Advanced procedure for oriented conjugation of full-size antibodies with quantum dots

Ideal nanoparticle-based nanoprobes should contain on their surface homogeneously oriented highly active affinity molecules, e.g., antibodies (Abs), and should not exceed 15 nm in diameter. Direct conjugation of quantum dots (QDs) with Abs through cross-linking of QD amines with the sulfhydryl groups resulting from the reduction of the Ab disulfide bonds is a generally accepted technique. However, this procedure yields conjugates where Abs are oriented irregularly. This decreases the number of functionally active Abs on the nanoparticle surface, because some Ab recognition sites face inwards and cannot interact with the target moieties. Here, we describe an advanced procedure of Ab reduction, affinity purification, and QD-Ab conjugation with optimized critical steps. We have developed a method for partially reducing the Abs yielding highly functional 75 kDa heavy-light chain Ab fragments. Affinity purification of these Ab fragments followed by their tagging with QDs results in QD-Ab conjugates with largely improved functionality compared to those obtained according to the standard procedures. The new approach can be extended to conjugation of any type of Abs with different semiconductor, noble metal, or magnetic nanocrystals.