sandwich elisa - high values in control wells

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Namalee
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sandwich elisa - high values in control wells

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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?

 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.
 
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. 
 
 
 

vanshita
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i think there must be a

i think there must be a problem related to non specific binding.use 150mM Nacl and 1%tween for washing.