| | |||||||
| Register | Search | Today's Posts | Mark Forums Read |
| Conferences , Symposiums and Meetings Post Information and News about Scientific Conferences , Symposiums and Meetings Here. |
| | LinkBack | Thread Tools | Display Modes |
|
#1
| |||||||||||
| |||||||||||
| I am developing an inhibition/competition elisa, Method: Coat plate with antigen (diluted in coating) (plate A) In different plate incubate monoclonal mouse ab with sample or standard (plate B) Block (1%BSA,0.05%tween)coated plate (A) Transfer contents of plate B to plate A, incubate wash Add hrp-anti mouse(diluted in block), incubate wash Add tmb however my problem is that I think I have matrix effect, but when I try to dilute my sample I go beneath the sensitivity of the assay, any suggestions? So far I've tried... -decreasing mouse mono antibody - background* does not decrease much, sensitivity of the assay decreases. -diluting sample in block buffer (1%bsa, 0.05%tween) - background decreases but the value is below linear part of standard curve (and as it is a competition elisa %b/b0 is ~100%) -the competition is for antigen bound to the plate, I've tried reducing the concentration of this antigen, - not much effect seen on sensitivity, signal decreases. -fiddled with lots of different incubation temps times - to no real effect *by background I mean that when I omit the antigen coating but run the assay I get pretty much the same signal as when antigen coating is present. Running the assay with no sample present but still both antibodies included I get a very low signal, therefore its not lack of blocking. Suggestions much appreciated! |
| Tags |
| competition , elisa , matrix effect , problem , sensitivity , troubleshooting |
| Thread Tools | |
| Display Modes | |
|
|
| | ||||
| Thread | Thread Starter | Forum | Replies | Last Post |
| ELISA Signal from Null-mutant Problem | WolfeMD | ELISA Assay Forum | 0 | 05-25-2010 03:36 PM |
| Another contimation problem | helpme | Cell Biology and Cell Culture | 3 | 05-18-2010 11:13 PM |
| Another contimation problem | helpme | Cell Biology and Cell Culture | 1 | 05-17-2010 09:53 AM |
| SDS-PAGE problem | Steve Nothwehr | Protocols and Methods Forum | 2 | 10-16-2009 08:59 AM |
| Normalization of Cell Death Detection ELISA? | Peter Frank | Cell Biology and Cell Culture | 1 | 08-25-2004 11:08 AM |