ELISAs Do Little? Your Guide to ELISAs Part Two
In Part One of our ELISA blog, we covered direct and indirect ELISAs; two of the four main types of ELISA based assays. In Part Two, we take a look at the other two main types; sandwich and competitive. Below is a guide to both the advantages and disadvantages of these two ELISA types and some of our top ELISA tips.
Sandwich ELISA requires the use of matched antibody pairs. The ELISA plate is coated with an antibody targeting the analyte of interest (known as a ‘capture’ antibody). The sample is then applied to the plate and a second antibody to the target analyte is added (known as a ‘detection’ antibody), which can then be detected in a direct or indirect manner.
It is crucial that the capture and detection antibodies are specific for different and non-overlapping regions or epitopes of the analyte of interest. If both antibodies bind to the same region of the target, the detection antibody will be blocked by the capture antibody.
Sandwich ELISAs are often performed with a detection antibody which has been conjugated, known as direct sandwich ELISAs. However, if a directly labeled detection antibody is not available, a secondary enzyme-conjugated detection antibody may be used. In this case, it is known as an indirect sandwich ELISA.
Sandwich ELISAs are more sensitive (~ 2-5 x) and more specific than direct or indirect ELISAs. This makes sandwich ELISAs a good choice if you wish to analyze samples where the analyte of interest has not been purified, or if the target is of relatively low abundance, such as prostate specific antigen (PSA).
For convenience, Bio-Rad offers matched antibody pairs validated in sandwich ELISA for many common targets. However if a matched antibody pair or kit is not available, a significant amount of time and investigation may be required to find two compatible antibodies, binding to different regions of your target (Table 1).
Table 1. Advantages and disadvantages of sandwich ELISAs.
Fig. 2. Steps in a competitive ELISA.
The last, but perhaps most complicated of the ELISA techniques, is the competitive ELISA (often referred to as an inhibition or blocking ELISA). In a competitive ELISA, the analyte in the sample competes with a conjugated analyte to bind to an antibody against that specific analyte bound in the well (Figure 2). A key difference to other ELISA types is that the signal produced is inversely proportional to the amount of analyte in the sample. That is the greater the concentration of analyte in the sample, the more it occupies binding sites on the antibody in the well, reducing the amount of conjugated analyte that can bind. Competitive ELISAs may be performed in direct, indirect, and sandwich ELISA formats. They are one of the few immunoassay techniques able to detect and quantify antigens with low molecular weights and therefore a limited number of antibody-binding sites, such as steroids.
Table 2. Advantages and disadvantages of competitive ELISAs.
Importantly, no matter which ELISA technique you choose, antibody selection, the use of the right buffers and the inclusion of the right controls is going to be critical in preventing your ELISA from doing little.
Our Top ELISA Tips
- ELISA assays are prone to two common errors: edge effect and hook effect. To prevent these, test all samples in duplicate, or better triplicate, in conjunction with a known standard to ensure the accuracy of the results and for quantification. Ideally, test several sample dilutions to check that final results fall within the linear portion of the standard curve
- When performing a sandwich ELISA, using a specialized buffer (like BUF037A) can reduce background noise and assist with matrix equalization. Differences between matrix components in plasma or serum samples can interfere with the antibody interaction
- Consistency and standardization of conditions will ensure the reproducibility and accuracy of your results. Factors such as humidity and temperature, as well as buffers used, should be consistent between experiments
- Use plates designed for ELISA as these have been manufactured to provide optimal conditions for data collection. Some enzyme substrates, for example, those that generate chemiluminescent signals, may require opaque plates
The Essentials of ELISA
Expand your understanding of the different types of ELISA assays with Bio-Rad’s ELISA guide. Available in the original in-depth version or a convenient, condensed pocket-sized guide.Download or Order Your ELISA Guide