Laboratory Applications of Poultry Lecture and Lab Overview ● Lecture Topics ● Caveats to the lecture / Philosophy ● Research and medical uses of chickens.

Laboratory Applications of Poultry Lecture and Lab Overview

● Lecture Topics● Caveats to the lecture / Philosophy● Research and medical uses of chickens● Immune system overview● IgY characteristics● Antibody generation● Antibody purification● Enzyme linked immunosorbant assay (ELISA)

● Lab Section● Calibration● Pipette use

Caveats to this Lecture / Philosophy of Science

● Challenge to present overview of immunity – details

● How deep and wide is the ocean? How vast the universe?

– Number of species, types of matter, subparticles per given atomic particle?

– Of biology, how much do we really know, cure a cancer yet?

● Radiation, 5-FU by Charles Heidelberger 1957

● JQ1 molecule by Jay Bradner 2010

● Arrogance Vs Humility of Science

– “Myself” is the easiest person to fool

– 97% of our DNA: we don't know what it's for

● Things are not always as they are presented to be

– Just because a scientist says it does not make it so

● Not an excuse not to know the basic information building blocks!

Research and Medical Uses of Poultry● Res. & Med. Uses

– Protein Production– gene insertion under ovalbumen promotor

– protein expression in egg white

– Food Supplement - IgY● Chicken IgY anti H. pylori (ulcers) - yogurt

– K. Horie, J. Dairy Sci 87 (12): 4073–9– Sold in Asia

● Chicken IgY - Puppy supplement

– anti parvovirus, coronavirus, rotavirus salmonella and E. coli K88 & K99

– PRN Pharmaceuticals

Research and Medical Uses of Poultry

● Res. & Med. Uses continued– Food Supplement - IgY continued

● Protective against Rotavirus in the pig model

– Henri Salmon, PlosOne, August 3, 2012

– Humanized antibody production purification from eggs● Who? Why? Uses- Arthritis, MS, Cancer, Infectious Diseases,

Antivenins● Human Ig subclasses purified from human serum and injected to

patients missing a given subclass, still face regulatory & safety issues

● Methods of - gene k/o replacement● Challenges: Glycosylation, Fc receptors for Ig packaging in eggs,

alteration of Ig genes

Research and Medical Uses of Poultry

● Res. & Med. Uses continued– Humanized antibodies current art

● Mouse monoclonals: J&J, Merck-SGP, BMS, Regeneron, Genzyme, Genetec, Roche, many others

● Often expressed with humanized frame work, mouse complement determining regions (CDRs), some Abs are completely humanized

● Arthritis (J&J Remicade®), Regeneron (Eylea®) “wet” macular degeneration, cancer targets ongoing

● Difficulties with post translational modification, immunogenicity, rapid clearance

Immune System Overview● Innate / Nonspecific Immunity

– Mechanical Barriers - skin, hair, mucous

– High body temp

– Chemicals● fatty acids in sweat, gastric secretions, acidic pH, lysozyme,

phospholipase, egg proteins

– Biologicals● normal bacterial flora on skin, gut and feathers

– Complement System● classic and alternative pathways● Perforate cell membranes● opsonize

Immune System Overview● Innate / Nonspecific Immunity continued

– Coagulation systems● Platelet derived beta-lysin anti gram neg activity (mammalian)

– Lactoferrin and transferrin ● bind iron to sequester it from bacteria

● bacterial use iron, use proteins called siderophores to sequester from surrounding matrix

– Phagocytic cells (some specificity)● Macrophages, Natural killer (NK) cells, lymphokine activated (LAK) cells,

neutrophils(mammals), heterophils(avian), eosinophils, polymorphonuclear cells, dendritic cells, etc

● Can argue some specificity due to opsonization and other cellular protein cues given to the cells.

