A501: Methods in Reproductive
Diversity (Fall 2006)
Oct. 31st Lab: Measuring immunity
So you think you want to
measure immunity?
Why measure immunity?
 Important physiological system in its own right
 Measure of fitness, somatic development
 To test for disease in your colony/population
 Tool for generating new technologies
(e.g., antibodies, viral vectors)
Types of Immunity
Overall Immunity
Physical
Barriers
Innate
Acquired (adaptive)
Humoral
Skin
Mucus
Macrophages
Complement
Natural Killer cells
B-cells
(antibodies)
Cell-mediated
T-cells
Types of Immunity
Overall Immunity
Physical
Barriers
Innate
Acquired (adaptive)
Humoral
Skin
Mucus
Macrophages
Complement
Natural Killer cells
B-cells
Cell-mediated
T-cells
Antimicrobial Peptides
Why: Determine antimicrobial protein concentration or
killing capacity of AMPs
How: Stimulate skin production of AMPs and swab skin surface
Need: skin surface; adrenaline to induce proteins
Equipment: Microplate reader or fluorometer or laminar flow hood
protein assay
In vitro assay
solvent
Types of Immunity
Overall Immunity
Physical
Barriers
Innate
Acquired (adaptive)
Humoral
Skin
Mucus
Macrophages
Complement
Natural Killer cells
B-cells
Cell-mediated
T-cells
Hemolytic Complement Assay
Why: To determine lytic capacity of serum complement
How: Assesses ability of serum complement to lyse RBCs
Need: Whole blood or serum; No species-specific antibodies
Equipment: Microplate centrifuge baskets; plate reader
Natural Killer (NK) Cytotoxicity
Why: To determine lytic capacity of NK cells
How: Assesses ability of NK cell to lyse
chromium-fixed cells
Need: Whole blood or serum; No specific antibodies
Equipment: scintillation counter
C
C C C
C
C
C
C
C
Chromium (Cr51)
fixed lymphocytes
NK
NK
NK
C
NK
C C C
NK C
NK
C NK C
C
C
Add isolated
NK cells
CC C C C C
C NK
C
NK
NK
Measure liberated
chromium
(NK Cytotoxicity)
Types of Immunity
Overall Immunity
Physical
Barriers
Innate
Acquired (adaptive)
Humoral
Skin
Mucus
Macrophages
Complement
Natural Killer cells
B-cells
Cell-mediated
T-cells
Lymphocyte Proliferation
Why: Measure ability of lymphocytes to undergo mitosis in
vitro
How: cells cultured in nutrient-rich media w/ or w/o mitogen
Mitogens:
– Concanavalin A (Cona A) [T cells]
– Phaseolus hemagglinin (PHA) [mixed B and T Cells]
– Pokeweed mitogen [B Cells]
Need: Whole blood or liberated lymphocytes (spleen,
thymus, bursa)
Equipment: Laminar flow hood (AB Core, Demas Lab)
Delayed-Type Hypersensitivity (DHT)
Why: measure T-cell-mediated inflammatory response
non-invasively
How: Prime with antigen and measure swelling on challenge
PHA
DNFB
Need: Measurable surface on animal
(pinna, wing web, fin, foot pad)
Equipment: hand-held calipers
seals
Types of Immunity
Overall Immunity
Physical
Barriers
Innate
Acquired (adaptive)
Humoral
Skin
Mucus
Macrophages
Complement
Natural Killer cells
B-cells
Cell-mediated
T-cells
Antigenic Challenge
Why: Determine specific antibody (immunoglobulin levels)
How: Immunize animal with antigen; bleed ~10 days later
SRBC
KLH
Diptheria/tetananus vaccine
Need: serum samples, specific antibodies (ELISA)
Equipment: microplate reader/washer (AB Core; Demas; IMBI)
ELISA
Agglutination Assay
Types of Immunity
General (Integrated) Immunity
Physical
Barriers
Innate
Acquired (adaptive)
Humoral
Cell-mediated
Hematological Parameters
Why: Assess specific components of small blood sample
(hematocrit, macrophages, lymphocytes)
How: smear drop of blood across slide or add to hemocytometer
Need: small fresh blood sample
Equipment: slides, light microscope
Standard tissue slides
hemocytometer
Flow Cytometry
Why: Count specific immune subtypes and sort viable cells
How: Shoot samples through FACS machine; laser activated
tagged cells and counts based on size & granularity
Need: Tagged antibodies specific to cell subtypes (commercial)
Side Scatter (granularity)
Equipment: FACS scanner or other cell sorter (JH Core)
Forward Scatter (size)
Cytokine Assays
Why: Assess circulating levels of specific cytokines
How: EIA using blood samples or homogenized tissues
Need: Blood/tissue samples, specific antibodies (kits)
Equipment: Plate reader/washer or gamma counter
Lipopolysaccharide (LPS)
Why: Assess sickness response (fever, sickness behavior,
cytokine production)
How: Immunize animal with LPS to mimic bacterial infection
Need: LPS from bacteria (Sigma)
Equipment: thermometer,
behavior recording equip.
