Conjugation of monoclonal antibodies
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 as well as the latest modification
date: August 15, 1996, plus any references listed with the detailed conjugation
protocol.
Address any questions, comments, or suggestions to Mario 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. Most of
these protocols will work just fine with any isotype--even IgM. 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 most IgM antibodies. Nonetheless, it is probably worth a try--we have
had stunning success conjugating some IgM antibodies with PE, APC, and Cy7APC.
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 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:
In addition, protocols for conjugation of Annexin V to the following molecules
are given as models for fluorescent conjugation of non-immunoglobulin proteins.
Conjugation of other fluorescent molecules would proceed very similarly
to the protocols listed below (use as a template the protocol Type that
is appropriate for the molecule you are interested int).
You may view the fluorescence spectra of the
fluorescent molecules listed above (all together), or view individual fluorescence
spectra for:
You can also view absorbance spectra for:
Credits:
Many people have contributed to the successful conjugations that we routinely
perform in the laboratory. In particular, Aaron Kantor designed and optimized
many of the basic protocols referenced in these pages, and made the chemical
conjugations simple for all of the biologists here. Randy Hardy and Alan
Stall devised the reductive cross-linking protocols used for all the protein-protein
conjugations. Other people who have been helpful in the development of the
more recent protocols include Masahiko Amano, Michael Anderson, Stephen
De Rosa, Rachel Gerstein, Peter Katsikis, and Gina Jager.
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CD ROM Vol 2 was produced by staff at the Purdue University Cytometry Laboratories
and distributed free of charge as an educational service to the cytometry community.
If you have any comments please direct them to Dr. J. Paul Robinson, Professor & Director, PUCL,
Purdue University, West Lafayette, IN 47907. Phone:(317) 494-0757; FAX (317) 494-0517; Web http://www.cyto.purdue.edu
EMAIL robinson@flowcyt.cyto.purdue.edu