1. ELISA (Enzyme-Linked Immunosorbent Assay)

2. ELISA Enzyme-linked immunosorbent assay ELISA kit components: Antibody
Allows for specific detection of analytic of interest
Solid phase (sorbent)
Allows one to wash away all the material that is not specifically captured
Enzymatic amplification
Allows you to turn a little capture into a visible color change that can be quantified using an absorbance plate reader

3. What is an ELISA? Measure antibody levels (allergies, vaccines)
Detect viruses (hepatitis, HIV, venereal diseases)
Detect hormonal changes (pregnancy)
Detect circulatory inflammatory markers (cytokines)

4. Advantages
Sensitivity
Quantitative
Reproducible

5. ELISA Plate - Solid phase A B C D E F G H 1 2 3 4 5 6 7 8 9 10 11 12 A B C D E F G H
96 well microplate

6. Types of ELISA Non-competitive Ab detection Ag detection Competitive

7. Non-competitive Y Y Ab detection Immobilize Ag Incubate with sample
Immobilized
Ab in Patient’s
sample
Labeled Anti-Ig Y
Ab detection
Immobilize Ag
Incubate with sample
Add labeled anti-Ig
Amount of labeled Ab bound is proportional to amount of Ab in the sample
Quantitative OD
Concentration

8. Non-competitive Y Y - Ag detection Immobilize Ab Incubate with sample
Immobilized
Ag in Patient’s
sample
Labeled Ab Y
- Ag detection
Immobilize Ab
Incubate with sample
Add labeled antibody
Amount of labeled Ab bound is proportional to the amount of Ag in the sample
Quantitative OD
Concentration

9. Sandwich Elisa

10. Competitive ELISA Ag Detection Y Concentration Labeled-Ag * Ag Ag*
Immobilized
Ag in Patient’s
sample
Labeled-Ag *
Ag*
Substrate OD
Concentration

11. Competitive ELISA Ab Detection Y Y Y Y Y Y Concentration Ag
Substrate
Labeled-Ab *
Ab in Patient’s
sample Y Y Y Y Y Y Ag Ag Ag Ag Ag
Immobilized OD
Concentration

12. Simple ELISA protocol 1. Coat antigen onto microplate
2. Allow protein adsorption and block unoccupied sites with neutral protein
3. Add antibody solution into each well
4. Add enzyme conjugated secondary antibody into each well and develop colorimetric reaction with appropriate substrate
5. Read absorbance in spectrophotometer

13. Enzyme label Horseradish peroxidase (HRP)
Alkaline Phosphatase (Alk-phos)
β-Galactosidase (β-gal)
Urease

14. Substrate Ortho Nitro Phenylene Diamine hydrochloride (ONPD)
Yellow, 450 nm
(p-Nitrophenyl Phosphate, Disodium Salt ( PNPP)
Orange, 492 nm
Tetra Methyl Benzidine (TMB)
2,2'-Azinobis [3-ethylbenzothiazoline-6-sulfonic acid]-diammonium salt
(ABTS)

15. How we will detect: read absorbance at 450 nm

16. Data analysis Total amount of plant tissue (mg) A450
Total amount of plant tissue (mg)
A450
NPTII concentration (ng/mL)
NPTII amount (ng protein /mg tissue)
Transgenic?
A 450
Your Sample
Your sample
Standard W E L
A Blank
N/A  0 2
B Blank 1
C Plant __
0.5
D Plant __
 Average
Average
Yes/No
0.25
E Plant __
0.125
F Plant __
0.0625
G Plant __
H Plant __