Immune System Overview● Specific / Adaptive Immunity

– Characteristics of Cellular and Humoral Immunity Branches● Cytokines● Positive and negative selection, self / non-self recognition● Antigen / peptide presentation

– Major histocompatibility complexes (MHC) I & II● Thymocytes (T-cell) & B Lymphocytes (B-cells)● T-cell receptors (TCRs) bind MHCs● Antigen recognition of peptides in MHCs by T-cells● B-cells produce antibodies that bind antigens / foreign “invaders”● B-cell affinity maturation – survival of fittest● B & T memory cell production

Immune System Overview● Specific / Adaptive Immunity continued

– MHC Presentation● All cells present intracellular and self peptides via MHC I

– Part of cellular manufacturing process– CD8+ T-cells bind MHC I– Some viruses & cancer cells down regulate MHC I production

● See Petersen, Morris, Solheim J. Immuno Nov 1, 2003, Vol 171, #9, pp4473-4478 - free text for MHC I cycling diagram

● Phagocytic cells present extracellular peptides via MHC II– Internalize matrix and plasma proteins, materials, debris, bacteria,

viruses via phagocytosis (Latin phagein for devour, kytos for cell, osis for process)

– Breakdown by both protease and acid in phagosomes– MHC II proteins incorporated into phagosomes and bind peptides

therein and are cycled to cell surface – CD4+ T-cells bind MHC II

Immune System Overview● Specific / Adaptive Immunity continued

– MHC Presentation Cont.● MHC with a peptide looks like hotdog bun holding a hotdog in it● Peptide AA length for MHC I & II varies, ~9-15 AAs● T-cell receptors (TCRs) bind MHCs containing peptides● TCR looks like the hand coming down to grab the hotdog bun

Immune System Overview● Specific / Adaptive Immunity: Cellular & Humoral

Components– Cellular Immunity: Thymocyte or T-cell based

● CD4+ T-cells (Helper)

– Binds MHC II– CD4+ cells are the “Quarterback” of specific immunity– Determine which branch of specific immunity is activated

● By cytokine release and protein protein cellular interactions– HIV K/Os the CD4s

Immune System Overview● Specific / Adaptive Immunity: Cellular & Humoral

Components continued– Cellular Immunity

● CD4+ T-cells (Helper) continued

– mechanism that triggers CD4+ determination of cellular or humoral response not entirely characterized

● CD4+ T-cell increases cellular or CD8+ activation via IL-2, 12 release.

● CD4+ T-cell activates B-cells via IL-2,4,5,6 release

Immune System Overview● Specific / Adaptive Immunity: Cellular & Humoral

Components continued– Cellular Immunity

● CD8+ T-cell (Killer)

– Binds MHC I– Can self activate by IL-2 release– Increased activation by CD4+ T-cells as described above– Recognizes non-self intracellular peptides– Releases perforin to kill infected cells

– creates pores / holes in cells● A small subset of activated CD4+ & CD8+ T-cells become memory T-cells

Immune System Overview● Specific / Adaptive Immunity: Cellular & Humoral

Components continued– Humoral

● B-cells, in chicken bursa bursa function discovered – B. Glick and his group discovered bursa function, 1956

● Produce antibodies– Opsonize (for phagocytosis), direct complement membrane attack

complex, inactivate molecules, viruses, cells– On surface of cell early in maturity

● Need CD4+ cytokines and Ig binding to activate● Maturation by cytokines and Ig binding affinity● Strongest binding cells survive as antigen levels decrease

– Survivors become memory B-cells

Chicken Antibodies● Ckns produce IgA, IgM & IgY (IgY is sometimes referred to as

chicken IgG)

● Only IgY is packaged in eggs

● Receptors mediate packing in ovarian follicle

– Mannose receptor family, Tesar, D. 2008

● IgY more similar to mammalian IgE - it can initiate an anaphylaxis response in ckns

– IgY binds basil, mast, heterophil(?) cells, they degranulate & release overload of histamines, immune & smooth muscle modulators,

– Respiratory and cardiac depression

● Glycosylated similar to mammalian IgA– IgY maybe early evolutionary precursor to mammalian IgG/IgA and IgE

Antibody Structure & Binding

IgA & IgMBinding / complement determining regions on Ig

IgG space filled model

hu IgG, hu IgE & av IgY

Images from various web sources

Duck

Chicken IgY● The name IgY has been proposed by Leslie and Clem in 1969. The authors

showed experimental data proving that IgY molecule is different from IgG ("Phylogeny of immunoglobulin structure and function" G. A. Leslie and L.W. Clem, Journal of Experimental Medicine, Vol. 130, No. 6: 1337-1352, 1969)

● Molecular mass [kDa] ~ 180 (light chain ~ 25 [kDa] each; heavy chain ~ 65-68 [kDa] each) Note: IgG MW ~ 150

● Isoelectric point 5.7 - 7.6 (6.6 +/- 0.9 Davalos-Patoja et al. 2000)

