Summer Science Academy Experiment:
The ELISA Immunoassay
TIME NEEDED: 2 ½-3 hours
In mammals, there are 5 major types of antibodies, called immunoglobulins. The five types, or classes, are known as IgA, IgD, IgE, IgG, and IgM. The major class of immunoglobulins in blood is IgG and this class constitutes about 10% of total proteins found in the serum (liquid) portion of blood. There is about 10 mg of IgG per ml of serum. The amino acid sequence of IgG varies for each species of animal. Therefore, when IgG from rabbit is injected into a goat, the goat will produce antibodies in response to the rabbit IgG molecules. These antibodies are called goat-anti-rabbit-IgG antibodies, and they will only recognize and bind to rabbit IgG molecules.
In this experiment, you will use a goat-anti-rabbit IgG antibody in order to study the specificity of an antibody-antigen reaction. In the analysis, you will compare the binding of the anti-rabbit IgG to IgGs in serum from rabbit, chicken, cow and horse. The anti-rabbit IgG has been chemically linked to peroxidase, which causes a color-producing reaction. This color-producing reaction will enable you to study the antibody-antigen interaction using an ELISA procedure. Please refer to the attached information describing the ELISA immunoassay.
Materials Needed (*materials in ELISA kit supplied from Modern Biology Inc.)
*TBS (tris-buffered saline)
*goat anti-rabbit IgG-peroxidase
*color development solution
0.1 N HCl
A. Adsorption of the Antigen
1. Using a microliter pipet, place 50 ul of TBS into the following wells of the microtiter plate:
A2 through A6
B2 through B6
C2 through C6
D2 through D6
2. Place 55 ul of chicken serum into well A1
Place 55 ul of cow serum into well B1
Place 55 ul of horse serum into well C1
Place 55 ul of rabbit serum into well D1
3. Dilution of the Sera:
You will now perform serial 10-fold dilutions of the sera. After you complete these dilutions, the concentrations of sera in the wells should be as follows:
Wells 1 1%
Wells 2 0.1%
Wells 3 0.01%
Wells 4 0.001%
Wells 5 0.0001%
Wells 6 no serum
To perform the dilutions, transfer 5 ul of the serum from well A1 to well A2. Mix the contents in well A2 thoroughly by drawing the sample into the pipet and expelling it back into the well a few times. Be careful not to splash the solution into any of the other wells! Transfer 5ul of the sample from well A2 into A3, then A3 into A4, then A4 into A5. Mix well between each transfer. Do not transfer from well 5 into well 6.
Using the same procedure, perform the series of dilutions in wells B1-5, C1-5, and D1-5.
Leave the plate on your lab bench for 20 minutes in order for the proteins to be adsorbed to the surface of the microtiter wells. Do not disturb the plates during this period of time.
4. After 20 minutes, add 2 drops of TBS-gelatin into each well using a transfer pipet. The gelatin is added to block sites on the plastic plates that are not bound to serum proteins.
5. Using a transfer pipet, carefully remove the liquid from the wells in the following order:
A6 through A1
B6 through B1
C6 through C1
D6 through D1
Use a new transfer pipet for each row. The discarded liquid should be placed into your discard beaker.
6. Add 3 drops of TBS-gelatin to each well. Wait 3 minutes and then remove the solution as described in the previous step above. Use a new transfer pipet for each row.
B. The Antibody Reaction
1. Add 50 ul of goat anti-rabbit IgG peroxidase to each well of your microtiter plate. Rotate the plate gently to make sure that all surfaces at the bottom of each well are covered by the antibody solution. Wait 20 minutes for the antibody to bind to the immobilized IgG.
2. Add 2 drops of TBS-gelatin to each well and then remove the solution as described in step 5 above. Use a new transfer pipet for each row.
3. Add 3 drops of TBS-gelatin to each well and immediately remove and discard the solution as described above. Use a new transfer pipet for each row.
4. Add 3 drops of TBS-NP40 to each well and immediately remove and discard the solution as described above. Repeat the TBS-NP40 wash 2 more times (a total of 3 washes with TBS-NP40).
5. Add 3 drops of distilled water to each well and immediately remove and discard as described above.
C. Color Development and Data Analysis
1. Add 50 ul of color development solution to each well. Wait 15 minutes for a color change.
2. Place the plate over a sheet of white paper and examine the intensity of the blue color. The blue product is the insoluble product of the peroxidase reaction. Record the relative intensity of the blue color in the table on the next page.
3. At low pH, the insoluble blue product is converted to a soluble yellow product which should be easier to detect. To enhance the sensitivity of the ELISA, add 0.25 ml of 0.1 N HCl to each well. Place the plate over a sheet of white paper and record the intensity of the yellow product in the table.
1. Which sera samples reacted with antibody and which did not? Explain why a difference was observed.
2. Estimate the lowest concentration of IgG that you detected in your assay. Hint: the serum concentration of IgG is about 10 mg per ml, and the concentration of serum that you started off with in well 1 is 1%.
3. Describe an ELISA procedure that you would use to determine if a person was infected with the human immunodeficiency virus (HIV), the virus that causes AIDS.
ELISA Relative Color Intensity
Well Number Blue Yellow A1 A2 A3 A4 A5 A6 B1 B2 B3 B4 B5 B6 C1 C2 C3 C4 C5 C6 D1 D2 D3 D4 D5 D6
Use "0" for no color, "+" for low color, "++" for moderate color, and "+++" for high color.