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Blank wells after development


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Hi D,

 

We have now carefully evaluated the plate you sent us and here are our conclusions:

 

From the start, it was evident that the blank wells did in fact contain very faint spots (see e.g. well G8 in plate P8 in the attached ppt), although not optimally developed as indicated by the whiteness of the membrane and faintness of the spots. Since actual spots were visible on the membrane, this proves that capture antibody must have been present from the beginning and that all detection reagents have been added correctly and in the right sequence by you guys.  

 

However, something has obviously gone wrong in the blank wells. Some part of the detection process was not performed in an optimal manner. By subjecting the blank wells to the recovery process described inside of the attached ppt, more spots were developed. In addition, the whiteness of the membrane was remedied. This would again indicate that the well did in fact contained capture antibody. 

 

Blank well investigation.pptx

 

So, what has gone wrong? Maybe not the full 100 microliters of either the detection antibody or the SA-ALP was added to the well? Perhaps there was a blow-out in the pipette and only 20ul was added in this well? Alternatively, maybe there was a bubble formation in either the antibody detection step, or during the addition of SA-ALP or substrate? We will never know. 

 

Kind regards,
Jens

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Hi Jens,

 

Thank you for your feedback. 

 

Based on your results I agree with your evaluation that the well apparently did contain detection antibody, but that somewhere during the detection process something has been suboptimal. And, as you say, it is difficult to pinpoint the reason for this. 

 

Do you have any further recommendations for avoiding such white wells in the future? Do you recommend following your “recovery” protocol if we experience similar blank or almost blank wells?

 

Regards,

D
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Hi D,

 

Yes, you can perform the recovery protocol if you encounter white wells in the future.

 

My recommendation for avoiding this is simply to be rigorous when it comes to pipetting. When using a multichannel pipette, always do a visual check that 100ul have been pulled up correctly in each tip. In addition, some pipettes come with a clip-on system so that tips are secure. A loose tip can in my experience generate a couple of suboptimal 100 ul pull-ups before it eventually falls off completely. That is, if you only judge the success of pipetting on the tip not coming off, you can easily have a few wells with less than 100ul.

 

After I have done my 100 ul dispensing I always take the plate, inspect volumes quickly by glancing through all rows and gently tap the plate on the side. This can take care of bubble formation. I don’t spend minutes glancing over the plate, I would say maybe 3-4 seconds. You quickly see if a well has less then 100ul as the “shadow” in the white well becomes different.

 

Kind regards,
Jens

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