ELISA EnzymeLinked Immunosorbent Assay ELISA Enzymelinked immunosorbent assay

ELISA (Enzyme-Linked Immunosorbent Assay)

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

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

Advantages • Sensitivity • Quantitative • Reproducible

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

Types of ELISA • Non-competitive Ab detection Ag detection • Competitive Ab detection Ag detection

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

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

Sandwich Elisa

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

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

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

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

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

How we will detect: read absorbance at 450 nm

Data analysis absorbance at 450 nm 0. 8 R 2 = 0. 995 0. 7 0. 6 0. 5 0. 4 0. 3 0. 2 0. 1 0 0 W E L L A Blank B Blank C Plant __ D Plant __ E Plant __ F Plant __ G Plant __ H Plant __ Total amount of plant tissue (mg) Your Sample N/A 0. 2 A 450 Your Sample 0. 4 0. 6 0. 8 1 1. 2 NPTII standard (ng/m. L) 1. 4 1. 6 NPTII amount concentration (ng protein (ng/m. L) /mg tissue) Transgenic? Your Sample 0 0 Average Your Sample N/A Your sample N/A Average Yes/No 1. 8 2 A 450 NPTII concentration (ng/m. L) Standard 2 1 0. 5 0. 25 0. 125 0. 0625 0. 03125 0. 015625