Hi,
I want to ask about Indirect ELISA. As you in pictures, some wells did not turn blue after adding solution B and also same wells did not turn yellow after adding stop solution. İs that normal or anormal situation?
Note: I used blood serum samples for ELISA and firstly, I added serum samples to each well, later I added anti- antibody before adding HRP conjugate and later incubated 1 hour. After incubation, I added solution A and B, respectively. After 10min incubation, I added stop solution.
You can any ideas about this situation. I wondering your answers.
Faithfully.
If no color develops, it means the antigen was not present in the well.
Hello Özgün Teksoy,
After looking at the ELISA plate, let me clear you "No problem with the ELISA"
By considering that:
The first two wells might be Blank, NSB and then increasing order of standards. The maximum green color you might have got where there is no or less antigen, while intensity of green color may get reduced due to high antigenic values. The stop solution (containing acid) converts green color into yellow (by stopping the reaction and change of pH in the solution.
The ELISA plate is giving signal that the green color development has occurred in Blank wells (first wells) and gradually declined due to presence of antigen (standards increasing down the wells). Several wells wherein you have added blood serum samples also showing less antigen as you can see color development (green followed by yellow after adding stop solution).
Hence ELISA is working fine but in my opinion, you have to do the following changes:
1. Strictly follow the company manual protocol.
2. Be careful and accurately load the Blank, NSB, standards and samples after bringing all at room temperature.
3. Vortex mix all solutions (standards and samples) before their use
3. Pipetting must be accurate, quick and change tips after each load (in duplicate)
4. Dilute the blood serum samples in order to bring the OD in the lower standard range (you can test it by having dilutions 1:10, 1: 50 and 1:100).
Key point: See the literature for the level of antigen in the blood serum that you are analyzing and dilute the blood sample accordingly before setting ELISA. Actually you should have done this before setting the ELISA.
You can make the use of this plate by taking OD to get information for rough estimation of the level of antigen in the blood sample, enabling you to dilute the blood sample accordingly.
Remember, just by looking the color development (green and yellow) give only rough estimation, our (human eyes) can only predict. Until you get OD after setting a particular wavelength (as given in the company manual protocol) in micro plate Reader (so called ELISA Reader in your case), its very difficult to say anything authentically.
Hope this helps
Best
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