Notes: Reductive cross-linking of antibodies

Reductive Cross-linking of Proteins to Antibodies

This procedure is useful for site-specific conjugation of proteins (such as the phycobiliproteins PE and APC, as well as their tandems) to antibodies. It is used in our antibody conjugation protocols for all phycobiliprotein-based fluorescent dyes, as well as conjugation of Texas Red BSA to antibodies. Finally, a version of it is also used to conjugate these fluorescent proteins to non-immunoglobulin proteins (such as Annexin V).

The concept of the conjugation is simple: Derivatize the fluorescent protein such that it has sulfhydryl-reactive maleimide group, and then reduce the hinge disulfide on the immunoglobulin to provide the reactive site. The first derivatization is accomplished using the heterobifunctional cross-linking reagent "SMCC". This molecule has two ends: one reacts with primary amines; the other end is a maleimide group that will react with free sulfhydryls.

SMCC is conjugated to the fluorescent proteins through simple amino-reactive conditions (i.e., basic pH), such that a few molecules are covalently conjugated to each protein. This derivatized protein is purifed away from the free SMCC. It is actually stable for extended periods of time; we have stored SMCC-conjugated phycobiliproteins in the MES buffer for several months in the refrigerator and then mixed them with the reduced immunoglobulins--with no notable loss in activity.

The immunoglobulin is treated with DTT to expose free sulfhydryls, and exchanged over a column to remove free DTT. Probably only one of the suflhydryls is then derivatized by the SMCC-protein conjugate (due to steric hindrance). The real advantages of this protocol thus becomes apparent: the conjugation is well-defined, and the conjugation does not interfere with the binding site of the antibody.

After the covalent coupling of the SMCC-protein to the immunoglobulin, NEM is added to block any more free sulfhydryls (on the immunoglobulin). Then, the reaction mixture is exchanged onto storage buffer, which also removes free NEM. Note that the final reagent can have unconjugated fluorescent proteins (e.g., unconjugated PE). Normally, this protein washes off after the cell-staining step. In case of excessive background or in conditions where washing may not be performed, it may be desirable to purify the product using Protein A or Protein G columns. Fractionation by sizing can be difficult, since the combined molecular weight (e.g.) of an IgG-PE conjugate is 400,000, compared to 240,000 for free PE.

Interestingly, many IgM's can also be derivatized by this protocol; each particular antibody should be conjugated by this protocol and tested for staining.