1. 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)

2. 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.

3. 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

4. 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.

5. Structure of Immunoglobulin (Ig)
Steve Harris
SPASH - Biotechnology

6. 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

7. 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

8. 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)

9. 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.

10. 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