A501: Methods in Reproductive Diversity (Fall 2006) Oct. 31 st Lab: Measuring immunity

So you think you want to measure immunity? Why measure immunity? Important physiological system in its own right Important physiological system in its own right Measure of fitness, somatic development Measure of fitness, somatic development To test for disease in your colony/population To test for disease in your colony/population Tool for generating new technologies Tool for generating new technologies (e.g., antibodies, viral vectors) (e.g., antibodies, viral vectors)

Types of Immunity Overall Immunity Physical Barriers Innate Acquired (adaptive) HumoralCell-mediated Skin Mucus Macrophages Complement Natural Killer cells B-cells (antibodies) T-cells

Types of Immunity Overall Immunity Physical Barriers Innate Acquired (adaptive) HumoralCell-mediated Skin Mucus Macrophages Complement Natural Killer cells B-cellsT-cells

Antimicrobial Peptides Why: Determine antimicrobial protein concentration or killing capacity of AMPs 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) HumoralCell-mediated Skin Mucus Macrophages Complement Natural Killer cells B-cellsT-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 C C CC C C C C C C C CC C C C C C C C C C C C C C Chromium (Cr 51 ) fixed lymphocytes Measure liberated chromium (NK Cytotoxicity) Add isolated NK cells Why: To determine lytic capacity of NK cells How: Assesses ability of NK cell to lyse chromium-fixed cells chromium-fixed cells Need: Whole blood or serum; No specific antibodies Equipment: scintillation counter NK NK NK NK NK NK NK NK NK NK

Types of Immunity Overall Immunity Physical Barriers Innate Acquired (adaptive) HumoralCell-mediated Skin Mucus Macrophages Complement Natural Killer cells B-cellsT-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) HumoralCell-mediated Skin Mucus Macrophages Complement Natural Killer cells B-cellsT-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) HumoralCell-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 hemocytometer Standard tissue slides

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) Equipment: FACS scanner or other cell sorter (JH Core) Forward Scatter (size) Side Scatter (granularity )

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 E. coli Serum added 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) rhinovirusPlasmodiumPneumococcus

Measuring anti-KLH antibodies with 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) Hypothesis: Meal-fed rats will consume less food and will display reduced humoral immunity Test:Measure serum anti-KLH immunoglobulin G Measuring anti-KLH antibodies with an Indirect Sandwich ELISA keyhole limpet

What was Done Before This Lab Y Y Y Y Ad lib & Meal-Fed Rats Immunized w/ KLH Blood samples (D10) drawn Sampled centrifuges, serum removed and stored

Step well microtiter plates coated with KLH KLH 96-well microtiter plate Single well Platewashed

Step 2. Plates blocked with a milk-blocking buffer to reduce non-specific binding. KLH-coatedmicroplate Remaining gaps coated

Step 3. Plates must be coated with your serum samples. Y Y Y Y Y Y Y Y YY Y Anti-KLH Antibodies in serum samples (rat anti-KLH) negative control No anti-KLH antibodies positive control A lot of anti-KLH antibodies YYY YYY Y Y Y Serum samples added Wash plates (removes unbound antibodies)

Step 4. After 3-hr. incubation, a secondary antibody (goat-anti rat IgG) will be added to plates. Y Y YY Y Y Y Y Y Y Y KLH Anti-KLH IgG in blood samples 2ndary antibody (goat anti-rat IgG) (conjugated to AP) YY Y Y

Step 5. Substrate buffer (pNPP) is added to trigger a biochemical colorimetric reaction. Y Y Y Y Y Y Color substrateadded Colorreaction

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