Preparing and purifying antibodyDNA conjugates

There are many heterobifunctional cross-linking agents suitable for conjugation of a DNA-label to a detection antibody. They have two reactive groups, one that reacts with the antibody, and a second that reacts with the DNA-label joining them together. We usually use SMCC (Figure 12.6), which reacts with an amino-group and a thiol-group. Thiol-groups can be added to antibodies by treating them with 2-iminothiolane (Traut's reagent), which reacts with primary amines introducing the sulfhydryl groups. The polynucleotide can be synthesized with an artificial amino-group in either end for coupling to SMCC. Conjugation is performed by first reacting the polynucleotide with SMCC and then reacting the SMCC-polynucleotide conjugate with the sulfhydryl activated antibody (Figure 12.6).

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