Antibodies for fluorescent molecular rotors.

We have prepared monoclonal antibodies for the fluorescent molecular rotors 9-(2-carboxy-2-cyanovinyl)julolidine (CCVJ) and 9-(dicyanovinyl)julolidine (DCVJ). Mouse monoclonal antibody (IgG2b) prepared against CCVJ-conjugated bovine serum albumin strongly bound CCVJ and DCVJ. The CCVJ (or DCVJ) binding to IgG and Fab was accompanied by a drastic increase in fluorescence quantum yield, suggesting the restriction of intramolecular rotational relaxation about the donor-acceptor bond of the fluorophores. Nonspecific IgG never changed the quantum yield of the fluorophores. From the Scatchard plots, the association constants of CCVJ to IgG and Fab were 6.8 x 10(7) and 5.4 x 10(7) M-1, respectively, and the numbers of moles of CCVJ bound per mole of IgG and Fab were calculated to be 2.0 (+/- 0.1) and 1.0 (+/- 0.05), respectively. The fluorescence spectra of the IgG-bound CCVJ were quite similar to those of Fab-bound CCVJ. The fluorescence lifetimes of the IgG-bound and Fab-bound CCVJ were 388 and 383 ps at 25 degrees C, respectively. They were 6.3 times as long as the fluorescence lifetime of CCVJ free in solution (62 ps). These results indicated that the drastic increases in quantum yields were due to the decreases of the nonradiative rate constants of the antibody-bound CCVJ, as well as due to the changes of the intrinsic radiative rate constant, and that the nonradiative internal rotations about the donor-acceptor bond of CCVJ were not dependent on the size of the bound antibody molecules.(ABSTRACT TRUNCATED AT 250 WORDS)