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

Macrophages, B-Cells & T-Cells Immunology Macrophages, B-Cells & T-Cells 1.) Macrophage (type of white Blood Cell) engulfs antigen particle. B - Cell T Cell 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. Macrophage B - Cell 5.) B-Cell is activated: B-Cell divides & divides. Antibodies are produced. T Cell T Cell 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. 2.) Macrophage displays antigen part of antigen particle on endosome (outer coat). T Cell Macro phage B - 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.

Structure of Immunoglobulin (Ig) Steve Harris SPASH - Biotechnology

Fragment Constant region (Fc) Hinge Region Fragment Antigen Binding region (Fab) (up to 1x106 different recognition sites possible) Light Chain (chromosome 12) -S-S- -S-S- Fragment Constant region (Fc) Heavy Chain (chromosome 6) -S-S- -S-S- Disulfide Bonds

1 TRILLION Antibody combinations 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) IgG (mammals) IgM (mammals) IgA (mammals) IgE (mammals) IgD (mammals) 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 Largest Mammalian classes IgG - Only Isotype able to cross the placenta membrane

Examples of Immunology in Action Rapid strep. Test (streptococcus (A)) Looking for antigen. 10-15min. Accurate ¾ of time. False negatives Strep. B, C, D, etc. Master gargling. Taking old medications. HIV – (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.

Concentration Gradient Centrifugation Small Molecules IGY Large Molecules 13,500 RPM pellet Supernatant Concentration Gradient [PPT A] IGY Low [PEG] High [PEG] pellet Supernatant [PPT B] IGY pellet Supernatant IGY Resuspended And Purified IGY