Application of click chemistry for signal amplification in IHC and ISH assays

A conjugate having Formula (IIa):

• wherein
• A is selected from the group consisting of dibenzocyclooctyne, trans-cyclooctene, azide, tetrazine, maleimide, thiol, 1,3-nitrone, hydrazine, and hydroxylamine;
• ‘Linker’ is a branched or unbranched, linear or cyclic, substituted or unsubstituted, saturated or unsaturated, group having between 2 and 80 carbon atoms, and optionally having one or more heteroatoms selected from O, N, or S;
• R1 is a group selected from phosphate, amide, urea, sulfate, ester, beta-lactam, or a sugar;
• R2 is a halide;
• R3, R5, and R6 are independently selected from hydrogen or an aliphatic group having between 1 and 4 carbon atoms;
• R4 is a hydrogen, an aliphatic group having between 1 and 4 carbon atoms, or the group —CH(R2)—R7-[Linker]-A;
• and
• R7 is selected from the group consisting of —(CH2)wNH—, —O(CH2)wNH—, —N(H)C(O)(CH2)wNH—, —C(O)N(H)(CH2)wNH—, —(CH2)wO—, —O(CH2)wO—, —O(CH2CH2O)w—, —N(H)C(O)(CH2)wO—, —C(O)N(H)(CH2)wO—, —C(O)N(H)(CH2CH2O)w—, —(CH2)wS—, —O(CH2)wS—, —N(H)C(O)(CH2)wS—, —C(O)N(H)(CH2)wS—, —(CH2)wNH—, —C(O)N(H)(CH2CH2O)wCH2CH2NH, —C(O)(CH2CH2O)wCH2CH2NH—, —C(O)N(H)(CH2)NHC(O)CH(CH3)(CH2)wNH—, or —N(H)(CH2)wNH—, where w is an integer ranging from 1 to 12.