Detection of Infectious diseases Using Antibodies Biotechnology I

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

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

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

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

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

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

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

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

How ELISA Works

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

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

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

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

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

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

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

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?