Developing your own sandwich ELISA may not be an easy task, since there are several factors that influence the correct functioning of the immunoassay, such as antibodies, buffers, diluents and even the plates themselves, among others.
To set up a sandwich ELISA, it is necessary to carry out the immunoassay, gathering all the necessary reagents and defining the working protocols, as well as a subsequent optimization phase by testing under different conditions, concentrations and variables until ensuring that the results will be robust and precise.
How To Develop A Sandwich Elisa
Factors To Consider When Developing A Sandwich Elisa
The different ELISA immunoassay formats offer different levels of specificity, sensitivity, complexity and time consumption, depending on the steps involved.
The sandwich ELISA is considered the most robust format of all, because the antigen is detected by two different antibodies. And precisely because of this peculiarity, it requires a pair of specific antibodies that are compatible with this yes. (Remember the entry Keys to select pairs of antibodies )
Materials And Reagents Necessary To Set Up A Sandwich Elisa
The main materials and reagents required for the set-up of a sandwich ELISA are the following:
- Microwell plates
- Disposable containers for reagents
- Washing device (microplate washer or manual washing bottles)
- Pipettes of various volumes
- Multichannel pipettes (8-12)
- Pipette tips
- Microplate reader
- Wash buffer
- Thinners
- Detection system (eg streptavidin-HRP
- Validated antibody pair
- Stop solution
Steps To Develop A Sandwich Elisa
The first step is to prepare the plate, covering it with the capture antibody. To do this, the following steps are performed sequentially:
- Transfer the capture antibody diluted in PBS to the microwells on the plate, seal and incubate at room temperature.
- Wash at least 3 times using the wash buffer.
- Block the plate by adding a blocking buffer (Remember the Tips entry for choosing the blocking buffer ), and incubate at room temperature for at least one hour.
- Wash again.
Once the plate is covered with the capture antibody, the assay procedure begins:
- The sample is added to the corresponding wells and the plate is incubated at room temperature for two hours.
- Wash at least 3 times using the wash buffer.
- Add the biotinylated detection antibody, and re-incubate for two hours at room temperature.
- Wash at least 3 times using the wash buffer.
- Add Streptavidin-HRP and incubate at room temperature for 20 minutes.
- Wash at least 3 times using the wash buffer.
- Add the solution with the substrate and incubate another 20 minutes at room temperature.
- Add the stop solution.
- Proceed with the reading of the results, determining the optical density (OD) of each well during the following 30 minutes.
How To Optimize The Performance Of A Sandwich Elisa
Once a sandwich ELISA has been developed that works, it is necessary to carry out some optimization procedures to improve its performance.
These procedures will be mainly aimed at testing different concentrations of antibodies, samples and buffers, until finding the combination that offers the best results.
1.- Optimize the concentration of the capture and detection antibodies and the enzyme conjugate:
Different concentrations of the antibodies (diluted in the upholstery buffer (capture antibody) and in the standard diluent (detection antibody)) and the enzyme conjugate will be prepared, and the same volume of each concentration will be applied to the corresponding wells, searching that with which a stronger signal is obtained at the same time as low background noise.
2.- Optimize the blocking buffer:
Different blocking solutions will be prepared and the same volume of each of them will be applied to the corresponding wells, looking for the one with which a stronger signal is obtained at the same time as a low background noise.
3.- Optimize the sample concentration:
The composition of the standard diluent should be as similar as possible to the matrix of the sample to be analyzed. If said matrix cannot be reproduced, different standard dilution solutions will be tested. The same volume of each one will be applied to the corresponding wells, looking in this case for the one with which a good dynamic range is obtained for the standard curve.
4.- Optimize temperature and incubation times:
Incubation times can be increased to room temperature, or replaced by incubations at 4º or 37ºC in order to achieve greater sensitivity, although without losing sight of the fact that this could also result in an increase in the background signal.