Cells & T-Cells 1.) Macrophage (type of white Blood Cell) engulfs antigen particle. 3.) Macrophage binds with correct B-Cell through matching Epitope (8 amino acids). 4.) T- Cells “Helper Cells” help B-Cells recognize part of antigin particle displayed on macrophage. T Cell B - Cell B - Cell Macrophage T Cell 5.) B-Cell is activated: •B-Cell divides & divides. •Antibodies are produced. B - Cell T Cell 2.) Macrophage displays antigen part of antigen particle on endosome (outer coat). 6.) Newly formed antibodies flood the body and begin attaching to the antigin particle. The attached antibodies draw attention to the antigen for engulfing by the macrophage, “Killer” T-Cells and Complement. T Cell T Cell T Cell 7.) Those B-Cells & T-Cells not involved in antibody production become memory cells & remain in the blood system on guard as part of the immune system against future antigens. While those B & T cells involved in fighting the antigen die out. T Cell Structure of Immunoglobulin (Ig) Steve Harris SPASH - Biotechnology Fragment Antigen Binding region (Fab) (up to 1x106 Hinge Region different recognition sites possible) Light Chain -S-S- (chromosome 12) -S-SFragment Constant region (Fc) -S-S-S-S- Disulfide Bonds Heavy Chain (chromosome 6) Hyper-Variable Region (400 genes) Attachment to specific Antigens Joining Region (4 genes) Fab Region Diversity Region (15 genes) Fragment Constant Region (Fc) Isotypes IgY (birds) Largest IgG (mammals) Mammalian IgM (mammals) classes IgA (mammals) IgE (mammals) IgD (mammals) IgG - Only Isotype able to cross the placenta membrane Fragment Antigen Binding Region (106) x Hyper-Variable Region (400) x Diversity Region (15) x Joining Region (4) x Fragment Constant Region (6 minimum) x Heavy & Light Chain (2) x Secondary organization levels of mRNA (sloppy mRNA splicing) = 1 TRILLION Antibody combinations Examples of Immunology in Action Rapid strep. Test (streptococcus (A)) Looking for antigen. 10-15min. Accurate ¾ of time. False negatives HIV Strep. B, C, D, etc. Master gargling. Taking old medications. – (ELISA – Microplate twice, then PCR if positive twice on micro-plate) Pregnant test. Vaccinated against: Hepatitis, Chicken pox, Polio Measles, mumps, rubella - (MMR) diphtheria, tetanus, pertussis (whooping cough) - (DTP) Questions Define immunology. Describe Antigen (ag). Describe Antibody (ab). Briefly describe an immune response to an antigen. Briefly describe the process of purifying antibodies using centrifugation and concentration gradients. Briefly describe the ELISA technique. Centrifugation Small Molecules Large Molecules 13,500 RPM IGY pellet Supernatant Concentration Gradient [PPT A] IGY Low [PEG] pellet Supernatant [PPT B] pellet IGY Supernatant IGY Resuspended And Purified IGY High [PEG]