Lab 10. Enzyme-Linked Immunosorbent Assay (ELISA)
The enzyme-linked immunosorbent (ELISA) technique is an extremely sensitive method for detecting antigens or antibodies, and can detect an antigen or antibody at a concentration as low as 1 ng/ml. The ELISA is commonly used to quantify antigens or antibodies, but is not good for detecting the presence of antigens or antibodies. The ELISA procedure has been applied in medical diagnosis and research, such as the detection of antibody to HIV or salmonella.
Antibodies conjugated to enzymes are used in ELISA. Horse reddish peroxidase and alkaline phosphatase are two enzymes that are commonly conjugated to antibodies. These types of antibody conjugates also can be used for Western blot and immunohistochemistry.
The ELISA technique takes advantage of the fact that most proteins adhere to plastic. When incubated with a 96-well plate, antigens will bind to the surface of wells. To cover the unbound sites of the plastic surface, the plate will be incubated with a non-specific protein (proteins unrelated to the test components), which will block any nonspecific binding of antibody in the next step. Then antibodies are added to each well. The antibody will bind if the appropriate antigen has bound to the plastic surface. The plate is washed to remove unbound antibody. The presence of any bound antibody can be detected by a secondary antibody that is conjugated to an enzyme. This is similar to the indirect immunofluorescence staining. The difference is that an enzyme-conjugated secondary antibody is used instead of a fluorochrome-conjugated secondary antibody. Finally the enzyme substrate is added. The enzyme conjugated to the antibody catalyzes a reaction that transforms a colorless substrate into a colored product. The optical density (OD) is measured by spectrophotometer to determine the amount of the antibody.
The ELISA technique can be modified to fit each analysis. When it is available, an enzyme-conjugated primary antibody can be used to directly detect its specific antigen. When the concentration of an antigen is low or the antigen does not bind to plastic well, its specific antibody can be coated on plastic first to capture the antigen.
In this lab exercise, you will learn to use ELISA to determine the concentration of anti-BSA (bovine serum albumin) antibody. Anti-BSA antibody with a known concentration will be used to establish a standard curve. Using the standard curve, the concentration of an unknown sample will be determined.
Three sets of controls are carried out to ensure the specificity of ELISA assays.
1. No antigen control: test the nonspecific binding of the primary antibody in the absence of the antigen.
2. Secondary antibody control: test the nonspecific binding of the secondary antibody in the absence of the primary antibody
3. Substrate control: test the color change of substrate in the absence of the enzyme-conjugated secondary antibody.
Materials:
Antigen BSA 1 mg/mlWashing solution PBS/T 0.05% Tween 20 in PBS (0.5 ml/L)
Tween 20 is a detergentBlocking solution 1% gelatin in PBS/T (10 ml/L)
Gelatin is a protein mixture.Primary antibody mouse IgG2a anti-BSA
1:100 dilution (10 mg/ml) in PBS/TSecondary antibody HRP-conjugated rabbit anti-mouse IgG2a
1:10,000 dilution in PBS/THRP substrate ABTS, 2,2’-azinobis(3-ethylbezthiazoline-sufonic acid)
0.1 M NaCitrate, pH 4.0 4 ml
ABTS 15 mg/ml in dH2O 0.2 ml
H2O2*, 0.3%, 40 mlProcedure:
March 8
1. Each group has a 96-well plate coated with BSA except for wells A1 and A2. The plate has been washed with PBS/T three times. Dump out PBS/T by inverting the plate over the sink, then blotting dry on a stack of paper towels. Add 200 ml of blocking buffer to each well (A1 to C12), cover the plate and incubate at room temperature for 30 min.2. Invert the plate over a sink to discard the blocking solution, then blot dry.
3. Wash the wells by filling them with PBS/T from a squirt bottle. Dump out the plate and repeat the wash twice more. Blot the plate on paper towels.
4. Label 11 eppendorf tubes from 2 to 12. Add 0.25 ml PBS/T to each tube. Prepare two-fold dilutions of the antibody by serially transferring 0.25 mls antibody through 11 0.25 ml PBS/T. This will give you 0.25 ml of each dilution.
5. Add 100 ml of each antibody dilution to the wells of rows B and C and wells A1, A2, A5 and A6. Add 100 ml of the antibody with unknown concentration to well A7 and A8. Cover the plate with plastic wrap and incubate at 4oC for 2 days.
March 10
6. Wash and drain the plate 3 times with PBS/T as in step 3.7. Add 100 ml of the secondary antibody to every well used except control well A5 and A6. Cover the plate and incubate for 30 min at room temperature.
8. Wash and drain the plate 3 times with PBS/T and blot as in step 3.
9. Prepare the substrate solution by adding 40 ml H2O2 to 4 ml ABTS solution.
10. Add 100 ml of freshly prepared substrate solution to each well used.
11. Read OD at 405 nm using a 96-well plate reader.
12. Processing data:
a. Subtract the OD values of wells B1 to C12 with the average of OD values of wells A5 and A6.
b. Calculate the average of OD values from duplicated wells.
c. Plot the average OD versus antibody concentration.
d. Determine the concentration of the unknown sample.
Reading
Immunobiology A-6Study Questions
1. What is the purpose of each of the control wells? Should they show any change in color? If they show changes in color, what do the changes indicate?
2. What is the purpose of the gelatin blocking solution?
3. Describe how to determine the concentration of IL-2 using ELISA. Please list names of the key reagents.
4. What would happen in an ELISA assay if:
a. there was no negative control?
b. the blocking step was missed?
c. insufficient amount of secondary antibody was added?
d. the primary antibody was left on the plate overnight?