2. ELISA Immuno ExlorerTM : Antibodies in Agriculture
From Mad Cow to GMOs

3. ELISA Immuno ExplorerTM Instructors
Stan Hitomi
Coordinator – Math & Science
San Ramon Valley Unified School District
Danville, CA
Kirk Brown
Lead Instructor, Edward Teller Education Center
Science Chair, Tracy High School
and Delta College, Tracy, CA
Sherri Andrews, Ph.D.
Curriculum and Training Specialist
Bio-Rad Laboratories
Essy Levy, M.Sc.

4. Why Teach ELISA? Hands-on Immunology Tangible results
Laboratory extensions
Real-world connections
Link to careers and industry
Standards-based:
One lesson integrates multiple standards
Health sciences
Immunology
Biodefense
Immune response – antibody/antigen interactions
Disease – infection, detection, transmission
Why Teach ELISA?

6. ELISA Immuno Explorer Kit Advantages
Lab completed in a 45 min period
Supplies for 48 students (12 workstations)
Comprehensive and flexible curriculum
Compelling real-world links
Striking results
Cost effective
Classroom Safe

7. Workshop Time Line Introduction Antigen Detection by ELISA
Ways the ELISA-Immuno Explorer Kit can be used
Real-World Examples

8. ELISA Enzyme-Linked Immunosorbant Assay
Mammalian immune system
Antibody specificity
Biology’s “magic bullet”
Evolved over millions of years
Harness nature’s tool kit
Imagine the applications!

9. Links to the Real World Mad Cow Disease, SARS, HIV GMO
Drug and steroid testing
Pregnancy / Reproduction
Biodefense
Cancer treatment

10. Immune Response C. Macrophage A. Pathogen D. Macrophage B. B cells
F. T cell
E. Macrophage
G. B cell
J. Antibodies attach to pathogen
H. Memory B cells
I. Plasma cells

11. ELISA Antibody Structure
Light chain
Heavy chain
Disulfide bonds

12. ELISA ANIMATION

13. ELISA Procedures Overview
Review activity steps

14. ELISA Kit Workstation Inventory
Reagents:
Yellow tubes Test samples 2
Violet tube (+) Positive control 1
Blue tube (-) Negative control 1
Green tube (PA) Primary antibody 1
Orange tube (SA) Secondary antibody 1
Lab Equipment and Supplies:
Microplate strips, pipettor, pipette tips,
transfer pipette, wash buffer, paper towels,
marking pen
Discuss materials at each workstation

15. Laboratory Quick Guide
Instruct participants to follow Quick Guide procedure at their own pace.

16. Step One Label and add controls
Obtain a test-sample
Label the 12-well strip:
First 3 wells: positive controls “+”
Next 3 wells: negative controls “-”
Remaining wells to identify test-samples
Add 50 ul of positive control to 1st 3 wells
Add 50 ul of negative control to 2nd 3 wells
Add 50ul of the student samples to the appropriately labeled wells
Wait 5 minutes for the antigen to bind

17. Microplate Strips Microplate strips are made of polystyrene
Hydrophobic side chains in amino acids bind to the polystyrene wells
No coating is needed

18. Step Two WASH
Remove samples from wells by firmly tapping them on a paper towel
Discard the top paper towel
Using a disposable transfer pipette wash wells with wash buffer
Remove wash buffer by firmly tapping the wells on a paper towel
Repeat wash step

19. Step Three Add (PA) Primary Antibody
Add 50 ul of the primary antibody (PA) to all 12 wells
Samples are left in wells for 5 minutes
After 5 minutes WASH 2X

20. Wash Buffer
Wash buffer contains phosphate buffer saline (PBS) to keep antibodies in a stable environment that helps keep their structure
Also contains Tween 20: a nonionic detergent removes non-specifically bound proteins and coats wells that acts as a blocking agent to reduce background
Antibody will only bind to the antigen

21. Step Four Wash antibody and add enzyme-linked secondary antibody (SA)
Wash the primary antibody from polystyrene wells as before
WASH 2X
Add 50ul of the enzyme-linked secondary antibody to each well
Wait 5 minutes

22. Antibody Specificity
Secondary antibody (enzyme-linked antibody) will only bind to the primary antibody (serum antibody)
Secondary antibody specifically recognizes the constant region of the primary antibody
In which wells do you predict this is happening?

23. Step Five Add enzyme substrate (SUB)
Wash the enzyme-linked secondary antibody from polystyrene wells as before
Using a disposable transfer pipette wash wells with wash buffer
WASH 3X
Add 50ul of the enzyme substrate to each well
Wait 5 minutes
positive samples
will begin to turn
blue

24. What are the reagents? Antigen: Chicken gamma globulin
Primary antibody (PA): Polyclonal anti-chicken antibody made by rabbits
Secondary antibody (enzyme-linked) SA: Polyclonal anti-rabbit antibody made by goats linked (conjugated) to horseradish peroxidase (HRP)
Enzyme substrate (SUB): 3,3’,5,5’ – tetramethylbenzidine (TMB) – a colorless solution that when oxidized by HRP turns blue

25. ELISA Kit Results
Repeat FLASH animation to solidify what they just did.

26. Ways The ELISA Kit Can Be Used
Protocol
Type of ELISA
Real-World Application I
Tracking outbreaks of disease
HIV, SARS, smallpox & anthrax II
Detecting antigens
GMO, BSE, pregnancy, drugs, (and all the above)
III
Detecting antibodies in serum
HIV, Lyme disease, smallpox and West Nile virus

