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Very high background in ELISA


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#1 shilpeejnc

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Posted 26 September 2010 - 09:42 AM

hi.I am screening HIV patient sera for Ab response against one antigen.I am doing indirect ELISA ,where coating of Ag is done for 2 h.I am using southern biotech isotyping kit for sec Ab.I get very high background in my no Ag control. Its almost equal to that in presence of Ag.I have tried blocking with different concentraions of BSA, FCS and horse serum.can any body please help me how to solve this problem.

#2 sgt4boston

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Posted 27 September 2010 - 02:56 AM

Either the patient's samples are reacting with the blocking agent or the secondary antibody is sticking. Does the 2nd ab react in absence of patient's sample? Eliminate this possibility first.

If the patient's samples are reacting you can pre-incubate them with the blocking solution before testing. Can you dilute your sample to any degree..say 1:10?

Do you have surfactant in the wash solution? You can also allow the wash to sit in the wells for 1 minute before decanting/blotting.

Lastly, and I don't think it applies at this time are 'heterophile abs' in sample.

#3 shilpeejnc

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Posted 27 September 2010 - 06:23 AM

2nd ab doesn't react in absence of patient's sample.I also diluted my patients sera 1: 100 in 1%fcs + pbs.What is the maximum incubation of secondary AB? I do at 37 d for one hour.I give washing with 0.1% PBST, which itself is very high.

Edited by shilpeejnc, 27 September 2010 - 06:28 AM.


#4 sgt4boston

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Posted 27 September 2010 - 11:24 AM

1. I have a question...how does the dose response curve look? Does the zero show any 'background'? Hopefully, you are using the least (lowest; most dilute) concentration of conjugate.

2. As you are diluting the patient's samples...1:100 which is good and using some fcs. Incubate the sample and diluent one hour is fine. The purpose being to adsorb out any interference. You could also use the same animal type as the secondary ab( conjugate) which could be goat or rabbit etc or mix all three.

3. I don't understand your question for incubation of secondary ab. this could be 15 min to one hour.

4. Can you describe your coating/blocking steps?

#5 shilpeejnc

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Posted 28 September 2010 - 09:43 PM

COATING OF AG I DO IN carbonate buffer pH-9.6, in Nunc strips for 2 h at 37.In my no Ag control I add only plain buffer.Blocking I do with 1% BSA, 1 h at 37.Response is not very high. It remains below OD 4.but the problem is that my no Ag gives equal od to that of Ag, so I am not able to conclude any thing.

#6 sgt4boston

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Posted 29 September 2010 - 02:37 AM

Do you have any positive 'control' sera to test. Known positives; or any commercial control material. Your test is to screen samples but what is serving as your positive and negatives?

#7 shilpeejnc

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Posted 29 September 2010 - 06:57 PM

Do you have any positive 'control' sera to test. Known positives; or any commercial control material. Your test is to screen samples but what is serving as your positive and negatives?

for negative I take HIV negative sera. for positive I dont take any thing sepsrately. But why this question?




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