Goat Serum and other doubts - (May/05/2011 )

Hello Everyone,

I have experienced a high background in my indirect ELISA, but a friend recommend me to add 3 to 5% of Goat serum (because my secundary is produced in Goat) in blocking solution. This worked very fine for me and i had low levels of background. From this now, my blocking solution is 10% non-fatty skinned milk, and 3% of goat serum in PBST 0,01%. I let the plates blocking for 2 hours at 37ºC.
So whats is the main importance of Goat serum in the blocking solution? Just milk is not enough?
My other question is about Tween 20: others tell to use 0,01% of Tween in washing solutions and others say don't use tween 20. What would you recommend me ?
The last one: proteases inhibitor and preservatives like azide and thiomersal works fine for in-house ELISA assay? Could they improve the reaction?

Thank you very much

Kind regards!

Reis, V.P.

The goat serum contains goat IgG that binds to and blocks the proteins that your goat antibody (also IgG) would bind to otherwise. The majority of the protein in milk is casein which will block some sites but not all. How effective milk is as a blocker is dependent on the assay and antibody specificity.

In theory tween or other detergents increase the stringency of the washing process - how stringent you want the washes is dependent on your assay. It sounds like you may need it to reduce background.

Protease inhibitors in your lysate should be a given, unless you have a specific reason for excluding them, so as to prevent proteases from degrading your samples. Azide is an anti-fungal and anti-bacterial, and is used in lots of these sorts of protocols, but I don't know if it enhances the ELISA at all. I know nothing about thiomersal, other than that it is used as a preservative in some immunizations and is the target of the anti-immunisation groups for supposedly inducing autism in children (which has been disproved, and the initial papers retracted for falsification of data).

The goat serum (or non specific goat IgG) is usually not added to the plate blocking solution but to the assay buffer or sample diluent or conjugate diluent. It is primarily used to block heterophilic reaction of antibodies in the samples to those used in the assay.

BSA, casein, etc in PBS are standard blocking solutions. I believe tween in blocking solution defeats the purpose of getting proteins adsorbed onto the plastic surface of the plate/wells.

Tween or triton x 100 are common in wash buffers...the higher the concentration the more stringent the wash. Some protocols also have a single dH20 wash after the PBS/Tween washes.

Azide is the most common preservative. Companies moving away from thimerosal as it contains mercury. Proclin and Kathon are other commonly used preservatives.

I forgot to include the preservatives do not enhance the reaction. If you wish to increase the reaction include PEG in the assay buffer.

After coating the plates, you also use the goat serum in the PBST (or just PBS) to block the plates? 37 degree 2 hrs? do you use the human serum? what kind of plate you use? Thanks.

hhyy