Dear Ana,

We tried Peprotech for our human cytokines ELISA 2 years ago. And we went through the same kind of troubles than you are facing now. We had high blank value, poor reproducibility between replicates (same sample on the same plate) and between experiment (same sample tested 2 different date).

We stopped using this brand quite fast and went back again to BD products, which are more expensive but also more robust.

Thank you Estelle for sharing your experience...the price is really impressive and so are problems ... Thanks.

Hello Ana

Even I had some problems with same company kit of human IL-12 CYTOKINE.Though you follow the instructions given by them you may not get quite good results. We started using eBIOSCIENCES kits for the same....and got through with good results. Peprotech kits for ELISA are not recommended.

I am currently using a Peprotech ELISA kit for mouse IL1b and I have exactly the same problem as you. Either the detection antibody or the avidin linked with HRP or even both are binding inespecifically to the plate. However I know a researcher who uses it and to date hasn´t encountered this problem.... I wish I could help you more. Good luck!

my friend used peprotech too for IL-4 and IFNg, he had the same problems first, but then he did some protocol modifications like prolonging block buffer time, incubation times etc... and he got then quite good results...

check the following links that he benefited from...

http://www.rndsystems.com/tsg_detail_objectname_elisa_development.aspx#TooMuchSignal

and these too....

http://www.abdserotec.com/elisa-troubleshooting.html

http://www.researchgate.net/post/Im_getting_high_background_signals_from_my_indirect_sandwich_ELISA_any_ideas_why

I have had similar problems. Besides the low intra-assay reproducibility, some times the standard curve often presents OD higher than those of the sample or blank value appears at higher values.

One thing to note is that the recommended dilution in the Peprotech site of the protocol is different from the recommended dilution in the documents accompanying the kits (on the site: 0.1 microgram / mL and protocol: 1,5pg / ml or 1 or 2 pg / mL ) ...

(see in: https://www.peprotech.com/Lists/PTProtocol/Attachments/19/Sandwich%20ELISA%20-%20web.pdf)

You resolve your issue?

I'm having exactly the same problems with a BMP-2 ELISA.  I followed the instructions and reconstituted, stored and applied the solutions exactly as specified and my standard curve is atrocious, absolutely no linearity and to make matters worse the blank has probably the highest absorbance values of the lot!  I emailed their tech support and have had to fill in a form to complain - hopefully I get the money back or at least a replacement batch.  Unfortunately it means the days spent doing the ELISA and the experiments are long gone, never to be seen again!  Nevertheless, my colleague has used a separate BMP-2 kit from Peprotech with no issues, seems to be very hit and miss.