Technique / Immunology / ELISA assay

Troubleshooting on establishing ELISA

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	By JASSIM on 31-Aug-2008 I am wark with indirect ELISA ,,I HAVE PROBLEM WITH background with negative serum control of sheep ,i titrate all concentration of Ag(recombinant protien,and dilution buffer also>blocking the concentration and incubation time also but no result OD FOR positive control 2-2.9 and OD for negative serum control 0.8-1.2 Rating: Fair Reply

By Sajani on 29-May-2008 I'm doing an indirect ELISA to detect antibodies that recognize specific peptides. I'm getting some values for my serum controls which shows that there is non-specific binding but i increased the NaCL to 1.5M and using 0.5% tween, my blocking buffer con is 5% non-fat milk. Do you think i need to increase the blocking buffer cause some have written that you can up to 10%... Cause of this i'm getting a higher value for the neg controls rather than for my positive controls. And the other problem is that i get very low values which makes it harder to calculate a cut off value..Hope anyones got an idea about this... Rating: Excellent! Reply

	By Georgina on 04-Apr-2008 I am trying to validate the indirect ELISA and I am getting low OD values for positive controls Rating: Poor Reply

	By Alfatih on 25-Mar-2008 I've low +ve control absorbtion? Rating: n/a Reply

	By cecilia on 03-Mar-2008 i gotta same problem. Have u resolved yours? if so, how did u do it? Rating: n/a Reply

	By aahma on 27-Sep-2007 I am running a direct ELISA and I get signal one day and no signal the other day. I am not sure what is causing I changed all buffers and am using HRP conjugated Sec. Antibody. I checked for Azide too and that does not seem to be the problem. Does any one have some tips? Rating: Excellent! Reply

	By Chanda on 15-Jul-2007 Iam trying to optimise my ELISA conditions and everytime I do the experiments I get s amll linaer portion and a larger non linear portion. I have tried to change my substrate but the situation has become even worse in that my first two dilution cannot be read by a microtitre palte reader. Rating: Excellent! Reply

	By magdy on 23-Apr-2007 I use ELISA in my lab. for qunatitation of Hormones and other Medicale matters I suffer from Temperture control for every test so If I do all test at 37 c begaining from calibrator s and measure the samples also at this temperature are this suugestion is right or not and what is your sugestion then Rating: Good Reply

	By DEBASHREE on 10-Jan-2007 I am using indirect ELISA to detect some proteins, and i am getting very high readings for the test while the control gives good result. My primary aswellas secondary antibody concentration is 1:5000, which worked well for ELISA for other related oroteins Rating: n/a Reply

	By DEBASHREE on 10-Jan-2007 I am using indirect ELISA to detect some proteins, and i am getting very high readings for the test while the control gives good result. My primary aswellas secondary antibody concentration is 1:5000, which worked well for ELISA for other related oroteins Rating: n/a Reply

	By Josephine on 12-Jul-2005 MY healthy cowpea control which looked healthy visually is reacting with the antibody while my samples gave the expected result, what cuold be wrong? Rating: n/a Reply

	By Virendra Pathak on 23-Mar-2005 Excellent Rating: Excellent! Reply

	By Cara Lunn on 17-Feb-2005 I am using indirect ELISA to detect a fern spore protein, and I am getting positive results in my preimmune serum-secondary antibody wells. What might be causing this? My secondary antibody concentration is 1:500, which worked well for ELISA with fern gametophytes. Rating: Good Reply