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High matrix effect upon using normal mouse serum. - (Apr/08/2010 )

I am developing an ELISA to detect immunogenicity reactions to a certain protein. My ELISA involves coating my antigen 1 ug/mlovernight at 4oC followed by blocking with 1% BSA. I then add different dilutions of my purified mouse anti protein Ab (primary) at concentrations 16 - 0.25 ug/ml. I then add my secondary which is goat anti-mouse IgG HRPO followed by TMB. My matrix in this case is lesser than 0.1. However, when I substitute my primary antibody with normal mouse serum at concentrations as low as 0.001%, I still end up with ODs as high as 0.3. Does this mean that my normal mouse serum has heterophile antibodies? If yes, can someone give me a solution. If no, I still need a solution.
Note : My coating buffer is 1X PBS, pH 7.2 and all other dilutions (except block) are in 0.1% BSA.
First wash post coating is with 1X PBS and all other washes are in PBS containg 0.05% Tween 20.

Heeeeeeeeeeeeeeeeelp!!!!!!!!!!!!

-Vinee d Pooh-

You are measuring mouse antibody to specific antigen. You immoblize ag on plates and have a dose response curve with purified ab. However, when you measure actual mouse sera (samples) you have a high background (non zero).

If you are developing a quantatative test your calibrator should be in the same matrix as the samples you are testing. If you take your purified ab and spike it into stripped or heat inactivated sera do you still get a curve?

If you run a panel of normal mouse samples do they give a tight distribution of results or is there a wide range.

Suggestions:
You should be doing several (3-4) washes in between assay steps with your PBS tween. You can allow the wash to sit in the wells for 15 seconds before decanting.
Titer the conjugate, time, volumes to account for the sample matrix instead of the buffer matrix you have been using.
If you think you have mouse ab binding to the BSA and to the conjugate ab (false postive) you can add some goat sera to the diluent of the samples and conjugate.

-sgt4boston-