An ELISA test cannot be run without a plate. The test depends entirely on the presence of welled plates that hold the substrates, buffer, antibodies, diluents, etc. The ELISA testing plates are either 96 or 384-welled, but the most commonly used plate is the 96-well plate. As a laboratory technician, medical researcher or a biochemist, you require the plates to run ELISA tests effectively. You should therefore keep the following factors in mind:
- The color of the plates
The ELISA tests are very specific and are widely used in medicine to detect presence or absence of antibodies or antigens. The tests depend on the color change detected. This means that the results should be visible clearly through the plates.
You should consider getting clear polystyrene plates for colorimetric signals. White or opaque plates are suitable for chemiluminescent or fluorescent signals.
A laser is shined through the plate wells so as to measure the absorbances of the colorimetric substrates being tested. Use of clear-bottomed plates results in concise results.
Aberrations result from scratched plates or plates with imperfections. To ensure that the correct data is collected, the plates should be in good shape, without any scratches and clear enough. This will give you the chance to obtain accurate results.
- Protein binding capacity
Before running the enzyme-linked immunosorbent assay, you must choose a plate with the minimum protein binding capacity. For instance, the most suitable one would have a minimum protein binding capacity of 400ng/cm2.
The protein binding coefficient of variation (CV) should be less than five percent. This limits deviation in values between the microplate wells.
- Material of plate
The type of material used on the microplate will affect the type of results and binding. The most common type of plate used is polystyrene. It is non-reactive thus will not alter results. The other types of materials that make up the ELISA microplates include nylon, polypropylene and polycarbonate.
- Coating buffer
The most common coating buffer used is acarbonate-bicarbonate buffer. Besides being non-reactive (no reactions with the polystyrene plates), it has a high pH of 9.4. This high alkaline level from the buffer makes it possible for the peptides and proteins to be protonated. The overall negative charge facilitates binding to the positively charged plate.
The other examples of buffers that may be used include phosphatase buffered saline (PBS) and tris-buffered saline (TBS).
- State of the plate
The plates used can be pre-activated or not. The pre-activated plates are necessary where a directed approach is necessary for attachment of the coating antibody to the plate. For instance, you may find plates coated with Protein A or Protein G when you require oriented binding of the coating antibody.
It is recommended that you use plates pre-coated with streptavidin when biotinylated samples are used.
In conclusion, the accuracy of your colorimetric analysis depends on the type of plate and the state of the plate. You must ensure that the plate is in perfect condition for the best results to be obtained.
Amanda Washington is a senior biochemist in the US. She is renowned for her efficient disease testing using the enzyme-linked immunosorbent assay. Read more of her work on the medical journal.