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high values in control wells - sandwich elisa


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#1 Namalee

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Posted 21 April 2010 - 06:10 PM

Hi All,
I am new to ELISA. I am trying to do a sandwich ELISA to capture an antibody from human serum. The capture antibody used to coat he wells is a anti-human mouse monoclonal antibody and the detection antibody is a anti-human rabbit polyclonal antibody conjugated with HRP. 5% w/v Non fat milk powder is being used as the blocking solution.
I am getting fairly high OD values in both the serum control wells and the antigen control wells (between half to one third of the positive control sample values). The conjugate control is OK, with very low values close to the values from the blank wells.
I have already tried using a very high NaCl concentration in the buffer solutions and a very high Tween concentration in the washing buffer to try to reduce non specific binding.
Could some one please advise me on what might have gone wrong, and what I should do about it?

#2 sgt4boston

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Posted 22 April 2010 - 03:22 AM

Please define what your

Serum Control....stripped serum no human IgG?
Antigen Control....human IgG in buffer?
Positive Control.....real human serum with IgG?
Conjugate Control...??

are?

If you run the test with all components and use buffer as sample do you have high signal?
If you run the test with all components and use stripped (no ab) human sera as sample do you have high signal?
If you run wells blocked only no capture ab with the above 2 samples do the wells have high signal?

#3 Namalee

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Posted 22 April 2010 - 07:34 AM

dear sgt4boston,
Thanks for offering to help me. I may have got the names of the controls mixed up.

By Serum Control I meant wells where,
capture antibody (anti-human mouse monoclonal) was NOT coated (phosphate buffer was added instead),
human serum (with IgG2 in human serum, which is what I need to detect) was added,
detection antibody conjugate (anti-human rabbit polyclonal conjugated to HRP) was added.
These wells have fairly high OD values, on average around 0.130 mostly.

By Antigen Control I meant wells where,
capture antibody was coated,
human serum was NOT added (dilution buffer was added instead),
detection antibody conjugate was added.
These wells have OD values around 0.100 on average.

By Conjugate Control I meant wells where,
capture antibody was NOT coated (phosphate buffer was added instead),
human serum was NOT added (dilution buffer was added instead),
detection antibody conjugate was added.
These wells have low OD values always less than 0.010, and almost the same as the OD values of the blanks.

For the Positive Control I am using pooled patients' serum.
For the Negative Control I am using pooled age and sex matched normal peoples' serum.

All wells exept blanks, were blocked with blocking buffer (5% non fat milk powder) for exactly one hour.

The patient' serum samples, normal control peoples' serum samples, pooled patient serum and pooled normal control serum give OD values ranging between 0.250 to 0.600 roughly, with the majority of the values being between 0.300 and 0.400, so the values in the control wells are too high to ignore, aren't they?

The method I am following has been used by other people in the past, but for a different group of patients. The records indicate that they have always had low OD values in all three types of control wells.

Could you please advise me on what the problem might be and what I should do about it.







Please define what your

Serum Control....stripped serum no human IgG?
Antigen Control....human IgG in buffer?
Positive Control.....real human serum with IgG?
Conjugate Control...??

are?

If you run the test with all components and use buffer as sample do you have high signal? YES
If you run the test with all components and use stripped (no ab) human sera as sample do you have high signal? I don't have stripped human serum
If you run wells blocked only no capture ab with the above 2 samples do the wells have high signal? YES


Edited by Namalee, 22 April 2010 - 07:40 AM.


#4 sgt4boston

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Posted 22 April 2010 - 11:49 AM

Ok here are some suggestions:

You described using patient and normal patient samples, pools etc. Did you confirm by a reference method what the patients' sample IgG2 values are? If not you can purchase IgG2 myeloma protein and create standards in buffer and 'stripped' serum. (I think Binding Site or Sigma may sell this ~~ $100-200.

You indicated OD values of 0.25 to 0.6 but did not provide what the concentrations are...that is why I think you need a purified reference material before jumping in with actual samples. Run a dose response curve over several logs in buffer first. ODs should run from 0.1 to 2.0. Confirm the analytical range of your assay to be sure it has the sensitivity you need for your samples.

It could be that your background 0.01 to 0.13 is OK but your samples have low levels of IgG2.

#5 Namalee

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Posted 23 April 2010 - 10:08 AM

Ok. Thanks. I'll try that and see. :blink:

Ok here are some suggestions:

You described using patient and normal patient samples, pools etc. Did you confirm by a reference method what the patients' sample IgG2 values are? If not you can purchase IgG2 myeloma protein and create standards in buffer and 'stripped' serum. (I think Binding Site or Sigma may sell this ~~ $100-200.

You indicated OD values of 0.25 to 0.6 but did not provide what the concentrations are...that is why I think you need a purified reference material before jumping in with actual samples. Run a dose response curve over several logs in buffer first. ODs should run from 0.1 to 2.0. Confirm the analytical range of your assay to be sure it has the sensitivity you need for your samples.

It could be that your background 0.01 to 0.13 is OK but your samples have low levels of IgG2.






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