COURSE
SCIE1046: Fundamentals Of Microbiology With Lab
- About the Lab
Learning Objectives:
- Explain the principle of different ELISA techniques.
- Apply sandwich ELISA to quantify protein samples.
- Analyze the standard curve of ELISA experiment.
- Understand the function of reagents and equipment used in ELISA.
- Describe the basic troubleshooting process of ELISA.
Estimated Length: 50 minutes to 1 hour
MAKE THE CONNECTION
The background information in section 2 was adapted from the following Microbiology lecture course Tutorials:
5.4.1 Antibody Production and Laboratory Testing
5.4.5 EIA, FEIA, and ELISA Testing
- Background Information
The following background information will be helpful as you prepare for the simulation.
In this lab simulation, the term hapten is used to reference the size of molecules. A hapten is a small molecule that binds with a protein carrier to elicit antibody production. Within the lab, four types of ELISA testing are covered. The type of ELISA testing used will vary based on whether you are assessing antibody or antigen activity and whether you are measuring small or large molecules.
There are conflicting definitions on what constitutes a small vs a large molecule. In the context of pharmacology, a small molecule is composed of less than 1,000 atoms, while a larger molecule is composed of more than 1,000 atoms.
TERM TO KNOW
This glossary term is important to know and will help you during the Activity.
Hapten
A small molecule that binds with a protein carrier to elicit antibody production.
2a. Data and Graphs
Raw data are the actual data values used in a set of scores used for assessment. During this lab simulation, you will be using raw data from the ELISA to create graphs. Graphs are mathematical models that plot data using points positioned above a number line. A graph will visually present data in mass numbers so that we can easily observe where data cluster and how they distribute around a mean, or average.
In a normal distribution, also called a standard distribution or standard curve, data are evenly distributed around the mean. A standard distribution often presents as a bell curve.
During an ELISA, multiple dilutions with a range of concentrations are conducted. This results in lots of raw data that are graphed to create a standard curve.
TERMS TO KNOW
These glossary terms are important to know and will help you during the Activity.
Raw Data
The actual data values used in a set of scores used for assessment.
Graphs
Mathematical models that plot data using points positioned above a number line. A graph visually presents data in mass numbers so that we can easily observe where data cluster and how they distribute around a mean, or average.
Normal Distribution
Data are evenly distributed around the mean, often presented as a bell curve. Also called standard distribution or standard curve.
2b. Serial Dilution
In order to create the multiple dilutions needed for an ELISA test, you will want to use a serial dilution technique. Serial dilutions are made by creating the same dilution step over and over using a previous dilution as the input for the next dilution to be conducted. Since the dilution fold is the same in each step, the dilutions create a constant ratio between any subsequent dilutions.
EXAMPLE
1/3 to 1/9 to 1/27 to 1/81
REFLECT
You will notice that each dilution is threefold relative to the previous one.
HINT
If you need to consider very large numbers that consist of several factors of ten, you will want to use a logarithmic scale. Refer back to the information from the Bacterial Growth Curve Activity for additional information on logarithmic scales.
The image below shows the steps of the pour plate method for estimating numbers of bacteria.
The following is a detailed breakdown of each step in the pour plate method.
STEP BY STEP
Step 1: The bacterial sample is mixed with warm agar (45 to 50 °C). The temperature must be cool enough for the bacteria to survive this step.
Step 2: The sample is poured onto a sterile petri plate.
Step 3: The sample is swirled to mix and allowed to solidify.
Step 4: The plate is incubated until bacterial colonies grow. The final plate in the image shows multiple dark spots representing colonies.
WATCH
The following video on serial dilutions is a valuable reference.
Play Video
References:
Parker, N., Schneegurt, S., Thi Tu, A., and Lister, P. (2016). Microbiology. Openstax. 9.1. Serial Dilution.
openstax.org/books/microbiology/pages/9-1-how-microbes-grow
2c. Introduction to EIA, FEIA, and ELISA Testing
There are various types of tests that use antibodies to detect the presence of antigens. These tests, called enzyme immunoassays (EIAs), are carried out in microtiter plates or in vivo and share a common characteristic: they use an antibody molecule with a variable region that binds to a specific antigen and a constant region that binds to an enzyme. The enzyme is important because it can undergo a reaction to show the presence of the antibody, allowing rapid visualization and/or quantification of test results.
Chromogens are colorless molecules that can be converted into a colored end product. EIAs often use enzymes that react with chromogens. Alkaline phosphatase and horseradish peroxidase are commonly used.
Enzyme-linked immunosorbent assays (ELISAs) are very commonly used examples of EIAs.
The Microbiology lecture focuses on direct, indirect, and sandwich ELISAs. The lab simulation discusses all three of these major ELISAs and also addresses the competitive ELISA. The Competitive ELISA is similar to the sandwich ELISA in that it measures the concentration of an antigen by detecting signal interference. Competitive ELISAs are commonly used for small molecules, when the protein of interest is too small to efficiently be sandwiched with two antibodies.
TERMS TO KNOW
These glossary terms are important to know and will help you during the Activity.
Enzyme Immunoassay (EIA)
An assay that uses antibodies to detect the presence of antigens.
Enzyme-Linked Immunosorbent Assays (ELISA)
Commonly used EIAs that detect the presence of antigens or antigen-specific antibodies or quantify antigen in a solution.
2d. Antibody Production and Laboratory Testing
You are encouraged to review the complete Microbiology lecture course Tutorial 5.4.1 Antibody Production and Laboratory Testing for background on this topic before you begin the simulation.
- Lab Manual
This Lab Manual gives a synopsis of the lab and the theory behind it. You’re encouraged to read or download the manual before launching the lab. This information will also be available during the simulation by selecting the “Theory” tab on the virtual LabPad.
- Launch Lab
You’re ready to begin! Review the helpful navigation tips below. Then click the “Launch Lab” button to start your lab. Be sure to answer all the questions in the simulation because they contribute to your score. Good luck, scientists!
- Exiting: To exit a lab simulation, press the ESC key on your keyboard. This key returns you to the objective screen for the simulation.
- Saving: You do not need to complete a simulation in one sitting. Labster saves your progress at predetermined checkpoints upon exit. To see your progress at any time, click on the “Mission” tab of the LabPad.
- Restarting: You are allowed an unlimited number of restarts for a simulation to improve your quiz score. Sophia and Labster will always store your best score.
- Just Browsing: You can restart a simulation to have a look around without completing it. The program will still retain your previous (and best) score.