The XENOBIND surface is a proprietary surface developed to covalently attach amino containing molecules to surfaces. It was originally prepared 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.
XENOBIND Covalent Binding Surface
The XENOBIND surface is available on standard polystyrene 96 well microwell plates and on microwell strip plates. The reactive surface is produced by a liquid phase process which results in a very uniform surface. Other types of surface treatment such as radiation or plasma treatment result in a more hydrophillic surface than the starting polystyrene, but are not as uniform plate to plate or well to well because there are positional differences in the treatment chambers.
The driving force for passive binding is the reduction of interfacial energy which results from covering a hydrophillic surface with a less hydrophillic molecule. Therefore, the presence of a more hydrophobic molecule in the solution will result in a displacement reaction with loss of the bound material. This can be a problem when a blocking step is included the protocol.
With the XENOBIND surface, the molecule is covalently and irreversibly bound to the surface through amino groups on peptides and proteins. Therefore, doing the required blocking step will not result in any displacement of the molecule of interest. This results in improved sensitivity and uniformity in ELISA assays.
We have also found that the results in ELISA assays when using monoclonal antibodies as the capture antibody are sometimes low and inconsistent. This occurs because the amino groups on the Fab part of the molecule is too close to the antigen recognition site on the molecule. Thus the antibody – antigen reaction does not occur due to steric hinderence and poor results are obtained.
In these situations we recommendthat the monoclonal antibody be biotinylated at the Fc end of the molecule and a XENOPROBE streptavidin coated plate be used as a base for the assay.
Clear, White and Black Plates
The XENOBIND surface is available on clear plates and strips as well as on opaque white and black plates. The clear plates are typically used when a colorimetric detection sysytem is used. An antibody linked to an enzyme such as horseradish peroxidase or alkaline phosphatase is used as the detection molecule. Then any of the available enzyme substrates that have a colored reaction product can be used to quantitate the amount of antigen present.
The same procedures are used on the white and black plates until the last step. In that step an enzyme substrate that produces a chemiluminescent signal or a fluorescent signal is used. This has the advantage of three orders of magnitude increase in the sensitiviy of the assay, but at substantially increased cost. These plates can also be used where the detection antibody has a luminescent molecule directly attached to it. No enzyme – substrate combination is needed, and the signal can be read directly. This will have somewhat lower sensitivity then the amplification obtained with the enzyme system, but is faster since it eliminates at least one step.
- 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.