Download Laboratory Applications of Poultry Lecture and Lab Overview

Laboratory Applications of Poultry
Lecture and Lab Overview
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Lecture Topics
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Caveats to the lecture / Philosophy
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Research and medical uses of chickens
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Immune system overview
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IgY characteristics
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Antibody generation
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Antibody purification
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Enzyme linked immunosorbant assay (ELISA)
Lab Section
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Calibration
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Pipette use
Caveats to this Lecture / Philosophy of
Science
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Challenge to present overview of immunity – details
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How deep and wide is the ocean? How vast the universe?
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Number of species, types of matter, subparticles per given atomic particle?
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Of biology, how much do we really know, cure a cancer yet?
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Radiation, 5-FU by Charles Heidelberger 1957
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JQ1 molecule by Jay Bradner 2010
Arrogance Vs Humility of Science
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“Myself” is the easiest person to fool
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97% of our DNA: we don't know what it's for
Things are not always as they are presented to be
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Just because a scientist says it does not make it so
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Not an excuse not to know the basic information building blocks!
Research and Medical Uses of Poultry
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Res. & Med. Uses
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Protein Production
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gene insertion under ovalbumen promotor
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protein expression in egg white
Food Supplement - IgY
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Chicken IgY anti H. pylori (ulcers) - yogurt
K. Horie, J. Dairy Sci 87 (12): 4073–9
– Sold in Asia
Chicken IgY - Puppy supplement
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anti parvovirus, coronavirus, rotavirus salmonella and E. coli K88
& K99
PRN Pharmaceuticals
Research and Medical Uses of Poultry
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Res. & Med. Uses continued
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Food Supplement - IgY continued
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Protective against Rotavirus in the pig model
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Henri Salmon, PlosOne, August 3, 2012
Humanized antibody production purification from eggs
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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
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Res. & Med. Uses continued
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Humanized antibodies current art
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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
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Innate / Nonspecific Immunity
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Mechanical Barriers - skin, hair, mucous
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High body temp
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Chemicals
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Biologicals
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fatty acids in sweat, gastric secretions, acidic pH, lysozyme,
phospholipase, egg proteins
normal bacterial flora on skin, gut and feathers
Complement System
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classic and alternative pathways
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Perforate cell membranes
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opsonize
Immune System Overview
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Innate / Nonspecific Immunity continued
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Coagulation systems
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Platelet derived beta-lysin anti gram neg activity (mammalian)
Lactoferrin and transferrin
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bind iron to sequester it from bacteria
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bacterial use iron, use proteins called siderophores to sequester from
surrounding matrix
Phagocytic cells (some specificity)
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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
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Specific / Adaptive Immunity
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Characteristics of Cellular and Humoral Immunity Branches
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Cytokines
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Positive and negative selection, self / non-self recognition
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Antigen / peptide presentation
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Major histocompatibility complexes (MHC) I & II
Thymocytes (T-cell) & B Lymphocytes (B-cells)
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T-cell receptors (TCRs) bind MHCs
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Antigen recognition of peptides in MHCs by T-cells
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B-cells produce antibodies that bind antigens / foreign “invaders”
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B-cell affinity maturation – survival of fittest
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B & T memory cell production
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Immune System Overview
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Specific / Adaptive Immunity continued
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MHC Presentation
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All cells present intracellular and self peptides via MHC I
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Part of cellular manufacturing process
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CD8+ T-cells bind MHC I
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Some viruses & cancer cells down regulate MHC I production
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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
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Internalize matrix and plasma proteins, materials, debris, bacteria,
viruses via phagocytosis (Latin phagein for devour, kytos for cell, osis
for process)
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Breakdown by both protease and acid in phagosomes
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MHC II proteins incorporated into phagosomes and bind peptides
therein and are cycled to cell surface
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CD4+ T-cells bind MHC II
Immune System Overview
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Specific / Adaptive Immunity continued
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MHC Presentation Cont.
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MHC with a peptide looks like hotdog bun holding a hotdog in it
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Peptide AA length for MHC I & II varies, ~9-15 AAs
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T-cell receptors (TCRs) bind MHCs containing peptides
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TCR looks like the hand coming down to grab the hotdog bun
Immune System Overview
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Specific / Adaptive Immunity: Cellular & Humoral
Components
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Cellular Immunity: Thymocyte or T-cell based
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CD4+ T-cells (Helper)
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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
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Specific / Adaptive Immunity: Cellular & Humoral
Components continued
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Cellular Immunity
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CD4+ T-cells (Helper) continued
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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
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Specific / Adaptive Immunity: Cellular & Humoral
Components continued
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Cellular Immunity
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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
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Immune System Overview
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Specific / Adaptive Immunity: Cellular & Humoral
Components continued
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Humoral
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B-cells, in chicken bursa bursa function discovered
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B. Glick and his group discovered bursa function, 1956
Produce antibodies
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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
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Maturation by cytokines and Ig binding affinity
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Strongest binding cells survive as antigen levels decrease
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Survivors become memory B-cells
Chicken Antibodies
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Ckns produce IgA, IgM & IgY (IgY is sometimes referred to as
chicken IgG)
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Only IgY is packaged in eggs
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Receptors mediate packing in ovarian follicle
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Mannose receptor family, Tesar, D. 2008
IgY more similar to mammalian IgE - it can initiate an
anaphylaxis response in ckns
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IgY binds basil, mast, heterophil(?) cells, they degranulate & release
overload of histamines, immune & smooth muscle modulators,
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Respiratory and cardiac depression
Glycosylated similar to mammalian IgA
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IgY maybe early evolutionary precursor to mammalian IgG/IgA and IgE
Antibody Structure & Binding
IgG space
filled model
Binding /
complement
determining
regions on Ig
IgA & IgM
hu IgG, hu IgE & av IgY
Duck
Images from various web sources
Chicken IgY
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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)
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Egg yields 80-100 mg/egg
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Equivalent total IgG antibodies in ~ 10 ml of serum
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Specific to epitope (reactive site) on antigen 1-2%
Chicken IgY
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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
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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.DavalosPantoja 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
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Antigen Preparation
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Antigen (Ag) is the molecule elicits immune response
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Epitope is site on antigen where antibody binds
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Modify antigen to be injected
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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
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Schedule
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Start at age 2mos
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Days 0, 14, 21, 40, 50, 60
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Age 2 mos, egg production at 4 mos of age
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Adjuvants: material antigen is mixed into
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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
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PEG or Ammonium Sulfate
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PEG method (most common)
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Precipitation of lipids and centrifugation in 3 or 3.5% PEG
(~PEG 6000)
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Supernatant transfer to new tube and adjust to 12% PEG
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Spin
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Resuspend pellet in PBS, then bring volume up and PEG to
12%
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Spin
IgY Purification from Eggs
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PEG method continued
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Resuspend pellet in PBS, dialyze
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Store -20C, not -70C due to stability issues
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http://www.jove.com/video/3084/igy-technology-extractionchicken-antibodies-from-egg-yolk
ELISA Formats
ThermoScientific
Capture/Sandwich ELISA Method
Sandwich ELISA is often the most sensitive format
Steps with wash in between:
1. Coat w/ Capture Ab
2. Block
3. Sample
4. Detection/Primary Ab
5. Secondary Ab labeled w/ HRP
6. Substrate
7. Read
1&2
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Epitomics
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5
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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
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Avian Immunology, Davidson, Kaspers & Schat, 2008
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Immunology, Abbas Lichtman, 2004
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Eppendorf Userguide Liquid Handling 21 No 020
(type into google search bar)
Lab Topics
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Calibration
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Pipetting techniques
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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