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Guest Sanda

LOW signal with plasma samples

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Guest Sanda

Hello,

I order 5 ELISA kits (IL-10,IL-6,TNF-alpha,IFN-gama and IL-1beta). I followed standard procedure from ELISA development kit but I am having very low signal with my plasma sample.

Samples were diluted in ELISA diluent 2x or 3x. My sample signal barely reaches the min standard concentration (the lowest conc recommended for std range).

Did anyone else experiance this problem and can I go with lower dilution of plasma?

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Dear Sanda,

How does you standard curve look like?

In normal healthy donors you typically have very low levels of IL-10, IL-6, TNFa, IFNg and IL-1beta in plasma. 

By contrast, in inflammatory diseseas like sepsis for example you can often get above 1000pg IL-6 and IL-10. 

/Christian

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Guest Sanda

Hello Christian,

Thank you for your answer. My plasma samples are patients diagnosed with PTSD. Here is a picture of my std and samples. HS43 would be a healthy subject. 

 

 

 

 

 

 

 

IL-10.jpg

 

IL-10.jpg

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Guest Sanda

Hi Christian,

Thank you for your answer. Plasma is from patients diagnosed with PTSD, so they should have higher cytokines concentration. I will attach a picture with std of IL-10. Sample R045 and R122 are diagnosed patients and HS43 is a healthy subject. Also, I read my plate after different incubation time with pNPP, hoping that my signal would increase. But my results are mixed... If anything my cytokine concentration is going down than up again... What would you suggest is the best incubation time with pNPP? I attached second picture with incubation time with pNPP 30,60,90 min. (My std range in this experiment is smaller because I realized that my sample has small concentration of cytokines so my standard range doesn't need to be that wide...)

I

IL-10.jpg

il-10-2.jpg

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Hi Sanda,

I think your standard curves look good, and the background levels are low. So the assays seem to have worked. The OD signal increases with development time, on both samples and standard, and I think 60 min is a good developing time for pNPP (30 min is a bit too short, why you shouldn't focus on those results). You have diluted the samples with a dilution factor of 2X and 3X, and 3X is often below the lowest standard point and you shouldn't use these measurements. Since you are analyzing plasma samples, you need to dilute the samples at least 2X in the ELISA diluent, so, therefore, you cannot dilute the samples less. The reason why you need to dilute the samples in ELISA diluent is that the diluent prevents false-positive read-outs which may be caused by interference of heterophilic antibodies found in serum and plasma.

My guess is that your samples have these very low levels of cytokines. I'm not an expert on PTSD but several factors might affect the levels, like timepoint for sampling, or disease progress etc? Also very important to use a diluent that prevents false-positive read-outs, I hope this has been done previously as well.

Best regards,

Lena

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