27. a b Prion Proteins (PrPres and PrPsens) PrPsens PrPres
ELISA test for Transmissible Spongiform Encephalopathies (TSEs)
Prion Proteins (PrPres and PrPsens) a b
Uses differences in diseased prions vs. normal prions to prepare sample.
Proteinase K only digests normal, not diseased, prions .
ELISA tests for any prion protein
PrPsens is composed mainly of alpha helicies
The conformational change that happens to make the disease protein (PrPres) causes the formation of many beta sheets. This protein becomes resistant to Proteinase K. Thus when the sample is mushed up (homogenised) in the homogeniser (the picture that looks like a centrifuge-but isn’t) and is then digested with proteinase K, all the normal prion protein is digested away (along with most of the other brain proteins) leaving only the resistant protein. The ELISA then tests for the presence of the PrP protein (sens or res, it doesn’t distinguish). The property of aggregation in detergent allows concentration of the sample.
TSEs include BSE, CWD (chronic wasting disease in deer & elk), Scrapie (sheep & goats) and (v)CJD (Creutzfeldt Jakob’s disease) in humans.
December 23, The first positive BSE cow in the US found in Washington state.
Since June 2004 to March, the USDA has screened 284,257 BSE samples.
Eight labs in the US: Washington, California, Colorado, Texas, Wisconsin, Georgia, New York, NVSL Lab (Ames, IA). Labs process 30 – 970 samples per day.
PrPsens
Proteinase K sensitive
Soluble in detergent
PrPres
Proteinase K resistant
Aggregates in detergent

28. TSE test sample preparation
Sample brain tissue
Homogenize brain tissue
Digest with Proteinase K (normal prions are digested, diseased prions are resistant)
Concentrate
Denature Proteinase K
Perform ELISA
Sample of brain taken from obex (brain stem)
Tissue sampling: 350 mg (40 mg) of obex
Sample homogenization :45 sec / 48 samples in ribolyzer (specialized homogenizer with in tubes beads that rip up and homogenize tissue)
Proteinase K treatment: Diseased prion is resistant to PK digestion
Normal protein is digested
Concentration of PrPres: Increased sensitivity
Denaturing treatment: The precipitate is redissolved and denatured in a solubilization buffer by heating for 5 minutes at 100°C

29. Protocol II: Antigen Detection ELISA
Real-World Application – TSE Test
Protocol - ELISA on simulated animal brain samples
Tube Description
Actual Tube Contents
Simulated Tube Contents
Student samples
Antigen or PBS
Processed brain
Primary antibody
Antibody against prion protein
Secondary antibody
HRP-linked antibody against primary antibody
Positive control
Antigen
Synthesized peptide with prion sequence
Negative control
PBS
Buffer
Picture is cleared sample ready for the ELISA test after proteinase K digestion, concentration and denaturation.
Controls: Some US labs for CWD use positive tissue for their positive control if they have it on hand. This is not done for BSE.
Confirmation on positive tests. 1) Repeat ELISA test in duplicate. 2) Perform immunohistochemistry (IHC) on brain tissue sample, i.e. look on microscope for the “spongiform” brain.

30. Real-world Applications of Antibodies
Agricultural Uses
– Crop-specific disease diagnosis
– Animal disease diagnosis
– Detection of GM crops
– Basic research
Applications
– Dipstick tests/ELISA
– Immunostaining
– Western blotting
Bio-Rad’s TSE
ELISA Kit

31. ELISA to test for GMOs DNA RNA Protein
“Genetically Modified Organism (GMO)"
an organism in which the genetic material has been altered in a way that does not occur naturally by mating and/or natural recombination
ELISA to test for GMOs
DNA RNA Protein
ELISA can help farmers separate their GMO grain lots from non-GMO grain lots.
ELISA tests are used to identify specific proteins
- Delta-endotoxin Cry1Ab from Bt11
- glyphosate from Round-up (RR)
Maize line Bt11 was developed through a specific genetic modification to be resistant to attack by European corn borer (ECB; Ostrinia nubilalis), a major insect pest of maize in agriculture. The novel variety produced the insecticidal protein, Cry1Ab, derived from Bacillus thuringiensis subsp. kurstaki (B.t.k.) HD-1 strain. Delta-endotoxins, such as the Cry1Ab protein expressed in Bt11, act by selectively binding to specific sites localized on the brush border midgut epithelium of susceptible insect species. Following binding, cation-specific pores are formed that disrupt midgut ion flow and thereby cause paralysis and death. Cry1Ab is insecticidal only to lepidopteran insects, and its specificity of action is directly attributable to the presence of specific binding sites in the target insects. There are no binding sites for delta-endotoxins of B. thuringiensis on the surface of mammalian intestinal cells, therefore, livestock animals and humans are not susceptible to these proteins. (
Technically safe to eat, so only those organisms that eat corn only would be affected by this endotoxin
How does round-up work? It block the ability to manufacture a specific AA, therefore (like in JP/Lysine) will kill off the plant by lacking that essential AA.

32. How to test for GMOs ELISA: PCR:
Test for presence of proteins expressed from genetic modifications
Pro: Quick, inexpensive, low tech
Con: Crop specific, protein stability
PCR:
Test for presence of inserted foreign DNA
Pro: ID different GM crops, DNA stability
Con: Expensive, timely
ELISA used only for fresh food (cheaper, faster). Photosystem II gene that all plants have: use as test for viable plant DNA.
There is also ELISA test for GMO.

33. Example: Pregnancy Test