● Extinction coefficient (i.e. absorbance of a 10 mg/ml solution at 280 nm) are: in 0.3 M KCl=13.18; in 0.1 N NaOH=14.4; in 5M guanidine=12.7(Leslie and Clem 1969)

● Egg yields 80-100 mg/egg

● Equivalent total IgG antibodies in ~ 10 ml of serum

● Specific to epitope (reactive site) on antigen 1-2%

Chicken IgY● Glycosylation similar to that of human IgE (“Site-specific N-glycosylation of

chicken serum IgG”, Noriko Suzuki and Yuan C. Lee, Glycobiology, vol 14, No3, pp 275-292, 2004)

● Does not bind to cell surface Fc receptor (Larsson 1992, Schmidt et al. 1993)

● IgY is capable of mediating anaphylactic reactions in chickens, a function limited to IgE in mammals.

● IgY has long term stability (Larsson 1992) - 10 yrs at 4oC no loss of activity!

● Does not bind to rheumatoid factor (an inflammatory response marker) in blood (Larsson et al. 1988) 

● Does not activate mammalian complement factors (Larsson et al. 1992)This can be of a great advantage in case of assay development for mammalian serum samples.

Chicken IgY● Does not bind to mammalian cell surface Fc receptor ( Schmidt et al. 1993)

● Does not bind to protein A (Kronvall et al. 1974) or protein G (Akerström et al. 1985)

● Latex particles sensitized by IgY molecules do not aggregate by means of the rheumatoid factor (as is the case of IgG antibodies). Moreover IgY-latex complexes have higher colloidal stability than IgG at pH 8 (L.Davalos-Pantoja et al. 2000)

● IgY antibodies are selectively, in large amounts passed to egg yolk and therefore NO IgM and IgA impurities can be found in IgY preparations (Schade et al. 2001)

● Might bind three to five times more secondary antibody (Horton et al. 1984)

Antibody Induction / Immunization in Chickens

● Antigen Preparation– Antigen (Ag) is the molecule elicits immune response

– Epitope is site on antigen where antibody binds

– Modify antigen to be injected● Haptens: molecules attached to antigen that activate a

delayed hypersensitivity response, i.e., really activates T-cells. Eg: Keyhole limpet hemocyanin (KLH), urushiol, the toxin found in poison ivy, biotin, digoxigenin, etc.

● By activating nearby T-cells, any B-cells w/ Ig that binds the protein will be activated by T-cell released cytokines

Antibody Induction / Immunization in Chickens

● Schedule– Start at age 2mos

– Days 0, 14, 21, 40, 50, 60

– Age 2 mos, egg production at 4 mos of age

– Adjuvants: material antigen is mixed into● Immune irritants● Freund's complete adjuvant 1st injection (contains oil and

killed mycobacterium)● Freund's Incomplete adjuvant (just oil) after the 1st injection● Named after Jules T. Freund

IgY Purification from Eggs● PEG or Ammonium Sulfate

– PEG method (most common)

– Precipitation of lipids and centrifugation in 3 or 3.5% PEG (~PEG 6000)

– Supernatant transfer to new tube and adjust to 12% PEG

– Spin

– Resuspend pellet in PBS, then bring volume up and PEG to 12%

– Spin

IgY Purification from Eggs● PEG method continued

– Resuspend pellet in PBS, dialyze

– Store -20C, not -70C due to stability issues

– http://www.jove.com/video/3084/igy-technology-extraction-chicken-antibodies-from-egg-yolk

ELISA Formats

ThermoScientific

Capture/Sandwich ELISA Method

Epitomics

Sandwich ELISA is often the most sensitive format

Steps with wash in between:1. Coat w/ Capture Ab2. Block3. Sample4. Detection/Primary Ab5. Secondary Ab labeled w/ HRP6. Substrate7. Read

1&2 3 4 5 6

ELISA Plate & Plate Reader

Example Standard Curve

Use sample OD and line equation to solve for concentration (x)

Note: the plate on left is a protein assay shown for illustrative purposes. An ELISA using TMB Substrate and acid stop solution is yellow.

Recommended Reading● Avian Immunology, Davidson, Kaspers & Schat, 2008

● Immunology, Abbas Lichtman, 2004

● Eppendorf Userguide Liquid Handling 21 No 020

(type into google search bar)

Lab Topics● Calibration

● Pipetting techniques

● Forward pipetting (+ tip blow out)● Reverse pipetting (- tip blow out, some liquid remains)● Eppendorf userguide 21 No 020 - on line

● ELISA plate and brief demo