PHOTOACTIVE METHYLENE-BLUE DYE DERIVATIVES SUITABLE FOR COUPLING TO PROTEIN

Methylene blue is a very strong photoactive dye that has an absorption peak (668 nm) that corresponds well to a popular low-cost diode laser . However, it has not been used in photodynamic tumor therapy and immu nodiagnostics because it cannot be covalently coupled to protein. Ther efore, methylene blue derivatives having a succinimido or maleimido fu nctional group were synthesized and coupled to antibody, serum albumin and transferrin proteins. Incorporation of dye into antibody protein at high ratios (more than three per molecule) caused precipitation and loss of antibody activity. Inclusion of one or more carboxylic acid r esidues in the methylene blue derivative before coupling to protein al leviated the precipitation problem, and up to 36 methylene blue dye mo lecules could be attached to an antibody fragment using bovine serum p rotein as a carrier. Methylene blue derivatives and protein complexes formed from them oxidized luminol when stimulated with red light. The new dye conjugates were used in an optically pumped chemiluminescence immunoassay for alpha-fetoprotein. These compounds and techniques shou ld also be useful for photodynamic tumor therapy where it is desired t o attach a red-absorbing photoactive dye to antibody protein.