Question:
What assay development accessories should be used for protein estimation?
The Protein Man Says:
Now that we have covered biotin-binding and protein/peptide binding plates, it's about time we talk about antibody binding plates. So, what are antibody-binding plates and what are their most common applications? Find out what you need to know by reading the rest of this article.
Antibody Binding Plates
Species-specific binding plates are activated plates designed to bind specifically with mouse or rabbit IgG antibodies. These plates are suitable for working with low quantities of antibodies or antibodies that are easily denatured upon direct binding to polystyrene plates, and can be used for direct, indirect, competitive and sandwich assays. Since these plates exhibit specificity to IgG, purified antibodies are not essential for the success of your research.
Plates that are designed to bind constant (Fc) domains of antibodies offer maximum exposure of the
binding site by ensuring that the antigen binding domain of the antibody is oriented away from the plate. These plates are coated with protein A/G which contains four binding sites from protein A and two from protein G to ensure maximum range of specificity and binding capacity.
Due to the unique orientation of the immunoglobulin in these plates, their antibody capacity is far more superior as compared to plates that are directly coated with antibodies. However, please take note that the plates are suitable for single antibody assays and not for multiple assays or sandwich ELISAs.
There are also some plates that are designed to bind the kappa light chains of immunoglobulins without interfering with the antigen binding site. These protein L coated plates bind to all classes of IgG (including IgG, IgM, IgA, IgE and IgD) and also bind to single chain variable fragments (scFv and Fab fragments). They only bind kappa I, III and IV in human and kappa I in mouse, have weak binding affinity for rabbit immunoglobulins and have no binding affinity for bovine, goat or sheep immunoglobulins. These plates are pre-blocked to reduce non-specific binding capability and are designed for single antibody assays.
Detection Substrates
While the ELISA principle is really quite simple, you may need some help in optimizing and perfecting your assay. As such, you may want to use complete ELISA kits for the accurate detection of horseradish peroxidase or alkaline phosphatase.
