Dear Anantharaj, if standad wells are developing colour normally it seems that you are right with the suggestion that all samples are have lower values than the limit of detection. Do you have some sample that can be used as a positive control? Are the antibodies/kit specific for bovine cytokines?

If the only problem is the threshold of detection you could try high sensitivity ELISA...

Hope this is helpful,

Ana 

Dear Ana,

Thanks for your helpful information. No i dont have any sample as positive control. I will try with some positive control. And also the Kit I am using is bovine specific. 

Regards

Anantharaj

Dear Anantharaj,

I agree with Ana about the sensitivity limits you are observing. I know from colleagues that work with bovine samples that reagents are limited. You may be able to improve the sensitivity of the ELISA by eliminating the top one or two concentrations on your standard curve and allowing the color to develop longer since the samples you are testing do not seem to register in that range. This may add to the background though. Another alternative would be to substitute your detection reagent for a biotinylated antibody along with strepavidin-HRP which would be better than avidin-HRP and much better than directly conjugated HRP.

Good luck!

Luis

Dear Luis,

Thanks for your kind information. Its really very useful to me. I will perform by removing two higher concentrations and let you know.

Regards

A. Anantharaj

I think you have errors in sample processing or you can missing in washing

May this link help you

http://www.abcam.com/protocols/elisa-troubleshooting-tips

Regards

i agree with Dr.Qassim

Dear All, 

Thanks for the suggestion. I am washing the plate manually. I dont think that I am having problem in washing the plate. Because the colour was developed in standard wells. So please can you elaborate how the sample may  miss while washing. Whatever antigen in sample it will be strongly bind to antibody coated with plate na. Or while tapping the plate will it go?

Regards,

Ananth