1. Detection of Infectious diseases Using Antibodies
Biotechnology I

2. Introductory Question
What is the purpose of antibodies in the immune system
What specific immunity mechanism is responsible for making antibodies?

3. Essential Question
How are antibodies used in ELISAs for detecting specific proteins?

4. Antibodies Specific type of protein
Function: Made by immune system that recognizes and tags foreign macromolecules, commonly proteins
Number of different antibodies circulating in the blood estimated between 106 and 1011

5. Variable Region of Antibody
Variable region have an antigen binding site that binds to the protein of interest
Variable region different for each protein
The antigen binding site binds to specific regions on the protein of interest.
The specific regions on the protein is known as the epitope

6. Specificity of Antibodies Useful For
Test kits – to detect specific proteins (e.g. commercial pregnancy test)
Enzyme-linked immunoabsorbant assay (ELISA) – used to determine the presence and concentration of particular protein in a solution

7. Development of Antibody that recognizes specific proteins
Antibodies produced injecting an animal (rabbits commonly) with the antigen (protein) of interest
Antibodies are harvested from the animal’s blood serum
Antibodies tested for binding to the protein of interest

8. Enzyme-Linked Immunoabsorbent Assay (ELISA)
Very specific- recognizes a specific protein in a mixture of others
Goal of ELISA is to recognize the antigen and measure its concentration

9. How Elisa Works
Antibody is added to the protein solution to form antibody/antigen complex
However, both antibody and antigen is colorless must add something to visualize the antigen/antibody complex
An enzyme is attached to the antibody that catalyzes a color change when a enzyme-specific substrate is added
Color change occurs at the antibody-antigen binding site
The presence and concentration of the antigen can be determined by the amount of color change

10. How ELISA Works

11. Two Enzymes Commonly Linked to Antibodies
Horseradish peroxidase (HRP)
Causes the colorimetric reagent tetramethylbenzidine (TMB) to change from clear to blue
Blue color unstable; add acid to stop reaction; turns blue to stable yellow color
Amount of yellow dependent on the amount of antibody-antigen complex

12. Two Enzymes Commonly Linked to Antibodies
Alkaline Phosphatase (AP)
Catalyzes the combination of two substrates – nitro blue tetrazolium chloride (NBT) and 5-Bromo-4-Chloro-3 indolyphosphate p-Toludine salt (BCIP) to produce a dark purple blue color
Amount of blue color proportional to antibody-antigen complex

13. PROPERTY OF PIMA COUNTY JTED, 2010
Think-Pair-Share
Explain how you know the specific protein is present with ELISA.
3. Think-pair-share
teacher presents a question
teacher gives wait time for student to form answer
teacher instructs students to share their answer with a partner
teacher calls on non-volunteers to share with the class
PROPERTY OF PIMA COUNTY JTED, 2010 3

14. Two different types of ELISA
Direct ELISA
Enzyme is attached to the antibody that recognizes the protein of interest

15. Two Different Types of ELISA
Indirect ELISA
Primary antibody attached to protein of interest
Add a secondary antibody that has the enzyme that produces the colorimetric reaction linked to it

16. Detection of Protein Can be done either
Qualitative- detecting presence or absence of antigen
Quantitative- measures how much antigen is present

17. Uses of ELISA Disease detection in people, animals, and plants
Detecting illegal drug use
Pregnancy tests
Look for contamination in samples- e.g. ground beef contaminated with pork meat
Testing allergens in foods e.g. presence of peanut contamination of food

18. Answer the following Questions IYOW
What region of the antibody binds to the foreign protein? Are all antibodies the same? Why or Why not?
How are antibodies made for use in test kits?
What creates the color in the ELISA test?
Explain the difference between indirect and direct ELISAs?
What are common uses for ELISAs?