Wound Healing
Why: Assess ability for wounds to heal (coordinated immunity)
How: Perform punch biopsy on skin surface
Need: animal
Equipment: Micropunch biopsy tool; digital camera
Skin Graft Rejection
Why: To determine integrated immocompetence
How: Transfer small graft of skin from unrelated animal; track
time to reject tissue (necrosis)
Need: skin surface; unrelated individuals
Equipment: template to estimate % rejection; digital camera
0%
25%
75%
Disease/Sickness
Assessment of Ectoparasites
Why: Assess levels of parasitism in natural populations
How: Trap/catch individuals, dust with insecticide and collect
parasites. Quantify using microscope
Need: animal; fast-acting insecticide
Equipment: microscope
In Vitro Bacterial Killing
Why: Assess ability of natural antibodies and complement to kill
pathogen in a dish
How: Coat plates w/ bacterial colonies & add serum
Need: animal; pathogen (Sigma), agar-coated plates
Equipment: incubator
Serum added
E. coli
24-hr
incubation
Infectious Disease Models
Why: Assess actual disease susceptibility in experimental setting
How: Inoculate animal with known amount of pathogen; quantify
(% infection, % parasitemia, immune responses, survival)
Need: pathogen (rhinovirus, plasmodium, pneumococcus)
Equipment: BSL 2 or 3 facility (pathogen-dependent)
rhinovirus
Plasmodium
Pneumococcus
Measuring anti-KLH antibodies with
Indirect Sandwich ELISA
Measuring anti-KLH antibodies with
an Indirect Sandwich ELISA
Goal:
Measure a specific antibody response
Study:
Effects of food schedules on antibody responses
Hypothesis: Food-limited rats that weigh less
will display reduced humoral
immunity compared with ad lib rats.
Manipulation: Rats fed ad lib or meal-fed (4 wks)
then immunized with antigen keyhole
limpet hemocyanin (KLH)
keyhole limpet
Hypothesis: Meal-fed rats will consume less food and will
display reduced humoral immunity
Test:
Measure serum anti-KLH immunoglobulin G
What was Done Before This Lab
Y
Ad lib &
Meal-Fed Rats
Immunized w/ KLH
Blood samples (D10) drawn
Sampled centrifuges, serum removed and stored
Step 1. 96-well microtiter plates coated
with KLH
KLH
96-well microtiter plate
Single well
Plate
washed
Step 2. Plates blocked with a milk-blocking
buffer to reduce non-specific binding.
KLH-coated
microplate
Remaining gaps
coated
Step 3. Plates must be coated with your
serum samples.
negative control
No anti-KLH
antibodies
Y
Y
Y
Wash plates
positive control
A lot of anti-KLH
antibodies
(removes unbound
antibodies)
YYY YYY
YYY
Y
Y
Serum samples
added
Y
Y
Anti-KLH
Antibodies in
serum samples
(rat anti-KLH)
Step 4. After 3-hr. incubation, a secondary
antibody (goat-anti rat IgG) will be added to
plates.
2ndary antibody
(goat anti-rat IgG)
(conjugated to AP)
Y
Y
Y
Y
KLH
Anti-KLH IgG
in blood samples
Y YY
Y
Y
YY
Step 5. Substrate buffer (pNPP) is added to
trigger a biochemical colorimetric reaction.
Color
Y
Y
Y
Y
Y
Y
substrate
added
Color
reaction
Step 6. Mircotiter plates will be are read at
405 nm to quantify reaction
plate reader
Step 7. Determining the antibody titer in
serum samples.
A % Plate Positive value will be calculated for each sample
The values for each groups used in statistical calculations to
test our hypothesis
Tuesday Immunology Lab
1. Start ELISA on rat study
DURING SECONDARY INCUBATION INTERMISSION
2. Visit to flow cytometry core facility
3. Learn to perform a DTH wing-web swelling
assay in birds
4. Finish ELISA/Analyze Results