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 immunodiagnostics because it cannot be covalently coupled to protein. Therefore, methylene blue derivatives having a succinimido or maleimido functional 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 residues in the methylene blue derivative before coupling to protein alleviated the precipitation problem, and up to 36 methylene blue dye molecules could be attached to an antibody fragment using bovine serum protein 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 should also be useful for photodynamic tumor therapy where it is desired to attach a red-absorbing photoactive dye to antibody protein.