Typical ELISA Protocol
Coating antibody or antigen onto the microplate
1.Dilute the protein to be coated to a concentration of 2-10 μg/ml in a buffer such as PBS or Carbonate-Bicarbonate and add 100 μl of this solution per well.
2. Incubate for 18-20 hours at room temperature or 4°C.
3. Block unoccupied sites with a blocking agent (200-300 μl/well) such as StartingBlock™ Blocking Buffer.
4.Store plate at 4°C with a dessicant for future use.
Perform assay
5. Add sample to be tested (50-100 μl/well) and incubate for 1 or more hours.
6. Wash using PBS with 0.05% Tween®-20 or TBS with 0.05% Tween®-20.
7. Add enzyme-antibody conjugate (100-200 μl/well) diluted in blocking buffer and incubate for 1 hour.
8. Wash again.
9. Add substrate.
10. Detect.
Types of ELISAs
1. The most commonly used ELISA assay format is the sandwich assay. This type of assay is called a “sandwich” assay because the analyte to be measured is bound between two antibodies – the capture antibody and the detection antibody. The sandwich format is used because it is sensitive and robust.
2. Competitive assays are often used when the antigen is small and has only one epitope, or antibody-binding site.
3. Often the antigen is labeled instead of the antibody. Unlabeled antigen and the labeled antigen compete for binding to the capture antibody and a decrease in signal indicates the presence of the antigen in the sample. ELISPOT assays are a form of sandwich ELISAs.
Either monoclonal or polyclonal antibodies may be used as the capture and detection antibodies in sandwich ELISA systems. Monoclonals have an inherent monospecificity toward a single epitope that allows fine detection and quantitation of small differences in antigen. A polyclonal is often used as the capture antibody to pull down as much of the antigen as possible. Then a monoclonal is used as the detecting antibody in the sandwich assay to provide improved specificity.
An important consideration in designing a sandwich ELISA is that the capture and detection antibodies must recognize two non-overlapping epitopes. When the antigen binds to the capture antibody, the epitope recognized by the detection antibody must not be obscured or altered. Capture and detection antibodies that do not interfere with one another and can bind simultaneously are considered a matched pair and are suitable for developing a sandwich ELISA.
Another design consideration in choosing antibodies is cost. A polyclonal antibody is generally less expensive (~five-fold) to produce than a monoclonal. The specificity gained by using monoclonals for both the capture and detecting antibody must be weighed against the cost and time required for producing two monoclonal antibodies. Preparing a “self-sandwich” ELISA assay, in which the same antibody is used for the capture and detection, can limit the dynamic range and sensitivity of the final ELISA.
Direct vs. Indirect Detection
Techniques for ELISA
The direct detection method originated in the 1940s when Coons and colleagues labeled antibodies with a fluorescent tag to mark tissue antigens.4 In this technique, a labeled primary antibody reacts directly with the antigen. Direct detection is not widely used in ELISAs, but is quite common for immunohistochemical staining of tissues and cells.
Advantages of direct detection
- Quick methodology, because only one antibody is used.
- Cross-reactivity of secondary antibody is eliminated.
Disadvantages of direct detection
- Immunoreactivity of the primary antibody may be reduced as a result of labeling.
- Labeling of every primary antibody is time-consuming and expensive.
- No flexibility in choice of primary antibody label from one experiment to another.
- Little signal amplification.
The indirect, two-step method uses a labeled secondary antibody for detection (Figure 1). This was first described by Weller and Coons in 1954 and is still a popular method.5 First, a primary antibody is incubated with the antigen. This is followed by incubation with a labeled secondary antibody that recognizes the primary antibody. For ELISA it is important that the antibody enzyme conjugate is of high specific activity. This is achieved when the antibody is affinity-purified and the enzyme conjugation chemistry preserves antibody specificity as well as enzyme activity. All Pierce antibody-enzyme conjugates fulfill these requirements.
Advantages of indirect detection
- A wide variety of labeled secondary antibodies are commercially available.
- Versatility, because many primary antibodies can be made in one species and the same labeled secondary antibody can be used for detection.
- Immunoreactivity of the primary antibody is not affected by labeling.
- Sensitivity is increased because each primary antibody contains several epitopes that can be bound by the labeled secondary antibody, allowing for signal amplification.
- Different visualization markers can be used with the same primary antibody.
Disadvantages of indirect detection
- Cross-reactivity may occur with the secondary antibody, resulting in nonspecific signal.
- An extra incubation step is required in the procedure
Developing an ELISA
Optimizing immunoreagent concentrations and dilutions The goals in developing an ELISA assay are 1) to achieve the best signal:noise ratio for the sensitivity level desired,
2) to have a robust, reproducible assay for the sample being tested and
3) to be able to measure the antigen over a biologically relevant assay range (dynamic range). Therefore, ideal concentrations of each assay reagent must be established empirically. The signal generated by a sample containing analyte, relative to the signal of the same sample without analyte, is the signal:noise ratio. As the signal:noise ratio increases, the assay becomes better at measuring small amounts of antigen.
Dilution ranges for assay reagents can vary widely, depending upon the detection system used. For example, most ELISA protocols based on enzyme-antibody conjugates using a colorimetric substrate recommend a 1:5,000 dilution (from a 1 mg/ml stock concentration) of the conjugate. But this ELISA may work equally well at 1:2,000 or 1:20,000 dilution. To establish the optimal dilutions, a checkerboard titration, also called a two-dimensional serial dilution, is performed. A checkerboard titration is a single experiment in which the concentration of two components is varied in a way that will result in a pattern. This method is used to optimize reagent concentrations
when performing an indirect ELISA that uses a labeled secondary antibody. For example, the primary antibody is serially diluted across the plate, and the enzyme-labeled secondary antibody is serially diluted down the plate. This design permits analysis of different concentrations of the two reagents in each well to obtain the best signal:noise ratio.