ELISA trouble

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Nov 16, 2017

Dear all,

I am having an issue with an ELISA for mouse IgG: samples return lower values on the right side of the ELISA plate when compared to the left side of the ELISA plate. The difference is about 50%.

Although it is not our core business, I have done different ELISA's before with good results.

We have done some trouble shooting:
- We tested an in-house developed ELISA as well as 2 different commercial ELISA kits.
- The plate reader was excluded as the source of error.
- Temperature effects were excluded as the source of error.

Could this somehow be caused by the mouse IgG we want to measure, for example instability/denaturation of the protein in contact with air? Or am I doing something wrong?

Best regards,



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shakeel 10 months ago

False signals can be generated as a result of non-specific binding of IgG present in the sample. Also, many biological samples are made of a complex mixture which consists of many different proteins and salts, these can also reduce the binding capability of the antigen to the antibody. I would also rule out blocking and washing steps since these are well known causes and also exposing the substrate to light. Some interesting information can be found on the following to webpages.


Hope this helps.

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Roberto Rosati 10 months ago

as per Forum policy, if you have commercial interest related to the links, please disclose it, so that we can monitor abuse.

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shakeel 10 months ago

No I do not have any commercial interest relating to the links I have posted.

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Roberto Rosati 10 months ago

I take it that you are not Shakeel Hussain, from Biosupply UK, the mothership company that created elisa-kits.co.uk.

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antibodyscience 10 months ago

It seems that you have an issue with the specificity of your staining that may have a number of reasons. I would try these before changing your target antigen.
One possible reason could be that you use higher than an optimal concentration of secondary antibody. Although you don’t see staining in secondary only controls, that only means secondary antibody needs primary antibody to stick to your sample. Having even trace amount of primary antibody on your sample (as hepatocytes or any other cells may carry low level of target antigen), using high amount of secondary antibody might saturate detection signal. Therefore, titrating secondary antibody and searching for optimal concentration should be the first strategy.
Another reason might be that you have a problem with blocking. Several strategies that you can use:
1) Increase blocking time. 
2) Prepare to block solution afresh.
3) Use stronger blocking solution, such as the one with higher BSA or serum concentration.
Lastly, your primary antibody might be ineffective, for example, due to suboptimal keeping conditions. You may try new antibody or you may even change your vendor