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Troubleshooting in ELISA assay development


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Guest AlineD

Hi everyone, 

I have to optimize an ELISA assay and as it's new for me, I'm not very familiar with how to interprate the data I get from my experiments. 
So far, I don't have a reproducible assay. Each time I run my assay with same materials, same dilutions, same plate, same substrate, same protocole, I always get different results... Either, I get a lower signal than the previous experiment, or my sigmoidal curve on the graph doesn't reach a plateau while I could see it before. Because I get different shape of curves on my graph, I'm not able to calculate the concentration of my sample compared to my standard, based on the EC50 value. 

It's a sandwich ELISA with anti-streptavidin antibodies, HRP conjugated 2nd antibody and chemiluminescent substrate. The results were quite ok using 96-well black plate (MaxiSorp), but since I've started to work with 384-well white plate (MaxiSorp), my results are not consistent anymore. 

I also use a plate washer and use as much as possible electronic multipipette. I incubate 1h with shaking for 1ry and 2ry antibodies. I keep my plate in foil from the moment I add the conjugated antibody, and I read it right away after adding the substrate. 

Is it possible that there is a competition between the capture and primary antibodies, or I use too much antibodies or not enough? 

Any tips of how to optimize an assay from scratch will be more than wecolme! 

Thank you very much in advance for your help. 

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Guest AlineD

Hi Christian,

Thank you for answering. 

Yes it's designed to detect Streptavidin, but we didn't purchase the ELISA from a commercial supplier. 

The results are better and a bit more consistent in the 96-well plate. We kept the same parameters on the plate reader when changing for the 384-well plate and use also the optimization reading from the machine.
The assay was first developped with TMB substrate before changing for chemiluminescence but I could see the same issue of non reproducible data...

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Hi,

Is the ELISA based on monoclonal antibodies or polyclonal antibodies? 

Have you developed these in-house?

There are many antibodies that generate background because they are "sticky". You screen for this during development. 

If you exclude strepatividin from your wells, but add all other reagents, do you still get a signal?

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Guest AlineD

Hi, 

We developed it in-house. There is a monoclonal streptavidin capture antibody coated on the plate (0.5 µg/mL), a polyclonal anti-streptavidin as primary detection antibody (diluted 1:30,000) and a polyclonal conjugated secondary detection antibody (diluted 1:200). 

No, I don't have signal from the blank when there is no streptavidin. 

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