Abnormally high TNFa in non-stimulated animals

[Questions from customers]

Hi,

 

I have faced some problems with your ELISA-kits and would really appreciate your help.

 

I used two kinds of ELISA kits to test the same samples, TNF-alpha (3510-1H-6) and IL-6 (3460-1HP-2). But the results were very strange. Please see attached table.

 

Sample 1,2,3 are normal monkeys, and the rat is also normal. When the results of IL-6 could not be detected, the results of TNF-alpha were very high, and TNF-alpha of normal rat was also very high. The standard curve is very good (see attached image). Why the TNF-alpha of different normal animals were all so high?

 

Looking forward to your reply.

 

Best regards,

Y  

 

[Jens@mabtech.com]

Hi Y,

 

Thank you for your question.

 

What species are the monkeys? And have you analysed serum/plasma or supernatants from cell cultures, and were the monkeys/cells stimulated in any way and if yes, then with what?

 

Generally for IL-6, given that you have analysed sample from a monkey species that our system reacts to (i.e. primarily “old world” monkeys), and given that you haven’t stimulated the cells, it is not strange to you don’t detect anything. Normally, the IL-6 levels are low (a reference value for healthy serum samples is <5-10 pg/mL).

 

In contrast, if the above assumptions are correct, the high level of detected TNFa is indeed strange. Like for IL-6, levels of TNFa are low in unstimulated conditions and if TNFa levels are increased due to some stimulation the IL-6 levels should also increase.

 

One key difference between the two kits is that the IL-6 is an ELISAPRO-kit whereas the TNFa is a ELISA development-kit. The ELISAPRO-kit includes an ELISA diluent buffer which block possible heterophilic antibodies. Maybe you have heterophilic antibodies in your sample that give rise to false positive signals in the TNFa-assay but are blocked by the ELISA diluent in your IL-6 assay?

 

Looking forward to your response.

 

Kind regards,

Jens

[Questions from customers]

Dear Jens,

 

Thank you so much for your kind reply.

 

Monkey is the non-stimulated Cynomolgus macaque and the rat is the non-stimulated SD rat (used to be another negative control). As shown in the table I posted above, IL-6 turned out low and the TNF-a signal detected from serum samples of these “blank” animals looks unusually strong. 

 

The IL-6 data were similar to our previous data while TNF-a appeared to be abnormal, compared to relevant data before.

 

Thus, there should be something wrong in the TNFa assay? Do you mean to use a standard ELISA diluent buffer in TNFa assay? Can you kindly tell us the recipe of this buffer? This would help us for trouble shooting.

 

Thank you in advance.

Y

[Jens@mabtech.com]

Hi again Y,

 

Thank you for providing the additional info. With unstimulated cynomolgus macaques, indeed there seems to be something wrong with the TNFa assay.

 

Yes, try to run the TNFa-assay again but use the ELISA diluent buffer to dilute your sample (the ELISA diluent buffer it is not only used for standard dilution but also for sample dilution). Unfortunately, I cannot reveal the recipe. But do you have anything left in the ELISA diluent-bottle from the IL-6-kit? If yes, then you could use that. If not, diluent is also sold as a supplementary item with the product-code 3652-D2. 

 

Kind regards,
Jens

[Questions from customers]

Hi Jens 

 

With your ELISA diluent buffer, the non-stimulated samples did not yield abnormally high value for TNF alpha. Detail data are attached below.

 

Thank you for your help.

Y

 

[Jens@mabtech.com]

Hi Y,
 

Fantastic news. Thank you for sharing them with us. 
 

Just let me know if you need any other help. 

 

Kind regards,

Jens