Transcript Slide 1

Immunology Field Trip
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BTCI (parent company Promega) – Madison
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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.
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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.
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Background to Immunology
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Antigen
not recognized as self
 Invasive/foreign to body
 Virus, Bacteria, protein, foreign material
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(allergies)
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Antibody
Protein
 Produced by B-cells
 Act against antigen
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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
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strep. Test (streptococcus (A))
Looking for antigen.
10-15min.
Accurate ¾ of time.
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False negatives
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 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:
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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.
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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]