The XENOBIND surface is a proprietary surface developed to covalently attach amino containing molecules to surfaces.  It was originally developed to improve results in ELISA assays.  The reactive groups on the surface spontaneously react with primary and secondary amino groups to create a covalent bond.  The binding capacity of the surface is about 250 ng/cm sq. for an IgG molecule.

 

Prepare a solution of the capture antibody at a concentration of 3 - 5 ug/ml in a buffer at a pH above the isoelectric point of the antibody.  This insures that the amino groups are in the free form and not in the acid form.

 

Incubate in the wells for 1 hour at room temperature or 37 degrees.  Since a chemical reaction is occuring,  trying to carry out this step in a refrigerator is usually not successful.

 

Wash three times with the same buffer.

 

Block with a freshly prepared 1% solution of BSA in water or PBS.  A solution more than a few hours old contains dimers and trimers which are not efffective in blocking.

 

Incubate for 1 hour at room temperature.

 

Wash three times withe same buffer.

 

Incubate with the antigen solution for 1 hour

 

Wash three times.

 

Incubate for 1 hour with the detection antibody.

 

Wash three times.

 

Incubate with the enzyme substrate.  The time depends on the speed of the reaction, but should be standardized for quantitative results.

 

Read the resulting color or signal on a plate reader

 

 

1

 

 

 

2.

 

 

3.

 

4.

 

 

5.

 

6.

 

7.

 

8.

 

9.

 

10.

 

11.

 

 

12.