Troubleshooting on establishing ELISA

gud. u try to make changes in ur diluent part.10-20% goat serum with 2% BSA can do better reaction. after using this diluents if u get background reaction u can add 5% sucrose to the same diluent. it can do better.
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I'm having two problems that I'd like some help with. First, I'm doing a series of serial 1:3 dilutions for my standard starting with 600ng/mL in A1-3 and ending with 0.274ng/mL in H1-3 and my OD values in the B wells are consistently higher than the ones in the A wells. What's happening to make the higher concentration A wells not perform as well? Second, for the past month I've been running the ELISA I'm doing just fine (with the exception of the first problem). However, since September started the values of my standards have gone down 0.3 OD values. This in turn has caused my known control samples to increase in value because the OD values have not gone down 0.3 for them. How do I fix this?
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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
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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...
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I am trying to validate the indirect ELISA and I am getting low OD values for positive controls
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I've low +ve control absorbtion?
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i gotta same problem. Have u resolved yours? if so, how did u do it?
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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?
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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.
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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
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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
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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
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MY healthy cowpea control which looked healthy visually is reacting with the antibody while my samples gave the expected result, what cuold be wrong?
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Excellent
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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.
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