Go Back   Science Forums Biology Forum Molecular Biology Forum Physics Chemistry Forum > Molecular Research Topics Forum > Protein Science > ELISA Assay Forum
Register Search Today's Posts Mark Forums Read

ELISA Assay Forum ELISA Forum. Discuss and post questions including optimization, troubleshooting, protocols and theory of ELISA assays using monoclonal, and polyclonal antibodies or peptides.


Weird Reuslts with Sandwich ELISA

Weird Reuslts with Sandwich ELISA - ELISA Assay Forum

Weird Reuslts with Sandwich ELISA - ELISA Forum. Discuss and post questions including optimization, troubleshooting, protocols and theory of ELISA assays using monoclonal, and polyclonal antibodies or peptides.


Reply
 
LinkBack Thread Tools Display Modes
  #1  
Old 01-06-2009, 09:34 AM
Pipette Filler
Points: 2,662, Level: 33 Points: 2,662, Level: 33 Points: 2,662, Level: 33
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Jan 2006
Posts: 27
Thanks: 0
Thanked 0 Times in 0 Posts
Default Weird Reuslts with Sandwich ELISA



Yoodles People!!
I have some issues with sandwich ELISA that I hope that this forum can help me clarify.. I depend on all of you

Here is my situation: When performing sandwich ELISA I am coating wells with NaHCO3/Na2CO3 buffer and monoclonal mouse antibody. Afterwards I apply blocking buffer followed by the antigen at varying concentrations, rabbit antibody, goat antibody, TMB, stopping reagent.

I prepared a blank on same plate where I place everything in above sequence excluding mouse antibody and antigen.

Results: blank and ALL sample wells with VARYING concentrations of antigen give the same colour no variation in optical density...no gradient in colour. I have repeated experiment more than once yet I get the same results.

An overseas researcher uses the same protocol as me but actually obtains good results using same antigen concentrations, same antibodies, same supplier for reagents and antibodies.

What the heck am I doing wrong? Please Help!!
:mf_surrend er:
Reply With Quote
  #2  
Old 01-06-2009, 08:00 PM
tgd tgd is offline
Volunteer
Points: 1,303, Level: 21 Points: 1,303, Level: 21 Points: 1,303, Level: 21
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Dec 2008
Posts: 45
Thanks: 2
Thanked 22 Times in 14 Posts
Default Re: Weird Reuslts with Sandwich ELISA

Hello,

I think we need a few more experimental details here. What do you block with and is the blocking reagent included in all antibody-containing solutions for example?

However, one thought springs to mind immediately. Is your 'goat antibody' anti-rabbit or (by any chance) anti-mouse? It should of course be anti-rabbit, for obvious reasons. I am presuming that by 'goat antibody' you mean goat (anti-rabbit) peroxidase conjugate or goat (anti-rabbit) alkaline phosphatase conjugate, or the equivalent. I am probably wrong, but the fact that you colleague gets the ELISA to work suggests some subtle mistake?

Good luck,

tgd
Reply With Quote
Reply

Tags
elisa , reuslts , sandwich , weird


Thread Tools
Display Modes

Posting Rules
You may not post new threads
You may not post replies
You may not post attachments
You may not edit your posts

BB code is On
Smilies are On
[IMG] code is On
HTML code is Off
Trackbacks are On
Pingbacks are On
Refbacks are On

Forum Jump

Similar Threads
Thread Thread Starter Forum Replies Last Post
ELISA Signal from Null-mutant Problem WolfeMD ELISA Assay Forum 0 05-25-2010 03:36 PM
high values in both serum and antigen control - troubleshooting sandwich ELISA Namalee ELISA Assay Forum 3 04-22-2010 04:21 PM
Sandwich ELISA Optimization HELP!? WolfeMD ELISA Assay Forum 3 12-05-2009 06:28 AM
right way to assemble the sandwich Anais1979 Western Blot Forum 2 09-13-2009 02:33 AM
Sandwich ELISA giving too many positive results fishgirl ELISA Assay Forum 2 02-12-2009 04:20 PM


All times are GMT. The time now is 05:48 AM.


Powered by vBulletin® Version 3.8.4
Copyright ©2000 - 2014, Jelsoft Enterprises Ltd.
Copyright 2005 - 2012 Molecular Station | All Rights Reserved
Page generated in 0.11696 seconds with 16 queries