Biomedical Engineering Reference
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quantitative experimental results have been reported. We previously reported derived
numbers of functional IgG antibodies conjugated to quantum dots based on calcula-
tions of quantitative electrophoresis experiments using two different conjugation
schemes: a common direct covalent conjugation using a reduced disulfi de maleim-
ide reaction and biotinylated antibodies bound to streptavidin-functionalized quan-
tum dots. Antibody-quantum dot complexes were run in a sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to separate the functional
component of conjugated antibodies from the quantum dots. We, then, blotted the
antibody fragments onto a membrane to determine the identity and amount of the
antibodies, and quantitatively compared the degree of functional binding to known
protein standards to derive the number of bound antibodies.
The number of functional antibodies covalently bound to commercially available
quantum dots was on average much less than one functional IgG molecule per quan-
tum dot (0.076 + 0.014) and therefore of limited utility for biological experiments.
In contrast, antibodies bound to quantum dots via the streptavidin-biotin system
resulted in higher numbers of functional antibodies, with 0.60 + 0.14 IgG molecules
per quantum dot for a 1:1 IgG:quantum dot molar ratio and 1.3 + 0.35 IgG molecules
per quantum dot for a 2:1 ratio. In addition to these specifi c results, our methods
may be of broader interest because our approach is easily extendable for experimen-
tally deriving the number of functional antibodies or peptides bound to other classes
of nanoparticles (e.g., magnetic nanoparticles).
We begin by considering the covalent conjugation of antibodies to quantum dots.
Prior to their conjugation, antibodies were reduced using dithiothreitol (DTT),
which generates three distinct fragments identifi able by their molecular weights: a
25-kDa light chain, which importantly includes the functional-specifi c epitope-
binding site for a particular IgG molecule; a 50-kDa heavy chain; and a 75-kDa
partially cleaved chain consisting of a heavy chain and a light chain held together by
an unreduced disulfi de bond (Fig.
3a
). Following this, individual fragments were
covalently bound to quantum dots via an
N
-succinimidyl 4-(maleimidomethyl)
cyclohexanecarboxylate (SMCC) linkage bond which cannot be broken by DTT
treatment, an important consideration for the interpretation of the experimental
results that follow. This gives rise to three possible binding scenarios to quantum
dots (Fig.
3a
): covalently bound light chains, covalently bound heavy chains, and
covalently bound heavy-light chain partial fragments, of which only the latter can
undergo further DTT reduction to remove the light chain fragment from heavy
chains that remain bound to quantum dots or heavy chains removed from light
chains bound to quantum dots.
We fi rst confi rmed that antibodies were indeed covalently bound to the quantum
dots by running IgG-quantum dot complexes though SDS-PAGE with and without
DTT. For DTT-reduced conditions, we observed light chains cleaved from cova-
lently bound partial fragments (Fig.
4a
, lanes 4-6). As expected, this separation
occurred minimally in lanes without DTT (Fig.
4a
, lanes 2 and 3). The presence of
a weak band at the 25-kDa position in nonreduced lanes (Fig.
4a
, lanes 2 and 3) was
due to low concentrations of reducing agents in the gel and running buffers.
Interestingly, we saw no heavy chains being dissociated from light chain-bound
