Prelude: You are free to copy and distribute these documents at will--but please do so in their entirety, complete with figures. To reference this material, please include the WWW address , shown below, as well as the latest modification date: August, 2004, plus any references listed with the detailed conjugation protocol that you use.
Typical reference: M. Roederer,
Conjugation of monoclonal antibodies (August, 2004). http://www.drmr.com/abcon/
Address any questions, comments, or suggestions toMario Roederer.
In this series of web pages, protocols, notes, and various illustrations are given to aid in the conjugation of proteins--principally
monoclonal antibodies--to fluorescent dyes. These conjugation procedures are commonly performed in our laboratory--we have conjugated
several hundred different monoclonals using almost all of the various dyes listed. The procedures are relatively straightforward and
require only minimal familiarity with standard laboratory techniques (gel filtration and spectrophotometry are the most difficult!).
Virtually
every protocol is designed for the conjugation of IgG (see below for conjugating other proteins). Most of these protocols will work
just fine with any isotype--even IgM--or any Ig from any animal species. However, the reductive cross-linking procedures, used for
conjugation of phycobiliproteins (PE and APC), their tandem derivatives, and Texas Red, may not work well for some IgM antibodies.
Nonetheless, it is probably worth a try--we have had stunning success conjugating IgM antibodies with PE, APC, and Cy7APC.
The conjugation
of immunoglobulins is very straight-forward--any laboratory should be able to make these procedures routine. For conjugation of PE
and APC, however, the raw material can be expensive when purchased in the small quantities needed for conjugation. If you intend to
conjugate many antibodies (e.g., 100 mg of various antibodies), consider buying the phycobiliproteins in bulk (1 gram lots) from a
vendor. If you are only conjugating small amounts, it is worthwhile considering the relatively inexpensive conjugation kits available
from ProZyme, Inc.--these kits come with all of the materials you need to prepare PE or APC conjugates, and let you perform the entire
conjugation in 2 hours. These conjugation kits were developed based on the protocols described here. Other conjugations (like FITC,
Cascade Blue, etc.) require relatively inexpensive raw materials--allowing you to optimize the conjugations in-house without great
expense.
There are also protocols given for conjugation of these fluorescent molecules to Annexin V. These protocols can serve as a
models for the fluorescent conjugation of nearly any protein.
No protocol is given for antibody purification after conjugation (e.g.,
Protein A or Protein G). In general, we do not purify our conjugates directed against cell-surface antigens, since unreacted dye is
removed during the washing steps. Our conjugates generally have low background levels. However, you may find it useful to purify conjugates,
especially if they are to be used for intracellular (cytoplasmic) staining.
The conjugations fall into four basic protocols: Type 1
(used for FITC, Cy5, and the initial preparation of the Cy5 and Cy7 tandem dyes); Type 2 (used for Biotin and Cascade Blue); Type
3 (used for conjugation of PE, APC, TR-BSA, and their derivatives); and Type 4 (used for the initial preparation of non-immunoglobulin
proteins like Annexin V). Buffers for all Type 1 reactions are identical, as for Type 2, etc.; some buffers are identical across the
different reaction protocols.
Procedures for the conjugation of immunoglobulins are provided for the following molecules: