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high values in both serum and antigen control - troubleshooting sandwich ELISA

high values in both serum and antigen control - troubleshooting sandwich ELISA - ELISA Assay Forum

high values in both serum and antigen control - troubleshooting sandwich ELISA - ELISA Forum. Discuss and post questions including optimization, troubleshooting, protocols and theory of ELISA assays using monoclonal, and polyclonal antibodies or peptides.


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Old 04-21-2010, 06:50 PM
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Unhappy high values in both serum and antigen control - troubleshooting sandwich ELISA



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?
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Old 04-21-2010, 10:40 PM
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Default Re: high values in both serum and antigen control - troubleshooting sandwich ELISA

For starters (make sure I'm right)

Blank ctrl(just antibody) works---blocking not a problem
Neg Serum ctrl--high signal--- should be to very low, similar to blank
Antigen control/quality control---too high--should work at 0.5-0.3X signal of a strong positive

Quick check:
Are you sure that capture antibody coated correctly?--can check coated wells with anti-mouse-HRP
If coating and sample volumes are ~100ul then blocking is at ~300ul.
Not saturating with HRP antibody 1:20K dilution or greater.
Capture and Detection antibody work in Western Blot, or cited in literature or product sheet.

If so:
1. Try minimizing incubation times, ELISA can be very fast, but then the background can creep up on you. Post incubation times,
2. Try adjusting temps (same principle) if doing at 37C, try RT.
3. If on a shaker try no shaker.
4. If doing no dilution then this should be #1 Optimize dilution.
Perform dilution series, check for hook effect, optimize for the correct dilution I'll attach sample pic in next post. Start diluting in Wash (with and without tween, for comparison; probably OK to run with tween).
5. Don't worry too much about the wash buffer, pick 1X PBS 0.5-1%Tween or thereabouts, use. You may need a sample diluent.

Since you are troubleshooting, minimize the samples you are running, pick 1-2 unknowns, 1-2 quality controls, keep stds if any. Don't waste your unknown samples on optimization.
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Old 04-21-2010, 10:50 PM
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Post Re: high values in both serum and antigen control - troubleshooting sandwich ELISA

Plot you serum/sample dilution like the attached pic.
Attached Images
File Type: jpg serum diln hook effect.jpg (56.9 KB, 29 views)
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Old 04-22-2010, 04:21 PM
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Default Re: high values in both serum and antigen control - troubleshooting sandwich ELISA

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.

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

For positive control I am using pooled patients' serum.
For negative control I am using pooled age and sex matched normal peoples' serum.

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.
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