1.17: ELISA

Learning Objectives

Goals:

• Demonstrate the power of an ELISA as a biomedical diagnostic tool.
• Perform an ELISA.
• Analyze the results of the ELISA and present a diagnosis based on the results.

Student Learning Outcomes:

Upon completion of this lab, students will be able to:

• Describe how an ELISA works.
• Given a set of data, interpret the results of the ELISA.

Introduction

ELISA (Enzyme-Linked ImmunoSorbent Assay) is an immunologic technique used to detect the presence and concentration of an antigen or antibody in a sample. The power of an ELISA is based on the extreme specificity of the antigen-antibody interaction. ELISAs have wide-ranging applications, especially as medical diagnostic tools.

Figure 1. ELISA reactions

This lab is a simulation of an ELISA performed on patients to determine if they may have been exposed to the HIV virus. Patients exposed to the virus (foreign antigen) will develop antibodies to the HIV virus, and the antibodies circulate in the bloodstream. By testing the patient blood samples, the presence or absence of these antibodies can be measured using the ELISA.

In the ELISA conducted for this lab, the antigen (from HIV virus) is adsorbed to the surface of the plastic wells (on the 8-well strip or 96-well plate). Patient blood serum samples (which may contain antibodies to the antigen) are added. If antibodies are present, then antigen-antibody complexes form (ImmunoSorbent Process). The detection of these complexes is accomplished by the addition of a secondary antibody that detects all human antibodies. For easier detection, the secondary antibody has been covalently linked to an enzyme. When the enzyme binds to its substrate, a reaction occurs to create a colored product. In summary, for patients with HIV, the antibodies in their blood bind to the HIV antigen, the secondary antibody will bind to the human antibodies, and the enzyme will produce a colored product that is easy to visualize. For patients that do not have antibodies to the HIV antigen, no antibodies bind in the first stage and no colored product is produced in the end.

Clinical Application

Scenario: You work in a clinic and two patients come in who have had possible exposure to HIV. ELISA is the first screening method for HIV antibodies because it uses less costly materials and machinery than other diagnostic procedures (i.e. Western Blot or PCR). You take a blood sample and centrifuge it to separate the blood serum from the red blood cells and will now be performing an ELISA, testing the serum for the presence of HIV antibodies.

Materials

Figure 2. ELISA lab materials

Reagents

Samples in Microfuge tubes

• (GREEN) – Positive Control: Serum with Antibodies to HIV antigen
• (YELLOW) – Negative Control: Serum with no Antibodies to HIV antigen
• (PINK) – Patient A’s Blood Serum (potential primary antibody)
• (BLUE) – Patient B’s Blood Serum (potential primary antibody)
• (CLEAR) – Secondary Antibody: Anti-Human Immunoglobulin linked to an enzyme
• (AMBER) – Substrate:
• Tetramethylbenzidine (TMB) chromogenic substrate
• 0.25M sulfuric acid (optional)

Equipment

• HIV protein-coated 8-well ELISA strips (antigen)
• P200 micropipette
• Box of P200 pipette tips
• Microcentrifuge tube rack with samples
• Squirt bottles with PBS Wash Buffer
• Waste bucket for tips
• Pan for washing strips
• Paper towels
• Microtiter plate reader with 450nm filter (optional)

Procedure

Step 1: Coat Antigen to Plate (Inactivated HIV Proteins) *This step has been completed for you*

1. 200-μL Inactivated HIV Proteins (antigens) were added to each well of the 8-well ELISA strip
2. The strip was covered with plastic wrap and incubated at room temperature for 1 hour.

Step 2: Block Non-specific Binding of Antibodies *This step has been completed for you*

1. The contents of the 8-well strip were emptied by turning upside-down and flicking until no more liquid was present.
2. Blocking Solution was added to each well. This step will prevent non-specific binding of the antibodies.

Step 3: Add the Sample (with possible Primary Antibody)

Begin the experiment here: Your antigen-coated ELISA strip is now ready for you to add the samples*

Note

IMPORTANT: Before adding the samples, mix the solutions by inverting the tubes. Remember to change pipette tips between each solution.

Figure 3. Marking to orient the well strip

1. Mark one side of the strip with a lab marker to keep it oriented during the procedure.
2. Add 100-μL of Positive Control (GREEN tube) to wells 1 & 2.
3. Add 100-μL of Negative Control (YELLOW tube) to wells 3 & 4.
4. Add 100-μL of Patient A’s Blood Serum (PINK tube) to wells 5 & 6.
5. Add 100-μL of Patient B’s Blood Serum (BLUE tube) to wells 7 & 8.
6. Let sit for at least 5 minutes.
7. Empty the contents of the 8-well strip by turning upside-down and flicking until no more liquid leaves the strip. Blot gently on paper towel to remove any remaining liquid.
8. Wash. Fill wells to the top with PBS Buffer. Empty the contents as above.
9. Repeat this wash step 3 more times.

Figure 4. Washing the wells

Step 4: Add the Secondary Antibody

1. Add 100-μL Secondary Antibody (CLEAR tube) to all wells. Let sit for 5 minutes.
2. Empty the contents of the 8-well strip by turning upside-down and flicking until no more liquid leaves the strip. Blot on a paper towel before washing.
3. Wash. Fill wells to the top with Buffer. Empty the strip and blot on paper towels.
4. Repeat this wash step 3 more times.

Step 5: Detect Presence of Antigen-Antibody Reaction

1. Add 100-μL Substrate (AMBER tube) to all wells. After a few minutes, some wells may begin to change color. A color change to blue indicates the presence of the antigen-antibody complex. The more antibody bound to antigen, the bluer the solution will be.
2. Fill in the presence of color in the chart below and answer the questions on the results page.

Step 6: (optional) Stop the Enzymatic Reaction and Read the Well on a Microtiter Plate Reader

1.      Add 100μL 0.25M sulfuric acid (color will turn from blue to yellow)
2.      Place your strip into the plastic frame carefully and read the absorbance (optical density) set to 450nm on the microtiter plate reader

Figure 5. Positive wells (varying shades of blue) and negative wells (no color)

Results

Using the following color key, record your results in the table below:

0 = no color + = very light blue (or yellow) ++ = light blue (or yellow) +++ = dark blue (or yellow)

Data Table 1. Presence of Antigen-Antibody Complex

Well #	1	2	3	4	5	6	7	8
Sample Added
Color
Absorbance (optional)

Data Analysis

1. Did your positive control exhibit color change? If not, how could this have occurred?
2. Did your negative control remain clear? If not, how could this have occurred?
3. Compare patient A to the positive and negative control. What can you deduce about patient A’s condition?
4. Compare patient B to the positive and negative control. What can you deduce about patient B’s condition?

Study Questions

1. Describe an ELISA assay.
2. Draw the Positive Control Well after Step 2.
3. Draw and label a diagram of the positive control well at the end of the procedure. What color is the product?
4. Draw the Negative Control Well at the End of the procedure.

Attribution

Adapted from Lab 17 ELISA by Sandra Slivka, PhD, Southern California Biotech Center, San Diego Miramar College, CA. Licensed CC-BY-NC-SA 4.0