RAPID SYNTHESIS OF BIOTIN-LABELED, DIGOXIGENIN-LABELED, TRINITROPHENYL-LABELED, AND FLUOROCHROME-LABELED TYRAMIDES AND THEIR APPLICATION FOR IN-SITU HYBRIDIZATION USING CARD AMPLIFICATION

A one-step procedure for the synthesis of different tyramide conjugate s, which can be utilized in the catalyzed reporter deposition (CARD) a mplification system, is described. Succinimidyl esters of biotin, digo xigenin, and of the fluorochromes fluorescein, rhodamine, aminomethylc oumarine acetic acid, and Cy3 were coupled to tyramine in dimethylform amide (DMF) adjusted to a pH of 7.0-8.0 with triethylamine (TEA). The coupling reaction can be performed within 2 hr and the reaction mixtur e can be applied without further purification steps. Furthermore, trin itrophenyl (TNP)-tyramide was prepared by adding 2,4,6,-trinitrobenzen esulfonic acid to tyramine dissolved in either MilliQ/DMF basified wit h TEA or in an NaHCO3 (pH 9.5) buffer. A subsequent precipitation of t he TNP-tyramide resulted in a high-yield isolation of this conjugate. The synthesized tyramide conjugates were applied successfully in singl e-and multiple-target in situ hybridization (ISH) procedures to detect both repetitive and single-copy DNA target sequences in cell preparat ions with high efficiency. The described approach provides an easy and fast method to prepare a Variety of tyramide conjugates in bulk amoun ts at relatively low cost.